CN102721814A - F protein detection reagent and detection method therefor - Google Patents

Abstract

The invention relates to an F protein detection reagent and a detection method therefor. The F protein detection reagent comprises rabbit anti-human polyclonal antibodies of an F protein, enzyme-labeled antibodies, color reagents and a stop solution. The enzyme-labeled antibodies are horse radish peroxidase-labeled antibodies. The color reagents comprise a solution A containing 0.01% of H2O2, and a solution B which is 0.1mol/L of a citric acid buffer having a pH value of 7.4 and containing 0.4mg/ml of TMB (3,3,5,5- tetramethylbenzidine). The stop solution is 2M of a sulfuric acid aqueous solution. After liver tissue is damaged, a large amount of F proteins are released from liver cells into blood circulation thereby inducing an organism to produce specific cellular and humoral immune responses. Based on the characteristic, an HIS-F protein prepared by a gene engineering technology and highly purified by Ni<2+> column chromatography is adopted so that the diagnosis precision and the diagnosis efficiency are improved and a detection cost is saved. Therefore, the F protein detection reagent has an important theoretical significance and a potential application value in clinical application.

Description

A kind of F protein assay reagent and detection method

Technical field

Patent of the present invention belongs to biomedical engineering and medical test technical field, refers in particular to a kind of F protein assay reagent and detection method.

Background technology

In the scientific research field and clinical medicine application of biomedical engineering about cell; F albumen is a kind of special protein that is present in the liver cell; Have only the liver parenchyma aggrieved Party of working as to be released in the blood, so modern medicine think that F albumen is a kind of special hepatocellular damage mark.The diagnosis of hepatopathy is important ingredient in the medical diagnosis always, and liver F albumen is as a kind of serologic marker that can directly reflect degree of liver, and its serum content becomes positive correlation with the degree of liver that a variety of causes causes.The hepatopathy no matter which kind of reason causes all shows as hepatocellular damage, and F antigen all can raise in the serum.The changes of contents of serum F antigen and liver histological change has close correlativity.Serum F antigen is that liver is peculiar, and susceptibility is very high, and normal human serum F antigen concentration does not have age, gender differences; The time that occurs in the blood when liver cell is impaired early; And can not induce, therefore, serum F antigen is a kind of new for the sensitiveer and special index of conventional liver function project.If process the immunoreagent of F albumen, it will play a significant role at the aspects such as judgement of the screening of hepatopathy early diagnosis and treatment, medicine and observation of curative effect, hepatopathy prognosis.

Obtain at present both at home and abroad the relatively standard of difficulty and lack of uniform of F albumen, be difficult to mutual checking, be unfavorable for going deep into of research work; There is the scholar to extract F albumen both at home and abroad, utilizes this method to be difficult to obtain a large amount of and purer F albumen through the method for purifying; Obtain the gene order report that only acquisition F albumen is cloned in rat of F albumen up to now with the method for gene clone, do not see the clone of people's liver F albumen and the bibliographical information of expression, also do not have corresponding sequence issue simultaneously on the Genebank.

Consulting on a large amount of pertinent literature bases both at home and abroad, the gene order of the rat liver F albumen that our reference and human homology's property are higher has successively designed 10 pairs of different upstream and downstream primers, successfully amplifies people's liver F protein gene sequence.We issue (sequence number DQ188836) with the gene order of human liver F albumen on the GENEBANK of NCBI; And this sequence carried out the Blast comparison; The result shows that the homology of the gene order of the gene order of human liver F albumen and rat liver F albumen is up to 99%.And then utilize the cDNA of acquired F albumen to give expression to F albumen at prokaryotic vector (e. coli bl21 (DE3) pLysS), and make a large amount of preparations of F albumen become possibility, solve it and come source problem; Through improving the purification article that the method for purifying obtains people's liver F albumen on the one hand, on the other hand the F albumen of purifying and the F albumen of clonal expression are verified through immunological characteristic each other; Prepare its polyclonal antibody and monoclonal antibody simultaneously; And through polyclonal antibody test section clinical samples; Come the feasibility of preliminary identification F albumen, for the clinical more solid foundation of establishing is moved towards in the research that makes F albumen as soon as possible from the laboratory as a kind of reagent for clinical diagnosis.

In numerous immunoassaies, (enzyme-linked immunosorbent assay, ELISA) application is the most extensive, the most representative in EUSA.ELISA is the end of the sixties, and is technological by a kind of enzyme mark solid-phase immunoassay of development such as Engvall and Perlmann and Van Weeman and Schuurs on the immunohistochemical basis of enzyme.After this immunoassay simply and easily occurs; Not only become a kind of very easy research tool; And promptly be applied to the Clinical detection of various bioactivators and mark, and in clinical practice, replaced gradually and have environmental pollution, waste liquid to be difficult to the radioimmunoassay of shortcomings such as handling.The basis of immunoassays is the specific binding reaction between the antigen-antibody, and ELISA is as the term suggests be exactly the solid phase adsorption assay method that serves as a mark thing, be the basis with the antigen-antibody immune response with enzyme.The organic component of the mensuration reagent of ELISA comprises three aspects, i.e. antibody or the antigen and the enzyme reaction substrate etc. of solid support and the antigen that encapsulates or antibody, enzyme labeling.The immobilization of antigen or antibody does not influence its immunity and combines activity, and the antigen of enzyme labeling or antibody also are so, and the activity of marker enzyme is not lost because of labeling process.In the whole mensuration; Antigen-antibody binding reaction carries out on solid support; Colour developing or fluorescence or the luminescence-producing reaction of generation of the judgement of reaction result after with enzyme and its substrate-function is criterion, colour developing or produce intensity of fluorescence and be directly proportional or inverse relation with the concentration of determinand in the clinical samples.The antigen that was used for immunoassays in the past is generally various purifying antigens; In recent years along with the development of Protocols in Molecular Biology; The use gene engineering method prepares various special antigens or immunoassay reagents such as antibody and enzyme conjugates thereof have become a reality; The assay method of various novel practicals constantly occurs, and has further widened the evolutionary path of immunoassay.

The application of gene engineering antigen in the ELISA method now obtained great expansion, and the trend that replaces artificial purified antigen is gradually arranged.Be that employed antigen is the viral antigen that is extracted behind the cell with scaling agent cracking HIV infection in the HIV antibody mediated immunity is measured the earliest, the antigen purity that obtains like this is relatively poor relatively, therefore influences specificity and the susceptibility of measuring.The ELISA kit that now is used for the HIV antibody test both at home and abroad all uses gene engineering antigen such as gp41, and gp120 and gp160 etc. have improved specificity and the susceptibility measured greatly.HCV at present still can not in vitro culture, can only obtain the various antigen fragments of HCV structural area and non-structural area with the method for genetic engineering or chemosynthesis.Known HCV genome is made up of 7 functional areas; Core, E1, E2/NS1, NS2, NS3, NS4 and NS5 district.Recently, the Chien of U.S. Chiron company etc. has developed the single fusion of 7 all immune decision bases of functional areas of a kind of HCV of comprising genome, is called MEFA7.1-6.The CLIA method of using this fusion to set up, the enzyme immunoassay (EIA) method that the fusion that its mensuration susceptibility is made up of NS3-NS4 core (C25) district than use is set up is wanted high 2-4 times.

In addition; Many in recent years researchers adopt technique for gene engineering recombinant expressed the membrane lipoprotein TpN17 with hyperimmunization originality of microspironema pallidum, TpN44.5 (TmpA) and TpN47 etc.; Set up the ELISA method that syphilis antibody detects with it; Good mensuration specificity and susceptibility have been showed; To become and get the test of your rapid plasma reagin annular lamina (Rapid plasma reagin circle card test is RPR) with the test of conveyor screw hemagglutination (Treponemal pallidum hemagglutination assay, the detection method that syphilis antibody TPHA) is desirable.

By above-mentioned visible, gene engineering antigen has irreplaceable effect to setting up the special method of immunity of highly sensitive height, has become the best source of the pathogen antigen that is difficult to cultivate, has also solved the problem of pathogen cracking extraction antigen purity difference.Take a broad view of future, the application of gene engineering antigen in immunoassays has its unique glamour and wide application prospect.

Summary of the invention

The objective of the invention is to overcome the weak point of prior art, provide a kind of both convenient, fast, safe, the economic again various hepatopathys of detection and the F protein assay reagent and the detection method of hepatocellular injury are filled up domestic blank at this detection range.

The objective of the invention is to realize through following technical scheme:

A kind of F protein assay reagent comprises the anti-people's polyclonal antibody of rabbit, enzyme labelled antibody, chromogenic reagent and the stop buffer of F albumen.

And said enzyme labelled antibody is the horseradish peroxidase labeling antibody.

And said chromogenic reagent is for containing 0.01%H ₂O ₂The WS and pH 7.4, contain the 0.4mg/ml TMB 0.1mol/L citric acid damping fluid of (3,3,5, the 5-tetramethyl connects aniline).

And, the aqueous sulfuric acid of said stop buffer flavor 2M.

And, also comprise damping fluid and cleansing solution.

And the said damping fluid that comprises is PBS 0.01M, and pH 7.4; Said cleansing solution is PBST, and collocation method is: 10 * TBS pH 7.6:Tris-alkali, 60.57g; 1M HCl, 420ml; NaCl, 85-90g, behind HCl accent pH to 7.6, constant volume is in 1L; 1 * TBST:, add 0.1% TWEEN-20 simultaneously with 10 times of 10 * TBS dilutions.

Utilize the F protein assay reagent to detect the method for F protein content, it is characterized in that: step is following:

(1) encapsulate: use the F protein polyclone antibody of purifying to encapsulate by 10 μ g/ml protein contents, 100 μ l/ holes, 4 ℃ are spent the night, 0.01M PBS pH 7.4 washings three times contain the PBS sealing of 1% hyclone, 110 μ l/ holes, 37 ℃ 1 hour, it is for use to abandon coating buffer;

(2) get 96 orifice plates that are coated with the F protein polyclone antibody, add test serum 50 μ l/ holes, reacting hole is sealed with the shrouding film in enzyme labelled antibody 50 μ l/ holes then, 37 ℃ of constant temperature oven incubation 60min;

(3) discard liquid, the thieving paper arsis is done, and PBST is filled it up with in every hole, leaves standstill 1min, gets rid of cleansing solution, and the thieving paper arsis is done, and so repeats to wash plate 3-8 time;

(4) every hole adds 0.01%H ₂O ₂The WS and pH 7.4 each 50ul of 0.1mol/L citric acid damping fluid of containing 0.4mg/ml TMB, 37 ℃ of lucifuges are hatched 15min;

(5) every hole adds stop buffer 50 μ l, measures the OD value in each hole in the inherent 450nm wavelength of 15min.

A kind of F protein assay reagent is used for enzyme and exempts from the purposes that appearance detects F albumen.

A kind of special-purpose F protein detection kit of immunoturbidimetry that is used for, it is characterized in that: reagent is formed as follows:

Reagent R1: contain the antiserum of mouse anti human F protein monoclonal antibody,

Reagent R2: increase turbid dose, antiseptic, surfactant, Tris damping fluid;

Definite value serum R3: the genetic engineering reorganization F albumen that contains quantitative purifying;

Said reagent R1 is a mouse anti human F protein monoclonal antibody, and antiserum concentration is 10 μ g/ml;

The polyglycol-6000 that increases turbid dose of employing 4%g/ml among the said reagent R2,

Said antiseptic is a potassium sorbate;

Said surfactant is 1%SDS v/v, Tris pH of buffer=8.2, and concentration is 20mmol/L.

Said reagent R3 is a F albumen dried frozen aquatic products, and every part of definite value F protein content is 1000 μ g, is dissolved in during use in the 10mlTris damping fluid, and being mixed with concentration is the definite value serum of 100 μ g/mL.

Advantage of the present invention and beneficial effect are:

1, the present invention is a kind of sensitive and special index that can reflect the hepatocellular injury degree according to F albumen; Adopt the method for biochemical purification article that separate the F albumen that the method for purifying obtains human liver and antibody thereof, employing RT-PCR to obtain the cDNA sequence clone of human liver F antigen; And carry out on the basis of prokaryotic expression F albumen; The ELSIA detectable of research and development F albumen is set up a kind of brand-new reagent and method for quick and precisely detecting due to the various hepatopathys hepatic injury.

2, the present invention is through the experimental exploring to each item methodology index of F protein assay reagent such as linearity experiment, sensitivity, repeatability, cross interference optimal reaction time, stability experiment etc.; The result shows, F protein reagent detection sensitivity reach 0.5 μ g/ hole, batch in repeated CV value 5.5%, batch between CV value scope about 10%, with this law in detection F protein process, confirm as 1 hour with no cross reaction, F Protein Detection optimal reaction times such as HbsAg positive control, HbeAg positive control, HAV antigen, HCV antigen, HDV antigen, HEV antigen, HSVI type antigen, HSVII type antigen, Escherichia coli, each ingredient of composition detection kit is put in 4 ℃ of preservations through P/N ratio decline about 15% in 6 months; 100 times of its P/N ratios of positive sample dilution are still in the range of linearity.

3, the present invention is discharged into the circulation from liver cell in the liver organization back that sustains damage according to F albumen in a large number, can induce body to produce specific cell and HI, adopts the genetic engineering preparation and through Ni ²⁺HIS-F albumen after column chromatography is highly purified has improved the degree of accuracy and the diagnosis efficiency of diagnosis, has practiced thrift the detection cost, makes it aspect clinical practice, have important in theory meaning and potential using value.

Description of drawings

Fig. 1 is a cloning vector pUCm-T synoptic diagram of the present invention;

Fig. 2-1 is an expression vector pET-15b synoptic diagram of the present invention, and Fig. 2-2 subordinate list reaches carrier critical sites locating information;

Fig. 3 is Western blot method result of the present invention;

Fig. 4 is used for the typical curve of F determination of protein concentration.

Embodiment

Below in conjunction with embodiment, the present invention is further specified, following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.

Consulting on a large amount of pertinent literature bases both at home and abroad; The gene order of the rat liver F albumen that reference and human homology's property are higher has successively designed 10 pairs of different upstream and downstream primers; Successfully amplify people's liver F protein gene sequence; The gene order of human liver F albumen is issued (sequence number DQ188836) on the GENEBANK of NCBI; And this sequence carried out the Blast comparison, the result shows that the homology of the gene order of the gene order of human liver F albumen and rat liver F albumen is up to 99%.And then utilize the cDNA of acquired F albumen to give expression to F albumen at prokaryotic vector (e. coli bl21 (DE3) pLysS), and make a large amount of preparations of F albumen become possibility, solve it and come source problem; Through improving the purification article that the method for purifying obtains people's liver F albumen on the one hand, on the other hand the F albumen of purifying and the F albumen of clonal expression are verified through immunological characteristic each other; Prepare its polyclonal antibody and monoclonal antibody simultaneously; And through polyclonal antibody test section clinical samples; Come the feasibility of preliminary identification F albumen, for the clinical more solid foundation of establishing is moved towards in the research that makes F albumen as soon as possible from the laboratory as a kind of clinical detection reagent.

One, the preparation method of F protein assay reagent is following:

1, the preparation of F albumen and purifying: total RNA of the normal person's of extraction traffic death fresh liver tissue, adopt the method for RT-PCR to obtain genes of interest, carry out agarose electrophoresis testing goal gene.With pUCm-T is cloned plasmids; PET-15b is an expression vector; E. coli bl21 (DE3) pLysS is final expression strain.Through IPTG abduction delivering destination protein, use Ni ²⁺-charged NTA His-binding column purifying F albumen adopts the purity of SDS-PAGE testing goal albumen then, and utilizes Western-blot to carry out specificity and identify.

Wherein, the extraction of people's liver F albumen and purification process are following:

The dead normal person's liver of the misfortune of picking up the car, after low-temperature homogenate, saturated ammonium sulphate precipitation are slightly carried again through DEAE-Cellulose 52, Sephacryl S-200 and CM-Cellulose-23 column chromatography, after polyacrylamide gel electrophoresis separate.Human liver organization obtains rough F albumen behind homogenate, centrifugal and ammonium sulphate precipitation; The F albumen that the purity that behind DEAE-Cellulose 52, Sephacryl S-200 and CM-Cellulose-23 column chromatography, obtains again is higher, the F albumen behind chromatography carries out the polyacrylamide gel electrophoresis.In the process that people's liver F albumen is purified; All through identify the specificity of the F albumen of putting forward with the anti-F protein antiserum of BALB/c mouse LH immunity C3H mouse, its purity was identified and was adopted sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) each step.Adopt IPTG abduction delivering people liver F albumen, use Ni ²⁺-charged NTA His-binding column carries out purifying.

2, the preparation of anti-F protein polyclone antibody:

Using physiological saline is 100 μ g/ml concentration with the F albumen dilution of purifying, gets solution 1ml and the abundant mixing of 1ml Fu Shi Freund's complete adjuvant, with the abundant mixing of Fu Shi Freund's complete adjuvant, gets the purebred White Rabbit of 2ml mixing liquid immunity New Zealand.First immunisation, the emulsification antigen of every rabbit injection 2ml is located subcutaneous multi-point injection behind both sides, back, ear etc.2 all backs are with same dosage booster immunization, back multi-point injection.Booster immunization once more after 10 days.Carry out booster immunization after 10 days the 3rd time.After the last immunity, antibody titer reaches requirement and promptly gets blood from jugular vein, obtains a large amount of antiserums.Adopt saturated sulfuric acid amine salt to analyse method, ion-exchange chromatography method and DEAE-Sephadex-A50 column chromatography successively polyclonal antibody is carried out purifying.

3, Monoclonal Antibody: after adopting F albumen that genetic engineering obtains as antigen immune BALB-C mouse; Measure antibody titer in the serum with the ELISA method; Choose 1: 72000 BALB-C mouse of antibody titer and prepare splenocyte; Merge with the myeloma cell, culture supernatant has 15 hole antibody positives after the Fusion of Cells, and antibody titer is up to 1: 25600.The Western-blotting test shows the monoclonal antibody of preparation and people's liver F albumen generation specific reaction of gene engineering expression, shows that the antibody of preparation has immunocompetence.

4,, adopt double fastener heart legal system to be equipped with ELISA reagent with polyclone, the monoclonal antibody of purifying people F albumen and horseradish peroxidase-labeled.

F Protein Detection ELISA reagent (double antibody sandwich method) preparation and operation steps are following:

(1) encapsulate: use the F protein polyclone antibody of purifying to encapsulate by 10 μ g/ml protein contents, 100 μ l/ holes, 4 ℃ are spent the night, 0.01M PBS pH 7.4 washings three times, 1% bovine serum albumin(BSA) PBS sealing, 110 μ l/ holes, 37 ℃ 1 hour, it is for use to abandon coating buffer;

(2) add sample and enzyme conjugates simultaneously: clinical samples adds by 50 μ l/ holes; Establish negative and positive contrast each two holes and blank one hole simultaneously; And add the enzymic-labelled antibody 50 μ l/ holes of right working concentration simultaneously; Hatched 1 hour for 37 ℃, PBST (the Tris borate buffer solution that contains tween) washes five times;

(3) colour developing: add tmb substrate A, each 50 μ l of B liquid, hatched 15 minutes for 37 ℃;

(4) add 2M sulfuric acid 50 μ l cessation reactions;

(5) the 450nm wavelength is surveyed the OD value;

(6) result judges: P/N >=2.1 are positive.

Two, the composition of F protein assay reagent is following:

(1) the anti-people's polyclonal antibody of the rabbit of F albumen (10 μ g/ml, 100 μ l/ holes);

(2) 10% (v/v) hyclone;

(3) self-control horseradish peroxidase labeling antibody;

The preparation method is following:

I. get 4mgHRP (horseradish peroxidase) and be dissolved in the 0.4ml deionized water, add sodium periodate 0.4ml, 4 ℃ of 30min, mixings gently.

Ii. add monoethylene glycol liquid 0.4ml 4 ℃ of 30min, mixings gently.

Iii. add antibody purification 4.8mg/0.4ml, 2h mixing gently under the room temperature.

Iv. sample is added bag filter, to the dialysis of 0.05M pH9.5 carbonate buffer solution, liquid is changed 4 times in the centre, more than the dialysis 18h.

V. add the equivalent saturated ammonium sulfate, 4 ℃ leave standstill the centrifugal 10min of 1000rpm behind the 30min, and deposition is dissolved to 1.6ml with 0.01M PBS pH7.4, add saturated ammonium sulfate 0.8ml and leave standstill 30min for 4 ℃, and the centrifugal 10min of 1000rpm precipitates and dissolves with 1-2ml PBS.

Vi. the deposition bag filter of packing into after the dissolving is to 0.01M PBS pH7.4 dialysis, till can not detecting sulfate ion with 1% barium chloride.

Vii. column chromatography: on the 2.5*30cm post, adopting Sephadex G200 is medium, and the homo-ion displacement chromatography of method, flow velocity are 1ml/min, and every pipe is collected about 3ml.

(4)PBS(0.01M，pH?7.4)；

(5) PBST (Tris 2.42g, NaCl 29.24g, Tween-202.5ml add water and be settled to 1000ml, transfer pH to 7.5);

(6) substrate A liquid (contains 0.01%H ₂O ₂The WS) and substrate B liquid (pH 7.4, contain the 0.4mg/ml TMB 0.1mol/L citric acid damping fluid of (3,3,5, the 5-tetramethyl connects aniline));

(7) positive control;

(8) negative control;

(9) stop buffer (2M sulfuric acid).

Three, the method for making of F protein assay reagent is following:

1, utilize the F protein polyclone antibody (10 μ g/ml) of purifying to encapsulate 96 orifice plates, every hole 100 μ l, 4 ℃ are spent the night, 0.01M, the PBS washing of pH 7.4 three times, with containing each hole of 1% bovine serum albumin(BSA) PBS, 110 μ l sealing, 37 ℃ are incubated 1h, and it is for use to abandon coating buffer;

2, adopt improvement sodium periodate labelling method to prepare the anti-F protein antibodies of horseradish peroxidase-labeled;

Modification method: get 4mg peroxidase (HRP) and be dissolved in the 0.4ml water, add sodium periodate 0.4ml, 4 ℃ of 30min, mixings gently.

3, preparation cleansing solution PBST:

10 * TBS (pH 7.6): Tris-alkali, 60.57g; 1M HCl, 420ml; NaCl, 85-90g, behind HCl accent pH to 7.6, constant volume is in 1L.

1 * TBST: with 10 times of 10 * TBS dilutions, the TWEEN-20 that adds 0.1% (v/v) simultaneously gets final product.

4, preparation chromogenic substrate: A liquid (contains 0.01%H ₂O ₂); B liquid (the 0.1mol/L citric acid damping fluid that contains 0.4mg/ml TMB);

5, reaction terminating liquid: sulfuric acid solution (2M).

Four, the detection method brief introduction of F protein reagent is following:

(1) get 96 orifice plates that are coated with the F protein polyclone antibody, add test serum 50 μ l/ holes, establish each two hole of yin, yang contrast simultaneously, reacting hole is sealed with the shrouding film in enzyme labelled antibody 50 μ l/ holes then, 37 ℃ of constant temperature oven incubation 60min;

(2) discard liquid, the thieving paper arsis is done, and 1 * PBST is filled it up with in every hole, leaves standstill 1min, gets rid of cleansing solution, and the thieving paper arsis is done, and so repeats to wash plate 5 times;

(3) every hole adds tmb substrate A liquid and each 50 μ l of B liquid, and 37 ℃ of lucifuges are hatched 15min;

(4) every hole adds stop buffer 50 μ l, measures the OD value in each hole in the inherent 450nm wavelength of 15min.

The oxidation reaction of horseradish peroxidase enzyme catalytic superoxide, the most representative superoxide are H ₂O ₂, TMB is an oxygen donator, H ₂O ₂Be hydrogen acceptor, reaction equation is: TMD+H ₂O ₂→ blue soluble product+H ₂O, all kinds of acid stop buffers (like sulfuric acid solution) then can make blueness be transformed into yellow, and available certain wavelengths this moment (450nm) is surveyed and read light absorption value.

Five, can also expand to dedicated test reagent thus:

1, is used for enzyme and exempts from the appearance specific enzyme and exempt from method F protein assay reagent, see for details aforementioned.

2, be used for the special-purpose F protein detection kit of immunoturbidimetry.

2.1 reagent is formed: reagent R1 (antiserum that contains mouse anti human F protein monoclonal antibody), reagent R2 (increasing turbid dose, antiseptic, surfactant, Tris damping fluid); Definite value serum R3 (the genetic engineering reorganization F albumen that contains quantitative purifying)

Mouse anti human F protein monoclonal antibody derives from above-mentioned steps (six) among the reagent R1, and antiserum concentration is 10 μ g/ml.The polyglycol-6000 (PEG-6000) that increases turbid dose of employing 4% (w/v) among the reagent R2, antiseptic is a potassium sorbate, and surfactant is 1%SDS (v/v), and Tris damping fluid (pH=8.2) concentration is 20mmol/L.Reagent R3 is F albumen dried frozen aquatic products (every part of definite value F protein content is 1000 μ g, is dissolved in during use in the 10mlTris damping fluid, and being mixed with concentration is the definite value serum of 100 μ g/mL)

2.2 kit operation instructions

2.2.1 by the antiserum that contains the F protein monoclonal antibody 100 μ l, the mixed that adds reagent corresponding R20.9ml becomes single agents (F albumen antibody mab liquid).Preferably face with preceding preparation consumption on the same day.

2.2.2 each reagent dosage such as following table

Each pipe of mixing, 37 ℃ of insulation 10min, wavelength 340min; On semi-automatic biochemical analyzer, suck blank pipe liquid earlier, suck standard pipe again, instrument draws a reduction coefficient according to optical density and reference value; Suck again and measure pipe, carry out computing according to optical density and coefficient in the instrument.

2.2.3 calibrating method according to the immunoturbidimetry principle, should be got multiple spot (3-9 point), 5 points are got in this experiment, press y=a+bx+cx ²+ dx ³The simple cubic equation regression curve calibrate, make the reference work curve.

2.2.3.1 the drafting of correction work curve:

Preparation antiserum dilution working fluid: press R2 reagent 0.9ml, add ratio (the F protein antibodies liquid) mixing of R1 reagent 100 μ l, for use.

Prepare 5 point calibration liquid: get definite value serum R3, become 5 concentration with 0.9% physiological saline doubling dilution, the 5th pipe is for former definite value serum-concentration, and other 4 pipe is respectively 1/2,1/4,1/8,1/16 (or concentration be 0 to be 0.9%NaCl, this gets concentration 0) of the 5th pipe.Like following table (unit is ml):

Each pipe of mixing is put 37 ℃ of water bath heat preservation 10min, and the machine workmanship makes curve on the follow procedure.

2.2.3.2 sample is measured: inhale test serum (measuring pipe) 10 μ l, add above-mentioned dilution antiserum working fluid 1.0ml,, go up machine-readable number in 37 ℃ of water bath heat preservation 10min.

2.2.3.3 surpass the working curve higher limit like measured value, instrument can be printed demonstration " too high ", at this moment, sample to be measured is done to survey behind the doubling dilution again.

2.2.3.4 the antiserum of every lot number should be done a multiple spot calibration, the antiserum of promptly measuring sample should be same lot number antiserum with the antiserum of calibration.

Six, the clinical practice instance of F protein reagent box

Each item methodology index of this kit such as sensitivity, specificity, repeatability, cross interference, optimal reaction time, stability experiment and the range of linearity etc. all reach the basic demand of medical test with kit.Use the serum specimen that this kit detects patient's (cirrhosis, hepatocellular carcinoma, acute hepatitis B, chronic hepatitis B) of being diagnosed as hepatopathy during No.3 Central Hospital of Tianjin City year September in April, 2005 to 2006, other non-liver diseases patients (angina pectoris, diabetes, cerebral infarction, hypertension) of part and health examination person.

The data that clinical practice detects are following:

Adopt two sandwich method serum analysis samples to find that the 39 routine liver cirrhosis patient F protein 19 example positives account for 48%, 15 routine tumor patient 100% positives, 21 routine acute hepatitis b patient 90% positives, slow hepatopath's 30% positive, it is all negative that 32 routine neonate's serum 50 routine reference serum specimen results remove weak positive all the other the F albumen results of 3 examples.

In the clinical samples that we did; Testing result by non-liver diseases (angina pectoris, diabetes, cerebral infarction, hypertension etc.) patient finds to have only 1 routine F protein positive in the middle of the 28 routine patients; What be worth to propose is, this example is the diabetic, and clinical diagnosis is suspected has lesions of liver and kidney.

Normal and the unusual patient F of the ALT protein positive result from ALT, unusual patient's 94 examples of ALT have 69 examples positive, about 70-80%; There are 8 examples positive in ALT normal patient 36 examples, about 20%.Can find out, ALT unusual with normally can not represent hepatocellular physiology or pathological state fully, especially hepatocellular damage.

From various hepatitis F Protein Detection result, the positive rate of serious hepatitis, liver cancer patient F albumen is almost 100%, and this is not last conclusion, on the one hand is because of reagent itself is because the test example number is many not enough on the one hand.In any case but, liver is caused the disease of bigger infringement, like oxyhepatitis, cirrhosis etc., the positive rate of F albumen is higher relatively, how more than 50%.And the lighter disease of infringement, like chronic hepatitis etc., positive rate is lower.

F albumen is a kind of mark of brand-new liver cell material injury, has very wide application prospect.Research shows: F albumen not only can replace reliable liver histological inspection, and the hepatic injury of its reflection is that the sero-enzyme inspection can not check a bit.Aspect the specificity and early detection of tumor in digestive tract such as liver cancer, cancer of the stomach and various diagnosing hepatisms, F albumen then is fabulous selection.The F protein detection kit adopts at first obtain pure article of electrophoretically pure F albumen and antibody at home biochemical the separation with the immunology means, has set up enzyme-linked immune analytic method, is about to be applied to clinical blood sample.The analysis result of evidence F protein detection kit clinical blood sample is satisfied.

Seven, the related experiment of the preparation of F albumen, detection and testing result

(1), the acquisition of people F albumen cDNA

The extraction of the total RNA of human liver organization and detection.Total RNA of the normal person's of extraction traffic death fresh liver tissue then, measures its OD value respectively under 260nm and 280nm, judge its purity then.Can know according to sequence homology method between the kind in the unknown gene searching strategy: the discovery of many sequences, make us obtain a common recognition, the coded sequence homology between promptly planting is much higher than noncoding region, and homology is higher between the kind of a lot of specific genes.Based on this strategy; We are consulting on a large amount of pertinent literature bases both at home and abroad; With reference to that provide on the GeneBank and the gene order higher rat liver F albumen of human homology's property, successively designed 10 pairs of different upstream and downstream primers, and in upstream primer, added the restriction enzyme site of Nde I.(being published in 2007 13 12 phases of volume of World Journal of Gastroenterology) primer is as follows:

1. the upper reaches 5 '-CGG CAT ATG TAC TGG GAC AAA GGA CCA AAG CCT GAG AGA-3 '

Downstream 5 '-TCC AGA CCT CAC ACC GTT GGT CTC CAG-3 '

2. the upper reaches 5 '-CTG GAT CCG GAG GTT TGA CTA AGA TCA ATC A-3 '

Downstream 5 '-GCG AAT TCT GGA TCT GGG ACT TCT TCT TG-3 '

3. the upper reaches 5 '-CTG GAT CCG GTG CAC ACG GAA TAT AGC TC-3 '

Downstream 5 '-GCG AAT TCT TTA ATC GGG AGG GCT GGA-3 '

4. the upper reaches 5 '-TAT CAT ATG GTA CTG GGA CAA AGG ACC AAA GCC T3 '

Downstream 5 '-TAT GGA TCC CAT CTG GGA AGG CAT CTT TAC TCC A3 '

5. the upper reaches 5 '-CAG GCT GCC TCC TTC TAT TG-3 '

Downstream 5 '-CAG GTG GTC ACC CAT CTC TT-3 '

6. the upper reaches 5 '-AAG AGA TGG GTG ACC ACC TG-3 '

Downstream 5 '-AGC ACA GCG AAC TTC ACC TT-3 '

7. the upper reaches 5 '-GTA CTG GGA CAA AGG ACC AAA GCC-3 '

Downstream 5 '-GAT CTG GGA AGG CAT CTT TAC TCG-3 '

8. the upper reaches 5 '-TCC CTG GAA CAA GAG ATG G-3 '

Downstream 5 '-AGC ACA GCG AAC TTC ACC T-3 '

9. the upper reaches 5 '-AAG AGA TGG GTG ACC ACC TG-3 '

Downstream 5 '-TTC ACC TTC CCG AAT TTG TC-3 '

10. the upper reaches 5 '-TCC CTG GAA CAA AGA GAT GG-3 '

Downstream 5 '-TTC ACC TTC CCG AAT TTG TC-3 '

After with above-mentioned primer the total mRNA of people's liver being carried out the RT-PCR reverse transcription and goes out cDNA, carry out PCR immediately, the product that amplifies is detected its base number with agarose gel electrophoresis.Wherein 1. 2. 3. 9. 10. do not see the product amplification is arranged; 5. 6. 8. product base number is about 250bp; 4. product base number is about 800bp; 7. product base number is about 1200bp, is close with the liver F protein gene sequence base number of bibliographical information mouse.Therefore select the upstream and downstream primer 7. to be used for protein rna reverse transcription of people's liver F and amplification, obtained the RT-PCR product that the base number is about 1200bp.Because the base number of this product is bigger, is connected with cloning vector after check order, sudden change has taken place in indivedual bases, and we have designed the upstream and downstream primer of being correlated with to this sudden change, and mutating alkali yl is repaired.Primer is following:

1. the upper reaches 5 '-CATATCCTACTGGGACAAAG-3 '

Downstream 5 '-CTTGATGACATGACTGACCACCTC-3 '

2. the upper reaches 5 '-TGGTCAGTCATGTCATCAAGCAAGGG-3 '

Downstream 5 '-TTCCCTCAAGTGGCGGATCGTTGTG-3 '

3. the upper reaches 5 '-CGATCCGCCACTTGAGGGAACGAC-3 '

Downstream 5 '-GGATCCGATCTGGGAAGGCATC-3 '

PCR product with three primers is a template at last, is the primer amplification total length with the upper reaches 1., downstream 3..After cloned plasmids was connected, its result conformed to PCR result through order-checking, obtains containing the cloned plasmids of target gene fragment.

From Ago-Gel, reclaim PCR product fragment, dig the piece absorption method by low melting-point agarose gel in " molecular cloning " handbook and carry out separation and purification.Concrete operations are following:

(1) cuts out one after plain agar sugar gel solidifies,, solidify the back point sample fully to wherein pouring into low melting point glue; (2) confirm that DNA gets in the low melting point glue zone, cuts out the DNA band under uviol lamp.A little centrifuge tube is put in the object tape cutting-out; (3) add 5 times of volume 20mM TrisCl (pH8.0)+1mM EDTA (pH8.0), 65 ℃ of warm down baths 5 minutes; (4) be cooled to room temperature, add equal-volume, be inverted 20s back and forth, centrifugal 10 minutes of 20 ℃ of following 4000rpm with the phenol of 0.1M TrisCl balance to pH8.0; (5) reclaim upper phase,, be inverted and mix 4000rpm extracting in centrifugal 10 minutes with equal-volume phenol and the mixed liquid of chloroform; (6) reclaim upper phase,, shift liquid phase in a new centrifuge tube with equal-volume chloroform 4000rpm extracting in centrifugal 10 minutes; (7) 4 ℃ of absolute ethyl alcohols of the precooling of 0.2 times of volume 10M ammonium acetate of adding and 2 times of volumes, room temperature was placed 10 minutes, centrifugal 20 minutes of 5000rpm; (8) abandon supernatant, the bottom is DNA; Drying at room temperature DNA after (9) 75% washing with alcohol is with the aseptic double-distilled water dissolving of an amount of volume.

2, the connection of dna fragmentation: the used cloning vector of this experiment is pUCm-T, and there is the single endonuclease digestion site of BamH I, Nde I in big or small 2773bp on its MCS.As shown in Figure 1.

Carrier DNA is connected with the donor dna fragment, and is big or small according to the molecular weight that inserts fragment and carrier, calculates the content that needs each dna fragmentation of adding in the linked system, and the ratio that fragment and carrier are inserted in general employing is 3: 1.

Reaction system is: 10X Ligation Buffer 1 μ l, and PEG4000 1 μ l, pUCm-T vector 1 μ l, the PCR product 6 μ l behind the purifying, T4DNA Ligase 1 μ l, 16 ℃ of connections are spent the night.

3, DNA connects the conversion and the screening of blue hickie of product:

The conversion of recipient bacterium DH5 α is according to said method carried out: 1 milliliter of recipient bacterium is got in (1), and 12000rpm abandons supernatant after centrifugal 30 seconds, the mixing deposition; (2) add the 0.1M CaCl of precooling ₂1 milliliter of sterile solution, behind the mixing, 12000rpm abandons supernatant after centrifugal 30 seconds, the mixing deposition; (3) add the cold 0.1M CaCl of 300 μ l ₂And 10 μ l connect product, mixing, ice bath 30 minutes; (4) 42 ℃ of heat shocks 90 seconds, ice bath is 2 minutes immediately, adds 800 μ l LB nutrient culture media, and 37 ℃ of shaken cultivation are 50 minutes behind the mixing; (5) getting 200 μ l bacterium liquid directly coats the Amp that contains final concentration 50 μ l/ milliliters and (contains X-gal, on flat board IPTG); (6) 37 ℃ of overnight incubation are selected transformant.

4, recon is selected and is identified: treat that blue white sieve is dull and stereotyped and go up the bacterium colony colour developing and fully select the white colony flap of some from flat board in the back that incubated overnight is to confirm as the white clone.Select the part white colony and change a new plate over to, liquid training is simultaneously spent the night, and is used for the extraction of plasmid.

Plasmid extracts and checks order as follows: 1.2 milliliters of bacterium liquid are got in (1), centrifugal 30 seconds of 12000rpm; (2) be resuspended in 100 μ l solution I, mixing; (3) add 200 μ l solution II (newly joining), light mixing left standstill 1-2 minute; (4) add 150 μ l solution III, gentle mixing, ice bath 3-5 minute; (5) the centrifugal 3-5 of 15000rpm minute, get supernatant; (6) add the saturated phenol/chloroform of equal-volume Tris/isoamylol (25: 24: 1), centrifugal 5 minutes in 12000rpm; (7) get upper phase, once with the chloroform extracting; (8) get upper phase, add 4 ℃ of absolute ethyl alcohols of precooling of 2 times of volumes, ice bath 30 minutes; (9) in 15000rpm centrifugal 3 minutes, outwell supernatant; (10) once, at air drying with 70% washing with alcohol; With an amount of TE dissolving.

Resulting plasmid pUCm-T-F is carried out double digestion with Nde I and BamH I identify, and be sent to Shanghai living worker bio-engineering corporation and check order, the order-checking instrument is ABI PRISM 377-96.The result shows that after cloned plasmids was connected, its result conformed to PCR result through order-checking, analyzed through Blast, and the homology of the gene order of the gene order of human liver F albumen and rat liver F albumen has reached 99%.

5, the structure of expression vector: the plasmid that sequencing result is correct carries out double digestion with Nde I and BamH I; And the small pieces (1.2kb) after enzyme cut is connected (plasmid essential structure and MCS district are as shown in Figure 2) with pET-15b carrier through same double digestion; 16 ℃ spend the night after; To all connect product and be transformed among Escherichia coli E.coli BL21 (DE3) plysS, screen transformant containing on the nutrient culture media of acillin.See Fig. 2 expression vector pET-15b synoptic diagram.

Extract the plasmid of each transformant respectively, it is carried out enzyme cut evaluation, enzyme is cut identified correct expression plasmid called after pET15b-F.

6, induction expression of protein

In the LB nutrient culture media that contains acillin (Amp+), expression bacterium BL21 (DE3) pLysS to OD600 that 37 ℃ of cultivations contain pET15b-F between 0.4～0.6, add this moment IPTG to final concentration be 1mmol/L, continues 37 ℃ of cultivations 5 hours.Whole bacterial protein carries out SDS-PAGE, detects its expression, as contrast, carries out abduction delivering with empty bacterial strain BL21 (DE3) pLysS that does not contain the pET15b-F expression plasmid and the bacterial strain that contains empty carrier pET15b but do not contain foreign gene F simultaneously.

(2), express the purifying of F albumen

1, preliminary purification: (1) is expressed bacterium liquid with 100 milliliters and was placed 12000rpm centrifugal 15 minutes, abandons supernatant.(2) get twice of an amount of PBS washing precipitation.(3) ultrasonic disruption cell.(4) 12000rpm is centrifugal 15 minutes, cleer and peaceful deposition in the separation.(5) the gained deposition is respectively washed once with 2mol/L and 4mol/l urea with 20ml 1Xbinding buffer washing several times again.(6) dissolve final sediment with the 1Xbinding buffer 6ml that contains 8mol/l urea.(7) room temperature 12, and 000rpm collects supernatant after centrifugal 20 minutes, are the F albumen of preliminary purification.

Ni2+-charged IDA His-bind column affinity chromatography: the 1Xbinding buffer of (1) preparation 13 bed volumes progressively is added on the chromatographic column.(2) the F albumen with preliminary purification progressively is added on the chromatographic column.(3) progressively wash foreign protein off with 8 milliliters of Washing buffer, and collect effluent, every pipe 0.5ml treats electrophoresis.(4) use progressively wash-out of 10ml Elute buffer at last, and eluent is divided into every pipe 0.5ml keeps to treat electrophoresis detection.

2, the purity of expressing F albumen is identified

Adopt polyacrylamide gel electrophoresis (SDS-PAGE): whole bacterial protein of expressing and the albumen behind the purifying are detected respectively.Adopt the discontinuous vertical slab electrophoresis of sex change, it is 5% that the upper strata concentrates gum concentration, and lower floor's resolving gel concentration is 15%.The preparation of gel is prepared separation gel and concentrated sol solution respectively by described in " molecular cloning " handbook.The concrete operations step is following:

(1) joins glue: two clean glass plates and partition are assembled be fixed on the encapsulating support.Prepare an amount of concentration and be 8% separation gel; Inject certain altitude along the interlayer between two blocks of glass; Be generally glass plate height ground about 2/3, on separation gel solution, cover the water layer of one deck 5mm then lightly, with the oxygen in the secluding air; And prevent that gel meniscus ground from forming, and makes gel surface smooth.After room temperature is placed 30min-60min.After gel polymerisation, an interface clearly will appear between separation gel and water layer, and the gel stent that can tilt slightly detects whether polymerization of gel.After treating the abundant polymerization of separation gel, inhale the layer that anhydrates, wash gel, and remove gel upper strata residual liquid with the thieving paper suction with damping fluid.4% the concentrated glue for preparing is flow on the separation gel, plug comb immediately, flush with the top of front glass panel until the bottom of comb tooth.The bottom of guaranteeing comb tooth does not have bubble.Wait to concentrate (about 30min) after the glue polymerization, in electrophoresis tank, add electrophoretic buffer, do not have concentrated glue, in case the gel drying shrinkage is carefully extracted comb.

(2) in the testing protein sample, add isopyknic 1X sample-loading buffer, boil 3-5min, after the cooling sample is added in the sample well, every hole 10-20 μ l avoids bringing into bubble, and bubble is prone to make sample to be blended in the adjacent well.

(3) connect power supply,, when Australia's phenol indigo plant gets into separation gel from concentrating glue, voltage is transferred to 120V, continue electrophoresis and stop electrophoresis apart from about 5mm place, gel bottom until Australia's phenol indigo plant earlier with 70V constant voltage electrophoresis.

(4) powered-down carefully takes out gel, is immersed in the container that fills a small amount of coomassie brilliant blue R250 dye liquor, dyes 2-3 hour.

(5) remove dyeing liquor, with gel rinsing several in distilled water.Adding destainer again decolours to obtaining clear blue band and clean background repeatedly.Analyze experimental result.

3, the specificity of expressing F albumen is identified

Adopt Western-blot to analyze: film is changeed in (1): albumen in the glue of SDS-PAGE is transferred on the nitrocellulose filter jump condition in transfering buffering liquid: 25 volts, and 2 hours.(2) Western-blot reaction: will shift good film with TBS rinsing 5 times, 5 minutes each; Add confining liquid (TBST+4% skimmed milk power) room temperature sealing 1 hour, TBST washes film 3 times, adds the suitably first antibody (the anti-people F of cavy protein polyclone antibody) of dilution, and room temperature is shaken first antibody is combined with antigen; TBST gives a baby a bath on the third day after its birth inferior, adds enzyme mark SA (the goat-anti cavy IgG antibody of horseradish peroxidase-labeled), and room temperature was shaken 2 hours; TBST gives a baby a bath on the third day after its birth inferior, and distilled water is washed once.Add DAB colour developing 5 minutes, use the water washing cessation reaction.

(3), the separation of F albumen is purified

Get the deadly liver of healthy male traffic accident wound, the complete liver of taking-up is transported the laboratory back for 0 ℃ and is put-70 ℃ of preservations in the 3hr.In physiological saline, thaw during use, be cut into small pieces, add homogenate under isopyknic physiological saline low temperature, with 4 ℃ of centrifugal 30min of following 8000rpm of homogenate.

Add solid (NH4) in the LH supernatant ₂SO ₄To 55% saturated solution, place 4 ℃ to spend the night.Potpourri is at 4 ℃ of centrifugal 30min of 8000rpm, and supernatant discarded is with a little redistilled water dissolution precipitation.Earlier to redistilled water again to 20mmol/L Tris-HCl Buffer, pH8.0 dialysed overnight.

With DEAE-Cell μ lose 52 posts on the dialysate (

) chromatography.Before 6hr with 20mmol/L THB, the pH8.0 wash-out, after this 12h carries out gradient elution, 0-1mmol NaCl-20mol/L THB, pH8.0.Collect the 1st peak ascending branch back segment and decent, ultrafiltration and concentration.

With Sephacryl S-200 post on the concentrate ( ) chromatography.With 80mmol/L THB, the pH8.0 wash-out.Collect the 3rd peak, ultrafiltration and concentration.

With CM-cell μ lose-23 post on the concentrate (

) chromatography.Before 5h with 10mmol/LNaAc-HAc, pH5.5-0.1mol/L NaCl wash-out, later 12hr carries out gradient elution, 0.1-1mol/L NaCl-10mmol/LNaAc-HAc, pH5.5.Collect the 5th peak, ultrafiltration and concentration.

The F protein frozen that will obtain is dry to be concentrated.

The pure article of F albumen of above collection are separated through polyacrylamide gel electrophoresis.Gel snap frozen behind the electrophoresis, cut the F protein fragments, the F albumen in the extracting gel obtains the pure article of a spot of electrophoretically pure F albumen.

(4), F determination of protein concentration (Coomassie brilliant blue method (Bradford method))

Coomassie brilliant blue (G-250) dyestuff with protein bound, makes the position of the maximum absorption band of dyestuff in acid solution, become 595nm by 465nm, and the color of solution also becomes blueness by brownish black.Think after deliberation, dyestuff mainly be with protein in basic amino acid (particularly arginine) and aromatic amino acid residue combine.The absorbance A595 that under 595nm, measures is directly proportional with protein concentration.

Operation steps is following: (1) BSA protein standard solution, doubling dilution is mixed with variable concentrations.200μg/ml，100μg/ml，50μg/ml，25μg/ml，12.5μg/ml，6.25μg/ml，3.125μg/ml。(2), play its OD value of mensuration with standard items with 20 times of dilutions of elution buffer testing sample.With the OD value is horizontal ordinate, and protein concentration is an ordinate, the drawing standard curve.Find the concentration of F albumen to be measured from typical curve, F protein standard curve is seen Fig. 4.

(5), the preparation and the purifying of the anti-people F of cavy protein polyclone antibody

1, the anti-people F of cavy protein antiserum preparation

Using physiological saline is 100 μ g/ml concentration with the F albumen dilution of purifying, gets solution 0.5ml and the abundant mixing of 0.5ml Fu Shi Freund's complete adjuvant, with the abundant mixing of Fu Shi Freund's complete adjuvant; Get 1ml mixing liquid immunity male guinea pig; At the two hindlimb muscle multi-point injections of cavy, totally three times, each week at interval.Injection back one all heart blood samplings are for the third time collected antiserum and are preserved subsequent use.

3, the anti-people F of cavy protein polyclone antibody purifying

Saltout: get the 4ml immune serum, add the physiological saline of equivalent, 4 ℃ are stirred down gently and dropwise add the saturated sulfuric acid amine of 8ml, left standstill 30 minutes, 10000 rev/mins centrifugal 10 minutes, abandon supernatant, resolution of precipitate in physiological saline to final volume 4ml.

4 ℃ are stirred gently down and dropwise add the saturated sulfuric acid amine of 2ml, left standstill 30 minutes, 10000 rev/mins centrifugal 10 minutes, abandon supernatant, resolution of precipitate is in physiological saline to final volume 4ml, repeats 3.1.2 step 3 time.

Deposition is used the 1-2ml physiological saline solution, and the dress bag filter is to normal saline dialysis, till can not surveying sulfate ion with 1%BaCl2.

Ion-exchange chromatography:

The polyclonal antibody that at first will use the salting out method purifying is to 0.01M PBS pH7.4 damping fluid dialysis equilibrium.

Will be through the polyclonal antibody of the salting out method purifying bag filter of packing into, it is subsequent use to be concentrated into the 1-2 milliliter with polyglycol (molecular weight 6000).

Column chromatography: get 10 gram DEAE-Sephadex-A50 and be dipped in 2000 ml deionized water, abandon water next day, add 0.01MPBS pH7.4, soaked overnight, the middle PBS twice that changes makes medium reach balance.Chromatographic column is 2.5*50cm, adds gel and uses the PBS balance.Slowly add the polyclonal antibody after saltouing in the time of last kind, use the PBS wash-out, flow velocity is 1 milliliter of a per minute, and every pipe is collected about 3 milliliters, and 280nm surveys the OD value, calculates content.

4, the anti-people F of cavy protein polyclone antibody enzyme conjugates preparation

Get 4mg peroxidase (HRP) and be dissolved in the 0.4ml water, add sodium periodate 0.4ml, 4 ℃ of 30min, mixings gently.

Added 4 ℃ of monoethylene glycol liquid 0.4ml 30 minutes, gently mixing.

Add antibody purification 4.8mg/0.4ml, following 2 hours of room temperature is mixing gently.

Sample is added bag filter, and to the dialysis of 0.05M pH9.5 carbonate buffer solution, liquid is changed 4 times in the centre, more than the dialysis 18h.

Add the saturated sulfuric acid amine of equivalent, 4 ℃ left standstill after 30 minutes 1000rpm centrifugal 10 minutes, and deposition is dissolved to 1.6ml with 0.01M PBS pH7.4, adds saturated sulfuric acid amine 0.8ml and leaves standstill 30min for 4 ℃, and centrifugal 10 minutes of 1000rpm precipitates and dissolves with 1-2ml PBS.

Deposition after the dissolving bag filter of packing into is to 0.01M PBS pH7.4 dialysis, till can not detecting sulfate ion with 1% barium chloride.

Column chromatography: on the 2.5*30cm post, adopting Sephadex G200 is medium, and the homo-ion displacement chromatography of method, flow velocity are 1ml/min, and every pipe is collected about 3ml.

Measure: with the light absorption of spectrophotometric instrumentation 403 and 280nm.

(6), the preparation of mouse anti human F protein monoclonal antibody and purifying

1, the collection of animal immune and mice serum

(1) choose 5 of 6-8 BALB/c mouses in age in week, female, body weight 18-23g indicates 1# with marking pen, 2#, 3#, 4#, 5#.

(2) F protein 60 μ g/0.25ml/ mouse mixes with isopyknic Freund's complete adjuvant, connects two syringes with sebific duct; Suck antigens mixed and adjuvant; Alternately pushing syringe is with emulsification antigen, and up to dripping potpourri on frozen water, the antigen of emulsification does not scatter.

(3) mouse is used iodophor disinfection, at the antigen of position multi-point injection emulsifications in subcutaneous minute such as nape portion, groin, foot pad, root of the tail.

The antigen of the emulsification equivalent that uses the same method after (4) two weeks adds incomplete Freund, subcutaneous multi-point injection, 30 μ g/0.25ml/ mouse.

After (5) one weeks, 30 μ g/0.25ml/ mouse, incomplete Freund's adjuvant, lumbar injection.

After (6) one weeks, strengthen 60 μ g/0.25ml/ mouse, lumbar injection eventually.

(7) after a week, eyelid is taken a blood sample in test tube, corresponding to 5 mouse, is numbered 1# respectively, 2#, and 3#, 4#, 5# leaves standstill 1h, and the centrifugal 15min of 3000rpm/min collects serum.

2, the cultivation of feeder cells

(1) in the previous day of Fusion of Cells, get a BALB/C mice that does not have immunity, cervical vertebra is put to death from disconnected, soaks 5min in 75% alcohol.

(2) mouse web portion of crossing soaking disinfection is placed in the aseptic 15cm plate up, on superclean bench, picks up mouse part skin gently with aseptic nipper, cuts off a horizontal osculum in the above with the aseptic operation scissors, does not break peritonaeum.

(3) clamp the skin of the last lower edge of osculum respectively with two aseptic nippers; Slightly firmly draw in the opposite direction, expose peritonaeum, get an asepsis injector and be filled 37 ℃ of PMI1640 nutrient solutions of heating in advance to tear skin of abdomen; Inject intraperitoneal to nutrient solution, back and forth pressure-vaccum several times.

(4) whole sucking-off intraperitoneal nutrient solutions inject in the aseptic 50ml graduated centrifuge tube, again with 5ml nutrient solution flushing abdominal cavity once.

(5) twice nutrient solutions pool together, and collect peritoneal macrophage and counting, and the centrifugal 5min of 1000rpm abandons supernatant, and the pipe floor cells is diluted to 2 * 105/ml with the HAT nutrient solution, mixing.

(6) join in 96 well culture plates, incubator is put in 100 μ l/ holes, and 37 ℃, 50%CO ₂Cultivate, subsequent use.

3, SP2/0 myeloma cell's recovery and cultivation

(1) takes out a frozen SP2/0 myeloma cell (2.2 * 106) in the liquid nitrogen container, directly put into 37 ℃ of water and thaw, move into the interior cell of frozen pipe in the one aseptic 50ml centrifuge tube after the equitemperature balance.

(2) get a droplets on glass sheet, microscopically observation of cell form and quantity.

(3) add 37 ℃ of RPMI1640 basic culture solution 30ml of temperature in advance in the past centrifuge tube, abundant mixing, the centrifugal 5min of 1000rpm abandons supernatant.Cell is used identical method washed twice more then, and last deposition adds the 20ml nutrient solution, mixing and counting.

(4) the inoculated and cultured bottle is put into incubator, and 37 ℃, 0.5%CO ₂Environment is cultivated down.

(5) test for fusion the last fortnight is used 8-AG nutrient culture media (30 μ g/ml) instead and is cultivated, to prevent myeloma cell's reversion.

(6) fusion is gone down to posterity once the previous day, and inoculum density is 4.5 * 105, and is changed to conventional nutrient solution.

4, preparation splenocyte suspension

(1) choose the highest mouse of serum antibody titer, the cervical vertebra dialysis is put to death, and in 75% alcohol, soaks 5min.

(2) in superclean bench, aseptic method is cut off the mouse stomach wall, tears skin of abdomen, exposes peritonaeum, and then cuts off peritonaeum, exposes abdominal viscera, takes out spleen, removes fat and connective tissue, washes with serum-free medium.

(3) the spleen immigration is filled in the 5ml serum-free medium, be cut into small pieces spleen.

(4) taking out the cell screen cloth is placed on the little plate; Pour the spleen tissue piece of cutting off in the filter screen into; With glass syringe inner core tissue abrasion's piece gently, make it pass through filter screen, with a small amount of nutrient solution from top flushing filter screen; The major part of organizing through filtering is divided into individual cells, adds a small amount of nutrient solution and makes cell suspension.

(5) use one deck strainer filtering again, remove little piece of tissue.Collect in the 50ml centrifuge tube, add 37 ℃ in advance the nutrient solutions of temperature to 25ml, counting, the centrifugal 5min of 1000rpm again with the incomplete nutrient solution washed cell of 25ml once, precipitates at last that to process the 5ml cell suspension subsequent use, cell concentration is 1 * 108.

5, Fusion of Cells

(1) with myeloma cell's mixing in the 50ml conical centrifuge tube of 1 * 108 splenocyte and 2 * 107-3 * 108, the centrifugal 5min of 1000rpm abandons supernatant, and with the suction pipe residual liquid that exhausts, the springing tube wall makes cell precipitation loosening and become pasty state gently.

(2) centrifuge tube is put 37 ℃ of water-baths, draws 37 ℃ of 50%PEG 0.8ml of temperature in advance with the 1ml dropper, slowly adds along the tube wall bottom; The limit edged slowly rotates centrifuge tube; Cell is fully contacted with PEG, in 1min, add, remake and use 2min; Add 20ml serum-free medium cessation reaction, centrifugal 1000rpm 5min.

(3) cell precipitation again with 20ml serum-free medium washing once notes not wanting violent liquid feeding or mixing, and the cell precipitation after centrifugal adds 10mlHAT and selects nutrient solution, mixes gently.

(4) join in 96 well culture plates that feeder cells are arranged in advance by every hole 100 μ l at last, put into 37 ℃ of 5%CO of incubator to culture plate ₂Cultivate, observe the fused cell growing state every other day.

6, hybridoma screening

(1) after cell was grown 4 days in the HAT nutrient solution; Can see at the bottom of the hole cell clone growth is arranged; Splenocyte also has survival; For the B emiocytosis antibody of eliminating survival possibly detect the influence that causes by antagonist as far as possible,, add HAT again and select nutrient solution 200 μ l/ holes the whole sucking-offs of the nutrient solution in the culture hole.

(2) cultivate and change HT after 3 days into and select nutrient solution to cultivate, after 3 days, hybridoma occupies above detecting antibody behind 1/10 the area at the bottom of the hole.

7, indirect elisa method detects the positive colony cell

(1) encapsulate F albumen in ELISA Plate, 0.5 μ g/ml, 4 ℃ are spent the night.

(2) lavation buffer solution is washed 3 times, 3min/ time, claps and does.

(3) 0.1%BSA confining liquid 100 μ l, 37 ℃, water-bath 1h, washing is clapped and is done.

(4) with the supernatant in all growth clones' hole in the culture plate, order adds ELISA Plate, and (washing is clapped and done for every hole 100 μ l, 37 ℃ of water-bath 1h.Positive control is an immune serum, and negative control is the SP2/0 culture supernatant, and blank is PBS.

(5) every hole adds goat anti-mouse igg-HRP antibody 100 μ l, 37 ℃ of water-bath 1h, and washing is clapped and is done.

(6) every hole adds freshly prepared substrate solution 100 μ l, 37 ℃ of water-bath 30min.

(7) every hole adds stop buffer 50 μ l cessation reactions.

(8) enzyme-linked immunosorbent assay instrument 490nm detects the OD value, treats gaging hole OD value 2.1 times more than or equal to negative hole, and is promptly positive.

(9) positive is detected hole doubling dilution (1: 200,1: 400,1: 800,1: 1600,1: 3200,1: 6400,1: 12800,1: 25600) order and add ELISA Plate, every hole 100 μ l, 370C water-bath 1h, washing is clapped and is done.Positive control is an immune serum, and negative control is the SP2/0 culture supernatant, and blank is PBS.

(10) every hole adds goat anti-mouse igg-HRP antibody 100 μ l, 37 ℃ of water-bath 1h, and washing is clapped and is done.

(11) every hole adds freshly prepared substrate solution 100 μ l, 37 ℃ of water-bath 30min.

(12) every hole adds stop buffer 50 μ l cessation reactions.

(13) enzyme-linked immunosorbent assay instrument 490nm detects the OD value.

8, the detection of antibody specificity (Western blot method)

Step by above-mentioned SDS-PAGE is carried out electrophoresis.

(1) after electrophoresis finishes, pull down offset plate, be put in the plastics plate that fills tap water, separately the layer glass plate takes out glue, and gets the part that needs.

(2) in little plate, soak gel with 1 of precooling * commentaries on classics film damping fluid.According to the size of glue, cut 6 Whatman3 filter paper and a pvdf membrane (size is all coincide with glue).In the commentaries on classics film damping fluid of precooling, soak the about 30min of gel, soak foam-rubber cushion, pvdf membrane, the about 20min of Whatman filter paper.Pvdf membrane is needing to soak into 10s with methyl alcohol earlier before the film damping fluid soaks with changeing.

(3) assembling filter paper/film/gel sandwich is followed successively by three layers of sponge by negative pole end to positive terminal, three layers of Whatman3 filter paper, gel, pvdf membrane, three layers of Whatman3 filter paper, three layers of sponge.Must guarantee when noting this sandwich of assembling that filter paper, film, the accurate alignment of gel do not stay bubble.Can wear gloves and drive the bubble between film and the glue away with glass pipet.Also should guarantee the tight of sandwich.Filter paper/the film that assembles/gel sandwich is put into commentaries on classics membrane electrophoresis groove, to change film damping fluid submergence sandwich, current stabilization 120mA, 1-2h.

(4) after the commentaries on classics film finishes, gel is dyeed with Coomassie brilliant blue, decolouring is changeed membrane efficiency all with conventional Protein Detection with check.Film is placed in the little plate, cleans 2-3 time, about each 5min with 1 * TBST.

(5) with confining liquid room temperature sealing 1h.Abandon confining liquid, add one anti-(the positive hole of cell culture fluid supernatant) of the new confining liquid liquid dilution of usefulness, 37 ℃, 1h.Clean 6 times 10min/ time with TBST.

(6) two anti-(the anti-mouse of rabbit) are with confining liquid dilution in 1: 10000, incubated at room 1 hour, room temperature 1 * TBST washing 6 times, each 10min.

(7) colour developing: an amount of DAB colour developing liquid is tiled on the blotting membrane after the two anti-hybridization, and room temperature is placed and is observed, and tangible sepia albumen colour developing band can occur.

(8) stop: get final product cessation reaction with Tris-HCl damping fluid or water rinse hybond membrane.

9, the monoclonal antibody detection (indirect elisa method) of tiring

(1) encapsulate: detect and to use antigen to be F albumen, wrapper sheet concentration is 1 μ g/ml, and every hole 50 μ l close every hole 0.05 μ g, and every hole adds 200 μ l, then flat board is placed the box that wets, and 4 ℃ are spent the night and encapsulate.Confide all the liquid in the dull and stereotyped shrinkage pool next day.

(2) sealing: every hole adds sealing damping fluid 200 μ l, 37 ℃ of sealing 2h.

(3) adding one resists: lavation buffer solution is washed plate 3 times after abandoning confining liquid.The different dilution antiserum that adding is diluted with PBS (dilutability is 1: 100,1: 300, and 1: 900,1: 2700,1: 8100,1: 24000,1: 72000), every hole adds 100 μ l, puts 37 ℃ and hatches 1h.Do blank (PBS) and negative control (non-immune mice serum) simultaneously.

(4) washing: liquid in the plate hole to the greatest extent, fill it up with cleansing solution, place 3min, 3 times repeatedly, at last reaction plate is upside down on the thieving paper, make that cleansing solution flows to end in the hole.

(5) add ELIAS secondary antibody: in each reacting hole, add the anti-mouse ELIAS secondary antibody 100 μ l of rabbit of fresh dilution, hatch 1h for 37 ℃.Liquid in the plate hole is to the greatest extent filled it up with cleansing solution, places 3min, and 3 times repeatedly, at last reaction plate is upside down on the thieving paper, make that cleansing solution flows to end in the hole.

(6) add the substrate solution colour developing: every hole adds 100 μ l substrate solutions and (contains 0.01%H ₂O ₂, the 0.1mol/L structure rafter acid buffer pH7.4 of 0.4mg/ml OPD), room temperature reaction 10min.

(7) cessation reaction: in every hole, add 2M sulfuric acid 50 μ l.

(8) result judges: survey its OD value in the 490nm place, survey each hole absorbance value with blank zeroing back.

(7), F Protein Detection ELISA kit top condition is explored and the checking of kit performance index

(I), the optimum condition of ELISA double antibody sandwich method

1, the enzyme conjugates working concentration is confirmed

(1) encapsulate: AC is 10 μ g/ml, 100 μ l/ holes, and 4 ℃ are spent the night, and PBS-T gives a baby a bath on the third day after its birth inferior, 37 ℃ of sealings of 10% hyclone 1h.

(2) application of sample: add the every hole 50 μ l of the known positive and negative control, add the enzyme conjugates of dilution in 1: 500,1: 1000,1: 2000,1: 4000 simultaneously, 50 μ l/ holes, 37 ℃ of 1h, PBS-T wash five times.

(3) add substrate: (3,3,5,5 tetramethyls connect aniline, and Sigma), A liquid, B liquid is 50 μ l/ holes respectively, 37 ℃ of 15min to add substrate TMB.

(4) add stop buffer: add 2M sulfuric acid, 50 μ l/ holes.

2, the coated antibody working concentration is selected

(1) encapsulates: by 1 μ g/ml, 5 μ g/ml, 10 μ g/ml, 15 μ g/ml, 20 μ g/ml, 25 μ g/ml, 30 μ g/ml, 35 μ g/ml; Every hole adds 50 μ l, and 4 ℃ are spent the night, and PBS-T gives a baby a bath on the third day after its birth inferior; 10% hyclone PBS-T, 100 μ l/ holes, 37 ℃ of 1h sealings.

(2) application of sample and HRP: each 50 μ l/ hole of positive and negative contrast, 37 ℃ of 1h, PBS-T wash five times.

(3) add substrate solution and stop buffer: A, each 50 μ l/ hole of B liquid, 37 ℃ of 15min, each 50 μ l/ hole of stop buffer.

(4) 450nm surveys the OD value.

3, double-antibody sandwich elisa experiment

(1) encapsulate: use the F protein polyclone antibody of purifying to encapsulate by 10 μ g/ml protein contents, 100 μ l/ holes, 4 ℃ are spent the night, 0.01M PBS pH7.4 washing three times, 1% bovine serum albumin(BSA) PBS sealing, 110 μ l/ holes, 37 ℃ of 1h, it is for use to abandon coating buffer.

(2) add sample and enzyme conjugates simultaneously: clinical samples adds by 50 μ l/ holes, establishes negative and positive contrast each two holes and blank one hole simultaneously, and adds the enzymic-labelled antibody 50 μ l/ holes of right working concentration simultaneously, hatches 1 hour for 37 ℃, and PBS-T washes five times.

(3) colour developing: add tmb substrate A, each 50 μ l of B liquid, hatch 15min for 37 ℃

(4) add 2M sulfuric acid 50 μ l cessation reactions.

(5) the 450nm wavelength is surveyed the OD value.

(6) result judges: P/N >=2.1 are positive.

(II), the methodology index of F Protein Detection

1, linear experiment

A positive serum by 1: 2,1: 4,1: 8,1: 16,1: 32,1: 64,1: 128,1: 256,1: 612 doubling dilution, is detected with double antibody sandwich method.

2, sensitivity

The F albumen of purifying is diluted with PBS-T, press: 32 μ g/ holes, 16 μ g/ holes, 8 μ g/ holes, 4 μ g/ holes, 2 μ g/ holes, 1 μ g/ hole, 0.5 μ g/ hole add, and detect with double antibody sandwich method.

3, repeatability

Repeated experiment in criticizing: detect 2 parts of positive samples with this law, every part is detected 20 holes, observes the repetition situation that detects in criticizing.

Repeated experiment between batch: divide 20 batches to detect 2 parts of positive samples with this law.

4, interference experiment

Detect F protein positive serum, HbsAg positive control, HbeAg positive control, HAV antigen, HCV antigen, HDV antigen, HEV antigen, HSVI type antigen, HSVII type antigen, Escherichia coli respectively with this law.

5, the optimal reaction time

Use this law test sample, after adding sample and enzyme conjugates, react 30min, 1h, 1.5h and 2h respectively, again through colour developing, cessation reaction and mensuration.

6, stability experiment

Each ingredient (comprise and encapsulate plate, negative and positive contrast, enzyme conjugates, antigen, substrate solution) of newly-built kit is put in 4 ℃ of preservations, detected once observing effect in every month.

(III), result

1, antibody titer detection-indirect elisa method

Exempt from method with enzyme and measure the anti-people F of gained rabbit protein antiserum immunizing potency, the result show the anti-people F of rabbit protein antiserum tire can reach 1: 25600 dry straight.The result sees table 1.

The anti-people F of table 1 cavy protein antiserum antibody titer

2, the detection of antibody specificity-Western blot method result

Behind the F protein electrophoresis in genetic engineering source, be an anti-immunoblot experiment that carries out with the anti-people F of cavy albumen (purifications) polyclonal antibody, the result is it is thus clear that a band clearly shows that specific immune response takes place for antigen and antibody.The result sees Fig. 3.

3, the enzyme conjugates working concentration is confirmed the result

Confirm the enzyme conjugates working concentration with the ELISA method, the result sees table 2.

Antigen-reactive result under different dilutability enzyme conjugates of table 2 and the certain condition

Dilutability	1∶500	1∶1000	1∶2000	1∶4000
					Positive control	1.82	1.54	0.86	0.51
Negative control	0.09	0.05	0.03	0.01

4, coated antibody working concentration selection result

Confirm the coated antibody working concentration with the ELISA method, by measuring the finally selected 10 μ g/ml of result for encapsulating concentration.The result sees table 3.

The different dilutability coated antibody of table 3 testing result

Antibody dilution	1μg/ml	5μg/ml	10μg/ml	15μg/ml	20μg/ml	25μg/ml	30μg/ml	35μg/ml
									Positive control OD	0.55	0.70	0.80	0.84	0.85	0.80	0.77	0.70
Negative control OD	0.03	0.05	0.05	0.05	0.05	0.05	0.05	0.05

5, F protein detection kit performance index checking

(1) the F Protein Detection range of linearity is measured

A positive serum by 1: 2,1: 4,1: 8,1: 16,1: 32,1: 64,1: 128,1: 256,1: 612 doubling dilution, is detected with double antibody sandwich method, and the result sees table 4.

The linear experimental result of table 4F Protein Detection

(2) sensitivity determination result

The F albumen of purifying is diluted with PBS-T, press: 32 μ g/ holes, 16 μ g/ holes, 8 μ g/ holes, 4 μ g/ holes, 2 μ g/ holes, 1 μ g/ hole, 0.5 μ g/ hole add, and detect with double antibody sandwich method, and the result sees table 5.

The sensitivity of table 5F Protein Detection

(3) repeatability is measured the result

Repeated experiment in criticizing: detect 2 parts of positive samples with this law, every part is detected 20 holes, observes the repetition situation that detects in criticizing, and shows that batch interior repeatability is good, and CV is less than 7%.The result sees table 6.

Repeated experiment between batch: divide 20 batches to detect 2 parts of positive samples, CV＜11% with this law.The result sees table 7.

Batch interior repeatability of table 6F Protein Detection

Table 7F Protein Detection batch between repeatability

(4) interference experiment result

Detect F protein positive serum, HbsAg positive control, HbeAg positive control, HAV antigen, HCV antigen, HDV antigen, HEV antigen, HSVI type antigen, HSVII type antigen, Escherichia coli respectively with this law, the result sees table 8.

The interference experiment of table 8F Protein Detection

(5) optimal reaction time result

Use this law test sample, after adding sample and enzyme conjugates, react 30min, 1h, 1.5h, 2h respectively, through colour developing, cessation reaction and mensuration, the result sees table 9 again.

The table 9F Protein Detection optimal reaction time is confirmed

Sample	0.5h	1h	1.5h	2h
					Positive control	15.8	17.9	18.5	18.8

(6) stability experiment result

Each ingredient of newly-built kit is put in 4 ℃ of preservations, detected once in every month, the result sees table 10.

Table 104 a ℃ preservation F albumen diagnostic reagent is formed the stability experiment result

Detection time (moon)	0	1	2	3	4	5	6
								P/N	18.5	18.5	17.5	16.9	16.5	16.1	15.5
Rate of descent (%)	0	2.16	5.40	8.65	10.81	12.97	16.22

(7) the serum F protein ELISA analyzing and testing coefficient of variation and recovery experimental result

Repeatedly analyze simultaneously with same sample, the detection coefficient of variation of F albumen is 4.2%, in same sample, adds the F albumen purification article of two kinds of variable concentrations, presses the sample determination method and handles, and the mensuration recovery of F albumen is 90.8%-94.1%.The result sees table 11.

The detection recovery (n=6 μ g/L) of table 11F albumen

Serum	Standard	Serum+standard	The recovery (%)
				124	186 372	?293 474	90.8 94.1

The present invention adopts a large amount of preparation of gene engineering antigen technology people liver F albumen; Prepare its polyclonal antibody simultaneously; And, come the feasibility of preliminary identification F albumen as a kind of reagent for clinical diagnosis through this antibody test part clinical samples, for the F protein detection kit gets into clinical laying the foundation.Through the experimental exploring to each item methodology index of F protein assay reagent such as linearity experiment, sensitivity, repeatability, cross interference optimal reaction time, stability experiment etc., the result shows that F protein reagent detection sensitivity reaches 0.5 μ g/ hole; In batch repeated CV value 5.5%, criticize between the CV value in scope about 10%; With this law in detecting the F protein process with no cross reactions such as HbsAg positive control, HbeAg positive control, HAV antigen, HCV antigen, HDV antigen, HEV antigen, HSVI type antigen, HSVII type antigen, Escherichia coli; The F Protein Detection optimal reaction time is confirmed as 1 hour; Each ingredient of forming detection kit is put in 4 ℃ of preservations and descends about 15% through 6 months P/N ratio; 100 times of its P/N ratios of positive sample dilution are still in the range of linearity.

Claims (9)

1. a F protein assay reagent is characterized in that: the anti-people's polyclonal antibody of rabbit, enzyme labelled antibody, chromogenic reagent and the stop buffer that comprise F albumen.

2. F protein assay reagent according to claim 1 is characterized in that: said enzyme labelled antibody is the horseradish peroxidase labeling antibody.

3. F protein assay reagent according to claim 1 is characterized in that: said chromogenic reagent is for containing 0.01%H ₂O ₂The WS and the pH 7.4 0.1mol/L citric acid damping fluid that contains 0.4mg/ml TMB.

4. F protein assay reagent according to claim 1 is characterized in that: said stop buffer is the aqueous sulfuric acid of 2M.

5. F protein assay reagent according to claim 1 is characterized in that: also comprise damping fluid and cleansing solution.

6. F protein assay reagent according to claim 5 is characterized in that: the said damping fluid that comprises is PBS 0.01M, and pH 7.4; Said cleansing solution is PBST, and collocation method is: 10 * TBSpH 7.6:Tris-alkali, 60.57g; 1M HCl, 420ml; NaCl, 85-90g, behind HCl accent pH to 7.6, constant volume is in 1L; 1 * TBST:, add the TWEEN-20 of 0.1% (v/v) with 10 times of 10 * TBS dilutions.

7. utilize the described F protein assay reagent of one of claim 1-6 to detect the method for F protein content, it is characterized in that: step is following:

(1) encapsulate: use the F protein polyclone antibody of purifying to encapsulate by 10 μ g/ml protein contents, 100 μ l/ holes, 4 ℃ are spent the night, 0.01M PBS pH 7.4 washings three times contain the PBS sealing of 1% hyclone, 110 μ l/ holes, 37 ℃ 1 hour, it is for use to abandon coating buffer;

(2) get 96 orifice plates that are coated with the F protein polyclone antibody, add test serum 50 μ l/ holes, reacting hole is sealed with the shrouding film in enzyme labelled antibody 50 μ l/ holes then, 37 ℃ of constant temperature oven incubation 60min;

(3) discard liquid, the thieving paper arsis is done, and PBST is filled it up with in every hole, leaves standstill 1min, gets rid of cleansing solution, and the thieving paper arsis is done, and so repeats to wash plate 3-8 time;

(4) every hole adds tmb substrate 0.01%H ₂O ₂The WS and pH 7.4 each 50 μ l of 0.1mol/L citric acid damping fluid of containing 0.4mg/ml TMB, 37 ℃ of lucifuges are hatched 15min;

(5) every hole adds stop buffer 50 μ l, measures the OD value in each hole in the inherent 450nm wavelength of 15min.

8. a F protein assay reagent as claimed in claim 1 is used for the purposes that enzyme is exempted from appearance detection F albumen.

9. one kind is used for the special-purpose F protein detection kit of immunoturbidimetry, and it is characterized in that: reagent is formed as follows:

Reagent R1: contain the antiserum of mouse anti human F protein monoclonal antibody,

Reagent R2: increase turbid dose, antiseptic, surfactant, Tris damping fluid;

Definite value serum R3: the genetic engineering reorganization F albumen that contains quantitative purifying;

Said reagent R1 is a mouse anti human F protein monoclonal antibody, and antiserum concentration is 10 μ g/ml;

The polyglycol-6000 that increases turbid dose of employing 4%g/ml among the said reagent R2,

Said antiseptic is a potassium sorbate;

Said surfactant is 1%SDS v/v, Tris pH of buffer=8.2, and concentration is 20mmol/L.

Said reagent R3 is a F albumen dried frozen aquatic products, and every part of definite value F protein content is 1000 μ g, is dissolved in during use in the 10mlTris damping fluid, and being mixed with concentration is the definite value serum of 100 μ g/mL.

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