CN1105181A - Immunoassay - Google Patents

Abstract

A method for determining antibodies to two or more different pathogens in a liquid test sample, which comprises capturing the antibodies on a solid phase on which are immobilized antibodies to one or more classes of immunoglobulins, especially a mixture of anti-IgG and anti-IgM antibodies, and determining any antibodies captured. The method may be applied to the determination of other non-crossreacting antibodies and may also be applied to the simultaneous but separate determination of antibodies to different pathogens.

Description

Immunoassay

The present invention relates to immunoassays.

Different biological from being present in, especially the humoral sample that obtains in the body of bacterium, virus, parasitic animal and plant and other (infectivity) biology that causes a disease and the check of solid sample such as cell or tissue thing are carried out routinely.Check is carried out in two main scopes.A kind of is check to humoral sample and solid sample, its objective is to diagnose the illness, and monitoring disease process and/or monitoring are to the therapeutic process of individuality.This check is commonly referred to as clinical examination, and a typical clinical labororatory tests to a large amount of different biologies." pathogen " this speech is with representing pathogenic organisms here.

Another kind of main check be for the blood that screens donation with supply that keeps blood and the pollution that blood product is not caused a disease.Each national Routine Management office is the clear and definite pathogen that need test of regulation all.All must carry out collective's inspection to human immunodeficiency virus (HIV), hepatitis C (non-A non-B hepatitis), hepatitis B and syphilis in most countries at present examines.Also require to check HTLV (HTLV) in some country.Most countries also must be checked HIV-1 and HIV-2 recently.Be used to transfuse blood to being about to or having accepted the patient who transplants or suffered the patients'blood of immunologic mjury generally will be verified the existence of cytomegalovirus (CMV).Just because of the new subspecies of finding new pathogen and known pathogen have caused threat to the supply of blood, make and to prolong and these pathogen the pressure of donated blood check.

Most widely used blood size analysis is immunoassays.For most of blood disease pathogen, the existence of antigen is than the required sensitivity of the more difficult acquisition of existence of check antibody in the check blood.In general, the check to HIV, HTLV, hepatitis C virus (HCV) and CMV is an antibody test.Hepatitis B there are two kinds of checks: to the antigen measuring of hepatitis B surface antibody (HBsAg) and to the TPPA of hepatitis B core antigen (HBc).At present, all must detect HBsAg in the most countries, in some country, also be essential to the check of hepatitis B core antibody, also may be adopted by more country.The check of antibody and antigen can both be used for syphilis.

The screening of blood is carried out on a large scale, the particularly important is to obtain the result very soon, because the shelf life of blood is shorter relatively.In order to save time and financial resources, proposed in a check sample, to measure the determination method of more than one pathogen.

Determine the assay for antibodies of two hypotypes of allogenic disease substance,, be supplied sale in particular for HIV-1 and HIV-2 (HIV-1+2) and HTLV-I and HTLV-II (HTLV-I+II).Yet must be pointed out very big cross reactivity is often arranged between the hypotype of the same race, for example, in fact the degree of the immune cross-reactivity between HIV-1 and the HIV-2 so big so that initial commercially available HIV-1 check has measured the blood that major part contains HIV-2 very much.Therefore, as in European patent application EP-A-0484787, being explained, the antibody test method that two kinds of hypotypes of virus of the same race are provided with two kinds of antibody testing methods that diverse pathogen provided that do not have immune cross-reactivity are basically had very big difference technically." joint detection method " speech is meant in the one-time detection to sample at this, can detect or measure the pathogenic organisms that two or more do not have immune cross-reactivity basically." different pathogens " speech does not have the pathogen of immune cross-reactivity basically in this expression.

Associating antibody testing method that the viral pathogen (blood disease pathogen) that may occur in the donated blood is carried out is proposed, especially to the joint detection method of HIV and HCV (hepatitis C virus) among European patent application EP-A-0484787.Resulting out-phase detection method relates to utilizes polypeptide to be coated on a solid surface to catch relevant antibody.Resultant antigen-antibody complex just can be measured.

The major defect of recently commercially available this joint detection method and the associating blood testing method of HIV-1+2 and HTLV-I+II is need be at a large amount of antigens of the collaborative coating of solid surface.Antigen (peptide and/or protein) not only must evenly coat, and also must can coat with the mode of antibodies can make antigenic determinant.In addition, must not influence each other between the synantigen.In order to ensure antigen with satisfactory way coat the quality control testing that must carry out, increased the cost of test itself and rejection.The antigen that coats is many more, and problem bothers cost also big more more.Whenever detect the existence that a kind of pathogen just need increase a kind of immobilized antigen at least more.Concerning some pathogen, multiple antibody must can be detected.For example, for HCV, present commercially available detection comprises four kinds of not synantigens.Then need six kinds of antigens at least for the HIV-1+2/HCV joint-detection.

The difficulty in the collaborative practical operation that coats, obtain required specificity and also have any problem.Point out to have strong antigenic high-purity antigen among European patent application EP-A-0484787.

Proposed to be used for the antibody/antigen joint detection method of HIV and hepatitis B surface antibody.For example mention among European patent application EP-A-0286264 and can be coated on the peptide of HIV antibodies on the solid phase, and will be coated on the solid phase the special antibody of HBsAg.These two kinds of solid phases can be different, perhaps also peptide and antibody can be coated on the same solid phase.WO91/10747 mentions the improvement to the sort of detection method described in European patent application EP-A-0286264.Improvement relates to the character of the peptide that is used to coat and detect HIV antibody and the character of detection system.

Yet, it should be noted that, based on disclosed principle among European patent application EP-A-0286264, be used for the unique commercially available joint detection method of HIV and HBsAg, coat relevant different difficult problems with collaborative, coat the HIV peptide and the antibody of the anti--HBsAg on the globule is used for combining with this test tube solved dexterously by inside surface at small test tube.And be not real joint survey method with regard to this detection method of present case because with sample with after globule in the test tube contacts, globule is removed, and detects step and carries out on globule and test tube respectively.Therefore, any about detecting an interactional potential difficult problem between used labelled antibody and the labelled antigen, and the potential difficult problem between detection system (mark) is avoidable in the process of the formed any immune complex of any detection.

According to the present invention, in the associating antibody testing method the collaborative difficult problem that coats multiple antigen of solid surface and by it difficult problem that causes with simply a kind of and perfectly use the method for immunoglobulin (Ig) acquisition mode to solve.

The immunoglobulin (Ig) acquisition mode is by for many years known.1979, people such as Duermeyer W. described the mensuration of specific IgM antibodies in the ELISA reaction of hepatitis A in medical virology magazine (Journal of Medical Virology) 4:25-32.The principle that described human sample detects is: will resist the IgM of human body to be coated on the solid phase, aperture as microtiter plate, added sample and incubation in the aperture that coats, the washing aperture adds antigen in the aperture and incubation again, finally can discern any combined antigen with the antibody of enzyme labeling.The author of this paper points out that this detection method can be used for any single IgM antibody, as wind resistance rash, herpes simplex, cytomegalovirus (CMV), epstein-barr virus (EBV), Toxoplasma gondii and hepatitis B core antigen or other antigen.

Utilize the detection method of single pathogen in the immunoglobulin (Ig) capture technique check blood, used several years as the detection method of rubella and hepatitis A.

Disclosed among the WO 88/07680 in the antibody of using in immunoglobulin (Ig) capture technique mensuration body fluid rather than the blood, particularly saliva and urine.What the commercially available HIV-1+2 that is used for saliva and urine detected utilization is described IgG capture technique.

Can be sure of immunoglobulin (Ig) capture technique ubiquity prejudice, this technology have the fact can prove this point: although just can be used from 1979, but, utilize the detection method of this technology to have only very limited amount available with respect to a large amount of commercially available antigen capture detection of antibodies methods of utilizing.Especially, can be sure of a kind of attitude of ubiquity: promptly this technology can not useful effect aspect specificity or sensitivity in practice.

Thereby utilize the immunoglobulin (Ig) capture technique will have the unwanted interaction of certain degree and cause specific shortage, the WO 89/12231 of the antibody capture detection method of measuring multiple antibody has for example been described, specificity that non-immune protein-protein interactions between the Fc zone of relevant antigen and immunoglobulin (Ig) is caused lacks has represented concern, and the antibody that proposes to be coated on solid surface in immunoglobulin (Ig) catch assay method is the antibody of anti-Fc.Therefore immunoglobulin (Ig) is hunted down in the Fc zone specifically, and this makes nonspecific proteins matter-protein interactions not obtain.

Sensitivity also is considered to a potential difficult problem when utilizing immunoglobulin (Ig) Acquisition Detection method, and for example, immunoglobulin (Ig) can not be caught selectively, does not therefore have the enrichment of the specific antibody kind that will study.If the amount that this strain specific antibodies exists under absolute sense and other antibody type that exists in blood serum sample is very little, the mensuration that then can predict the antibody of required research will be had any problem and is unreliable, especially detects to lack sensitivity.

Opposite with prediction curiously present inventor finds to utilize the immunoglobulin (Ig) capture technique, and the antibody capable of two or more different pathogen is detected simultaneously with fabulous sensitivity and specificity in the sample of being studied.

The invention provides the antibody that utilizes the immunoglobulin (Ig) capture technique to measure two or more different pathogen in the individual sample.

Therefore, the present invention also provides the method for the specific antibody that is used for measuring two or more different pathogens of liquor sample.This method comprises:

(i) sample is contacted with solid phase, fixed a class or the antibody of multiclass immunoglobulin (Ig) more on this solid phase, like this, all kinds of or multiclass immunoglobulin (Ig) that exists in the sample can be hunted down on solid phase.

(ii) that two or more are different antigen simultaneously or contact with solid phase successively, the immunoglobulin (Ig) from sample on this solid phase is hunted down.Each antigen can optionally combine with the specific antibody of a certain pathogen of being studied, but each antigen is provided so that the mode of detecting signal can be provided directly or indirectly.With

(iii) measure the final any immunoglobulin (Ig)-antigenic compound that forms on the solid phase.

Fixing antibody can be direct anti-IgG, IgM or IgA, or the mixtures of antibodies of the anti-inhomogeneity immunoglobulin (Ig) that may use.Especially preferably the mixtures of antibodies of anti--IgG and anti--IgM.

Detection method of the present invention " joint detection method " has unlimited versatility, because used general solid phase to come capture antibody.Which kind of antibody the suitable antigen reagent that the user is used for detecting by use just can select and which kind of pathogen of sample of being studied thereby just can be measured simply.This and common detection method are diverse, this common detection method utilize antigen capture antibody and the antibody studied each time in conjunction with needing a kind of different antigen to coat solid phase.

The versatility of using general solid phase all is a main advantage for the user of manufacturer and multiple pathogen detection especially blood size analysis and clinical detection.As mentioned above, general antibody coats solid phase all can be used as any He all pathogen combinatorial antibody checks as globule that is coated or the microtiter plate aperture that is coated, and is determined because the selectivity of any distinct antibodies check is combination by antigen-reactive agent used in the detection.

This versatility is favourable for the manufacturer of detection method, can be united the detection of antibodies use by all because general antibody coats solid phase, no matter be blood screening or clinical examination.At present the mandatory requirement for the antibody test of donated blood is different between country and the country, is different to different regional required antigen coating solid phases therefore.The solid phase that provides a kind of general antibody to coat for all regions can reduce manufacturing cost largely.Concerning clinical examination, each different associating antibody test needs different antigen to coat solid phase.Provide a kind of general antibody to coat solid phase and can reduce manufacturing cost once more largely.

Thereby for manufacturer further benefit be to be coated on solid phase the composition decreased number manufacturing cost is reduced.Utilize antigen capture detection of antibodies method, need multiple antigen to few to the associating antibody test that two kinds of pathogen are only arranged, for example the joint-detection to HCV/HIV-1+2 needs six kinds of different antigens at least.Pathogen to be determined is many more, and the antigen that is coated on the solid phase is also many more.Yet according to the present invention, need not on solid phase, to fix more antibody, only have the antibody existence of anti-IgG just can measure multiple pathogen in order to measure more pathogen.In addition, be used to the antibody that coats than to coat required high-purity antigen cheap usually in order successfully to coat especially multiple antigen.

The diversity of joint detection method of the present invention is also very favourable to the user, can be only by utilizing suitable antigen combination of agents because for example detect any special pathogen combination.This is particularly useful in that clinical trial is indoor, coats solid phase and just can detect any required pathogen and make up because only need buy and store a general antibody.

Just need not know the organic character of pollution for the purpose of sieving.In blood screening, if blood sample all is positive to the antibody of any forbidden pathogen, then this blood is unsuitable for as blood transfusion or is used for the production of blood product, for example, blood plasma or Factor IX, and this blood should be dropped.

The whole blood antiviral antibody that can measure defined in any special national regulations in one-time detection to a sample of donated blood is a great progress, and it provides can big time saver and the blood storehouse of financial resources.It is very important to save time, thus the time of not only having saved the technician further provide cost savings, and see also very important from the relatively short aspect of the shelf life of whole blood.

Immunoglobulin (Ig) Acquisition Detection method of the present invention has fabulous specificity and sensitivity.We have obtained to contain the result of the sample of interested mixtures of antibodies, and this is the summation of gained result in corresponding serum or the plasma sample basically, and each only contains a kind of antibody these two kinds of samples.Can not predict in joint-detection of the present invention that to react in the organic lot of antibodies of difference each just look like only to exist a kind of antibody the same.The added influence of observing about the number of measured antibody type does not reckon with, but important practical advantage is arranged.Do not reckon with yet and of the present inventionly can utilize the conventional determining mode for example microtiter plate and determination method are so sensitive unexpectedly.

Mensuration of the present invention can be that IgG measures, IgM measures or be specific purposes IgA mensuration.Alternatively, the potpourri of the antibody of variety classes immunoglobulin (Ig) can be used to coat solid phase.The preferred especially mixtures of antibodies that uses anti--IgG and anti--IgM.

Determination method of the present invention is compared another important advantage with the determination method of the antigen capture antibody that utilize to coat be by using fixing anti--IgM, and early stage infection can be measured reliably.This is all very important for blood screening and clinical research.

IgM replys the first kind of immunoglobulin (Ig) that produces when infecting, and infects that specific I gM antibody rises in the first few week of back, and slightly later, specific I gG has produced, and the reaction of IgG antibody response is continual, can lasting for years, even throughout one's life.IgM can measure with routine inspection in theory, promptly interested antibody be captured to the surface that antigen coats, utilize then and measure the existence of captive antibody as the Anti-Human's class-IgM antibody of mark.Yet in the practical operation, this IgM check lacks specificity usually, because IgM is " viscosity ", in other words, IgM trends towards being incorporated on the capture antigen non-specificly.Anti--IgM the antibody through mark that is used to measure antigen-IgM compound can not be distinguished the IgM of specific antigen-IgM compound and non-specific binding.For this reason, utilize the routine inspection of immobilized antigen to measure with anti--IgG antibody usually, promptly this check can only be effective to IgG.

In the mensuration of the present invention, anti--IgM antibody can be coated on solid phase.Then, those antibody are caught the IgM immunoglobulin (Ig) specifically from sample, and captive IgM is detected specifically by the mode of the antigen of mark.We find that this system is not " viscosity " curiously, and measure the existing specificity problem of IgM antibody with conventional antigen capture detection method and also no longer exist.As mentioned above, the preferred especially anti--IgG antibody and the potpourri of anti--IgM antibody of using coats solid phase, the existence of anti--IgM antibody can guarantee that any early antibody that exists in the sample is measured on the solid phase, and simultaneously, the existence of anti--IgG antibody can guarantee that lasting antibody is measured.

Antibody fixing on the solid phase can be polyclonal antibody such as the anti-human antibodies of polyclone, and this antibody contains all antibody of planting immunoglobulin (Ig).Alternatively, also can use the directly polyclonal antibody of anti-certain particular types immunoglobulin (Ig), as polyclone anti--IgG and polyclone be anti--IgM antibody.Polyclonal antibody can be purified, and for example passes through affinity chromatography.If desired, also can use monoclonal antibody.Anti-immunoglobulin can be respectively is special to immunoglobulin (Ig) ν, μ or the α chain of IgG, IgM and IgA.

The polyclone and the monoclonal antibody that are used to coat solid phase can prepare by known method.Commercially available different anti-immunoglobulin, as the polyclone of rabbit, sheep and goat anti--monoclonal anti-IgG and anti--IgM immunoglobulin (Ig) of IgG and anti--IgM immunoglobulin (Ig) and mouse.If coating used is the potpourri of different antibodies, the different antibodies proportion also can change on demand in the potpourri so.The present invention is not limited to coming from the research of human sample, also is applicable to animal doctor's application.Therefore, coat used antibody and should directly resist the sort of immunoglobulin (Ig) that obtains from institute's study sample, for example for the mankind's sample, coating antibody should be anti-human antibody.

As mentioned above, an especially favourable feature of determination method of the present invention is: no matter the number of the pathogen of being studied or character how, all can use a kind of general antibody to coat the method for solid phase.As directly anti-one or more the kind immunoglobulin (Ig)s of the antibody that coats antibody, especially anti-IgG and IgM, and also it can capture the representative part of various immunoglobulin (Ig)s from sample.By selecting any specific combination of specific antigen, the user of this determination method can detect any combination of pathogen.Antigen reagent can mix again and provide, and perhaps the user uses antigen reagent respectively in any required combination.If use separately, antigen reagent can simultaneously or contact with the solid surface that carries captive immunoglobulin (Ig) with any order successively.Usually the potpourri that adds required reagent in a step is more convenient.

Used antigen can be anyly can interactional antigen entity optionally take place with the antibody of being studied in the detection method of the present invention.For example, antigen can be peptide or polypeptide, can be antigen that synthesize or reorganization, perhaps from the cell culture antigen of purifying from the virolysis thing for example.Particularly useful in some occasion with the recombinant fusion polypeptide that contains certain special organism and more than one antigen zones, as HCV.

Antigen self can be labeled, usefulness be can directly or indirectly provide recognizable mark so that any immunoglobulin (Ig)-antigenic compound can be measured method.The antigen that is labeled is known as " antigen bond ".

Discernible sign can be on optic, radioactive or the physical chemistry, can directly provide by labelled antigen, for example use dyestuff, color grains, radioactive label, electroactive class, magnetic resonance class or fluorophore, perhaps provide indirectly with enzyme-labelled antigen, this enzyme self can cause various variations of measuring.Alternatively, sign can produce from the agglutination relevant with the antigen bond, diffraction or birefringence effect.

More particularly, can provide the method for recognizable mark to comprise an antibody that combines with antigen indirectly.This antibody (Ab1) can offer antigen (monoclonal antibody body measurement system) with above-mentioned direct or indirect mark mode, perhaps the mode with another kind of antibody (Ab2) is labeled, this antibody combines with Ab1, and it self is labeled as stated above directly or indirectly to supply with antigen (double antibody mensuration system).If be difficult to produce suitable antigen bond, the list or the double antibody system that measure the antibody-antigenic compound that is hunted down are just particularly useful.Be tending towards stable inadequately when for example some antigen bonds use as commodity, and some antigens in conjunction with the time lost antigenicity.

The advantage of monoclonal antibody body measurement system is to relate to less washing and incubation step, but shortcoming is all must produce an antibody conjugates (the antibody A b1 that is labeled) to each antigen that need measure.The advantage that double antibody is measured system is suitably to select Ab1 and Ab2, just can measure all captive antibody by antibody conjugates of as described below usefulness (the antibody A b2 that is labeled): when produce every kind of antibody (Ab1) in same animal kind, then second kind of antibody (Ab2) is the antibody of anti-this animal kind.If for example each Ab1 antibody is the antibody of sheep, the antibody of the anti-sheep that is labeled so just is used as Ab2.The advantage that only needs a kind of antibody conjugates just can measure all analytes has surpassed the shortcoming of extra incubation of needs and washing step usually for the user, especially when using automated system.For manufacturer, only needing a kind of antibody conjugates is another significant advantage.

The Mk system that is particularly preferred for antigen or antibody conjugates is the enzyme labeling system, particularly a kind of like this system, and in this system, antigen or antibody combine with enzyme, and when suitable substrate existed, this enzyme can the detectable change color of catalysis.This enzymoimmunoassay or ELISA method are widely used as commodity, and the automation equipment that can obtain to carry out this determination method use, as microtiter plate, have " pearl " or " IMX " (trade mark) system of patent rights, perhaps use hollow bar or head of pipette.Also can obtain being used for handling gained result's computer software.

Used enzyme system is well-known, for example can be referring to Kemeny D.M.﹠amp; " ELISA and OtherSolid PhaseImmunoassays, the Theoretical and Practical Aspects " that Challacombe S.J. is compiled.

The example of typical enzyme system is that those use alkaline phosphatase, beta galactosidase, urase or peroxidase, for example system of horseradish peroxidase.

The solid phase of capture antibody can be the aperture or the cuvette of for example globule or microtiter plate, also can be other form, strictly according to the facts or hollow bar or transfer pipet, or diameter is the particle of 0.1 μ m to 5mm.(no matter what the raw material of making is, this particle all is known as " latex " particle usually.)

Solid phase can be plastics or polymeric material, for example nitrocellulose, Polyvinylchloride, polyphenyl ethylbenzene, acid amides, Kynoar or other synthetic polymkeric substance.In addition, particle can also be the polymkeric substance of nature, for example latex or protein.Microtiter plate and globule are widely used in blood screening and clinical examination, and can extensively be bought as commodity.

The also spendable solid phase of another kind comprises film, thin slice and band, they are by material porose, fiber or suction, for example nylon, Polyvinylchloride or other synthetic polymkeric substance, cellulosic natural polymkeric substance, as the derivant of cellulose acetate or nitrocellulose class nature polymkeric substance, or glass fibre is made.Stationery such as diazotising paper also can use.Film and coating that material suction or fiber is made also can be used as solid phase as previously mentioned.

The example that should be appreciated that above-mentioned listed solid phase is only as illustration, and the present invention is not limited to only use these solid phases.The present invention can carry out being applicable on any solid phase of immunoassays.

As the solid phase of film, thin slice, band, film or coating can be installed into measure multiple, more often be a kind of device of sample.

" determinator " speech carries out the instrument of immunoassays in this expression, comprise a solid phase in this determination method, be generally the thin layer solid phase,, be fixed with the antibody of a class or multiclass immunoglobulin (Ig) on it as film, thin slice, band, coating, film or other lamelliform thing.The antibody that is fixed preferably exists in the zone that limits, and is called " antigen trapping area " here.

Pick-up unit is equipped with solid phase in hard holder or cover, the reagent of some or all that it comprises also that testing process is essential.Usually sample should be placed on preassigned sample and use the district in pick-up unit, for example, sample is poured or splashed into into this zone, and the relevant portion in perhaps will installing dips in sample.If sample uses the district different with the antibody capture zone position, the arrangement of device makes the antibody capable in the sample move to the antibody capture district usually.Then, required reagent is added into the use district of appointment again by suitable order, and this use district and sample use the district can be the same or different.Moreover if this or any one reagent use the district different with the antibody capture zone position, then the arrangement of this device makes reagent can transfer to the antibody capture district usually, and any formed antigen-antibody complex all can be determined in this distinguishes.Required whole or some reagent of immunoassays can place in the device with liquid or dry form.If like this, install general such arrangement: the interaction between the device different piece makes different reagent contact so that immunoassays are carried out with correct order each other.This interaction can also can be caused by the device user in the device operation in automatic generation.

Big amount determining device has been described in the document of immunoassays.United States Patent (USP) the 4th, 623,461 and 4,693, the example of film device has been described in No. 984.According to their design and responsiveness, some determinator is called as " gage ", and some is called as " fast measuring " device." fast unit " generally just can provide the result in 10 minutes adding sample.(typical microtiter plate or globule detect and need incubation step, and need one hour just can go out the result usually at least.) therefore, although determinator generally than the mensuration mode costliness of microtiter plate or globule, they have special use in clinical detection, when for example needing to provide the result fast in urgent operation.

Determinator has a special advantage, i.e. their use does not need complicated laboratory equipment or even do not need any laboratory equipment.Therefore they can be used to " then and there " check, for example in the first-aid room, in doctor's the operation, in the pharmacy or be used for family's check in some cases.For the mutual again less area a long way off of laboratory equipment, this device is particularly useful.

As mentioned above, joint detection method of the present invention is particularly useful for the screening of donated blood.Therefore, this detection method can be used for detecting the antibody of at least two kinds of viruses, and these two kinds of viruses are selected from HIV, as HIV-1, HIV-2 and HIV hypotype such as HIV-1 hypotype 0; HCV; Hepatitis B (antibody of cAg); HTLV is as HTLV-I and HTLV-II; CMV; EBV (Epstein-Barr virus); Can also choose detection syphilis wantonly.The combination of preferred two or more viruses, these viruses are selected from HIV, as HIV-1+2; HCV; HTLV is as HTLV-I+II; And hepatitis B virus core antigen (HBc); For example HIV and HCV; HIV and HTLV; HTLV and HCV; HIV, HCV and HTLV; HIV, HCV and HBc; HIV, HTLV and HBc; HTLV, HCV and HBc; HIV, HCV, HTLV and HBc.In the combinations thereof any can comprise syphilis.The selection of this combination is influenced by the regulations of particular country.In some cases, also need detect other pathogen in the blood of donation, for example the rubella pathogen.

In addition, when finding new pathogenic thing, (the new biology and the new subtype of known organism), and the requirement of blood screening will be examined again, and enforceable check should expand to those pathogen.For example, in most countries HIV-1 and HIV-2 are needed check now.The discovery of last HIV-1 hypotype 0 makes to require to measure and also must detect this hypotype.The pathogen combination that therefore, detect almost will inevitably enlarge in time.The present invention includes the detection of this pathogen combination.

As use test tube/globule two component systems can produce further versatility, for example, with test tube and globule with after sample contacts, remove globule and make it with one or more not the antigen of pathogen of the same race contact, test tube then with the combination contact of different antigen or antigen.

Test tube/globule two parts detection modes further are modified to: one or more anti-immunoglobulin are fixed on one of them parts, resistance of hepatitis B surface antigen (anti-HBsAg) is fixed on another parts.For example, the inside surface with anti-IgG and optional anti-IgM are coated on small test tube is coated on anti-HBsAg on the globule, otherwise also can.Globule and test tube can be used to HBsAg/ many kinds of detection of antibodies, particularly HBsAg, HIV, the HCV that unites and one or more analytes that further detect of choosing wantonly simultaneously from hepatitis B (core), HTLV and syphilis then.

With HIV-1 and HIV-2 interactional peptide of specificity and polypeptide also description to some extent as everyone knows take place, for example in European patent application EP-A-0347148.We have found that to check the sulfydryl of the halfcystine family that has repressor with synthetic HIV peptide particularly useful, referring to European patent application EP-A-0307149, the peptide that can cause the peptide, the especially enzyme labeling that are labeled has bigger immunocompetence than the peptide that is not checked accordingly in detection.

The antigen that is used to discern hepatitis B core antibody is cAg.The DNA of different serum of hepatitis B types and the protein sequence of prediction are disclosed, and for example people (1983) such as Ono is at Nuc.Acids.Res 11, the description in 1747 can obtain the peptide and the polypeptide cAg of suitable synthetic and reorganization simultaneously from disclosed sequence.

At present, hepatitis C looks does not also have the dominant antigenic determinant of a kind of immunity, preferably checks the antibody in zone more than.For example, will comprise that the fusion of an above antigenic determinant is favourable as antigen, as derive from the amino acid sequence of at least one structural area and at least one non-structural area, as UK Patent Application GB-A-2, described in 239,245.Antigen from core and adventitia district is preferred construction antigen.Non-structural antigens can be selected from for example NS3, NS4 and NS5 zone.

HTLV antigen can be for example p21e or gp46 recombinant protein or from the peptide (for example referring to United States Patent (USP) the 4th, 743, No. 678) of p21e or gp46.Purified p21e can be from Cambridge Biotech Corporation, 1600 East Gude Drive, Rockv-ille, Md 20850-5300, U.S.A. obtains, gp41 can be from Repligen Corpora-tion, 1 Kendal Square, Building 700, Cambridge, Ma 02139, and U.S.A. obtains.

In order to detect CMV antibody, can use the CMV core protein of purified cultivation, as p66 or reorganization pp150, it is preponderated in west spot (Western blots).In order to detect EBV antibody, can use capsid or early stage antigen.Purified Spirochaeta pallida antigen can be used for detecting syphilis antibody.

Joint detection method according to the present invention can be used as the preliminary size analysis of several pathogen in clinical diagnosis.If test positively, then carry out the independent experiment of every kind of pathogen.If the result is negative, just need not to carry out again.Generally speaking, saved time and money.The example of this associating is to be used for the screening that the pregnant woman is called as " TORCH " in many countries.Require similarly to blood screening, it is different that the pathogen of being studied is combined between the various countries, but normally examines, selects toxoplasmosis, CMV and the bleb from wind.Utilizing through the fixing anti-IgG and the potpourri of anti-IgM antibody is very favourable to guarantee detecting recent infection.

In the pick-up unit mode, particularly the joint detection method of the present invention represented of " fast detecting " device mode be when pressing for screening result, as when carrying out urgent operation, is of great use.For example, to the fast detecting of two or more different blood disease venereal disease substances, warning especially should be notified the surgeon if desired.These pathogen as mentioned above, for example HIV-1+2 and HBc.

A kind of to the pick-up unit of interested pathogen combination be useful, for example, " scene " of doctor's Surgical Operating Room detected with auxiliary diagnosis; And, generally speaking, detecting and avoid repeating into aspect the Surgical Operating Room in clinical labororatory, this device can both save time and money.Moreover pick-up unit is useful for the detection that the laboratory equipment condition is difficult for the area of acquisition, for example in rural area and developing country.The example of pathogen combination is CMV and HIV, TB (tubercle bacillus) and HIV.

The versatility that the solid phase of utilizing general antibody to coat detects a large amount of antigens is able to further application in clinical examination.In clinical examination, this purpose is normally discerned the pathogen in the sample.At present, if a large amount of sample must be checked the not existence of pathogen of the same race with the mode of common microtiter plate or globule, then need to prepare the aliquot of different samples usually, and carry out a series of checks, each is checked at a kind of different pathogen.This is because the mode that conventional check utilizes antigen to coat is caught the specific antibody of pathogen.Therefore, be necessary to utilize a kind of different antigen to coat mode, normally globule or microtiter plate for every kind of antigen.So just increased the time that particular sample is obtained the result, also produced risk, promptly the aliquot of sample may be misplaced or obscure when all different tests are carried out.

Moreover the instrument that antigen coats generally occurs with complete form with other reagent, as the positive and negative control, and cleansing solution and dilution.Therefore the user must have the different instrument of many covers.

Utilize determination method of the present invention, a general antibody coats instrument and microtiter plate, only need use suitable antigen reagent just can simultaneously and measure every kind of a large amount of pathogen respectively.Different sample series can be verified simultaneously, and some are in order to check this pathogen, and some are in order to check another.Particularly advantageous is can be simultaneously and check the aliquot of the single sample of different pathogens respectively, for example, each aliquot of check is to measure different pathogen in each aperture of microtiter plate, the result has saved time and money, has also reduced owing to the careless omission of sample or obscures the risk of makeing mistakes.

Therefore, the present invention also provides simultaneously but has measured the method for the specific antibody of two or more different pathogens in the liquid check sample respectively, and this method comprises:

(i) one in every kind of sample and a large amount of unit is contacted, for example in single assembly, each unit comprises a solid phase, this solid phase carries the class that is fixed or the antibody of multiclass immunoglobulin (Ig), all kinds of or each the monoid immunoglobulin (Ig) that contains in each sample so just is hunted down on solid phase, and the antibody that is fixed in all unit of assembly is same class or monoid.

(ii) each unit that will contact with sample contacts with a kind of antigen again, and this antigen can optionally combine with the specific antibody of a kind of pathogen that will study; Each antigen be provided in the mode that recognizable mark can directly or indirectly be provided and

(iii) measure the final immunoglobulin (Ig)-antigenic compound that forms on the solid phase.

The number of used different units and therefore and the antigen number that uses is by the pathogen number decision of being studied.Each unit can be an aperture of for example microtiter plate or a container that contains globule.

Can a localized area on film, thin slice, band, film or the coating when selectively, each unit especially occurs in verifying attachment.Known that some device has the presumptive area of catching composition from the sample of being studied.According to of the present invention the simplest a kind of be used for simultaneously but separately the band verifying attachment of independence test comprise that the strip of making as a water-absorbing material has a band of catching immunoglobulin (Ig) across the width of this strip, is called the antibody capture district here.To obtain the Region Segmentation that immunoglobulin (Ig) coats be a plurality of parts by at large with best for this.Each part of having fixed immunoglobulin (Ig) can be considered to a unit in antibody capture district.

In the time of above-mentioned but independently to check required verifying attachment and joint survey separately be related.

For manufacturer and user, the versatility of verifying attachment of the present invention provides special facility, independently detects when this device can be used for different pathogens but separately.Main advantage is that the device in an antibody capture district that provides many qualifications can be provided for together with most of antigen reagent and checks a large amount of different antibody in the sample simultaneously.As previously mentioned, the facility of manufacturer is only need make a kind of device to all analytes, and user's facility only need also to be a kind of device, and further advantages are that the user need can freely select the analyte combination of mensuration.

As mentioned above, for any special pathogen combination in the study sample, can implement of the present invention to different pathogens the time but independently detect separately.For example, the antibody and the hepatitis C virus (HCV) of anti--cAg of hepatitis A, hepatitis B can be verified simultaneously, gonorrhoea, chlamydiaceae and Mycotoruloides can be verified NUS patient's sample to the sample of hepatitis patient.

While of the present invention but separately independently the check modification be: each unit no longer has the sessile antibody of same class or monoid, can have inhomogeneous fixedly immunoglobulin (Ig) on one or more unit.For example, can provide a device that is with two unit, a unit has fixing anti--IgG, and another has fixing anti--IgM.

Be used to check a series of checks simultaneously of the distinct antibodies of the sample that obtains from same individuality to carry out a period of time the verifying attachment of this two unit, this detects for measuring early infection and later lysis particularly useful, especially later serum conversion is as HIV or HCV.The device of one cover Unit three, unit has fixing anti-IgG, a unit has fixing anti-IgM, the 3rd unit has fixing anti-IgA, this device is for checking whether the baby has HIV and is particularly useful: existing himself antibody also has the antibody from maternal in baby's the blood, therefore be difficult to determine with routine inspection whether the baby has the HIV antibody of himself, this antibody is the sign that infects.

The further example that utilizes more than one antibody is what is called " TORCH " test of the pregnant woman being carried out in a lot of countries.Pathogen is selected from rubella, toxoplasmosis, CMV and bleb.The combination of selecting is different because of country.The rubella check should comprise that IgM detects to measure proemial recent infection.Therefore, detect for " TORCH " that relate to rubella, at least one unit should be coated anti--IgM in the used device.

The assembly itself that comprises two or more unit also is a part of the present invention, and these unit have the antibody of the anti-different monoid immunoglobulin (Ig)s that are fixed.Therefore, the present invention also provides the assembly with two or more unit to be used for to different pathogens simultaneously but detect respectively, these unit comprise the antibody of the anti-immunoglobulin that is fixed, at least one unit has, and sessile antibody directly resists inhomogeneity immunoglobulin (Ig) next in the immunoglobulin (Ig) that is fixed in another unit, for example, in the unit fixing anti-IgM can be arranged, and in one or more unit fixing IgG can be arranged.Another program is that assembly comprises three unit, and there is fixing anti--IgG a unit, and there is fixing anti--IgM a unit, and there is fixing anti--IgA the 3rd unit.

Also the assembly that comprises two or more unit can be arranged in pick-up unit, for example, arranging has two or more antibody captures district, as IgG trapping region and IgM trapping region.This pick-up unit can be as the different pathogens time but is independently measured separately, any as above-mentioned special method.

Assembly optionally comprises two covers or overlaps unit (every cover comprises identical unit) more.Different covers unit series can be used as an assembly and is provided.For example a cover unit can be used as capillary strip and is provided.This capillary strip can be installed on demand and comprise that two overlap or overlap in the assembly of unit more, and for example, an assembly can comprise two capillary strips, and the anti-mankind of a usefulness-IgG coats, and another coats with the anti-mankind-IgM.Article three, having the fixing anti-mankind-IgA also can be added into.Capillary strip is that complete unit can be installed on demand to form conventional microtiter plate.For example, the micropore that anti-on the plate-IgM and anti--IgG coated is staggered in order to subsequently blood conversion with row or column.Moreover, be favourable to manufacturing cost and versatility, especially capillary strip or similar complete unit.

Unless otherwise defined, below describe and can be applicable to simultaneous determination method of the present invention and determination method respectively simultaneously but separately:

To clinical examination, the liquid check sample can be any body fluid, as blood, blood plasma, serum, saliva, urine, celiolymph, milk liquid, lymph liquid or tears.Selectively, solid sample such as the cell or tissue form that can become liquid is used for check as organizing transudate.

For blood screening (carrying out better with the simultaneous determination method), interested pathogen is so-called blood disease venereal disease substance (HIV, hepatitis B and hepatitis C also can be chosen HTLV, CMV and EBV wantonly) and syphilis.For clinical examination, the scope of interested pathogen is very wide, comprises the pathogenic microorganisms of above-mentioned organism and other virus, bacterium and other kind, and following is for example, and not as restriction:

Rubella, measles, bleb (simple with reproduction), chlamydiaceae, gonorrhoea, hepatitis A, varicella, mumps, the human parvovirus, Much's bacillus, Mycobacterium leprae, mycobacterium avium, staphylococcus aureus, monocyte Listeria monocytogenes, Bacillus anthracis (antigen/toxin), actinomyces are (as streptomycete, Nocardia, red coccus), salmonella typhi, yersinia enterocolitica, pylorus convolution bacillus, campylobacter jejuni, pseudomonas mallei and pseudomonas pseudomallei, pseudomonas aeruginosa, legionella pneumophilia (Legionella pneumophila) and subspecies thereof, soil draws hot francis fungus, Bacterium melitense, Mycoplasma pneumoniae, question mark shape Leptospira, the subspecies of Borellia, Spirochaeta pallida, Candida albicans and by the caused disease of protozoan pathogen, for example amcbiasis, babesiasis, Chagas' disease, leishmaniasis, malaria and toxoplasmosis.

Through fixing anti-immunoglobulin can be a class or multiclass, that is to say, and can be IgG class, IgM class and IgA class.As above mention, IgG is an immunoglobulin (Ig) the abundantest in the serum, and IgM replys to infect the first kind of immunoglobulin (Ig) that produces, so it more can provide the early indication of infection than IgG.For blood test (blood plasma or serum), usually according to the purpose of checking, IgG or IgM can use separately, perhaps for simultaneous determination anti--potpourri of IgG and anti--IgM can use in a unit, perhaps for simultaneously but divide other check, anti--IgG and resisting-IgM can be placed in the different unit.Detect for saliva, also preferably catch IgG and/or IgM.For urine test, the preferred use resists-IgG, chooses wantonly to combine with IgM and/or IgA.

For those detection modes of utilizing globule/test tube two component form among the present invention, by fixing one or both anti-immunoglobulin at the inside surface of plastic test tube, fix one or both different classes of anti-immunoglobulin on the globule that in test tube, uses, can further increase the applicability of detection of the present invention.For example, can coat anti--IgG on of these two parts, another coats anti--IgM.

In addition, can use, test tube and globule with after sample contacts, remove globule it is contacted with a kind of antigen, and test tube are contacted with another kind of antigen, so that carry out two kinds of antibody tests in a sample in conjunction with globule/test tube form.

Although blood screening described above and clinical examination, the type that detects used principle or material therefor does not have any difference.The special analysis thing scope of blood screening is to be limited by specific national management office, and only because a large amount of identical check makes it trend towards full automation more than clinical examination.Yet, be to be understood that in the whole instructions that unless otherwise defined, all descriptions are all relevant with detection method of the present invention substantially, both have been included in the detection of carrying out in the pick-up unit, also comprise the detection that those carry out on for example microtiter plate or globule.

Detection of the present invention can be carried out according to a conventional method, " the ELISA and Other Solid Phase Immun-oassays Theoretical and Practical Aspect " that is compiled referring to Kemeny D.M. and Challacombe S.J. for example, immunoglobulin (Ig) can be fixed on the solid phase, for example solid phase is contacted with the immunoglobulin solution of debita spissitudo with the pH value, can be 7 to 11 as the pH scope, particularly 9 to 10.The preferred damping fluid that uses is as the sodium carbonate/bicarbonate damping fluid.As mentioned above, suitable immunoglobulin preparation can have been bought, and commercial anti liquid solution suitable dilution degree is that for example, 1: 200 to 1: 4000V/V.

Antigen reagent (bond) through mark can be by any generation in the big metering method, and for example referring to Kemeny and Challacombe, loccit generally uses antigen-enzymatic compositions.

Sample can be diluted, and for example half-and-half dilution of blood or plasma sample is as adding 50 μ l samples in the 50 μ l dilutions in the micropore.Must be pointed out that used any sample diluting liquid can not contain that class or those class human immunoglobulins that will catch to some extent.In the detection of carrying out with microtiter plate or globule form, sample generally then will be with the solid phase incubation.The time of incubation and temperature are complementary, and the time required when temperature is low is longer.Typical incubation conditions be 37 ℃ following 1 hour.Behind the incubation, after titer plate or globule will thoroughly wash again with antigen reagent (bond) incubation.Be used for typical incubation conditions in conjunction with the stage and be 37 ℃ following 30 minutes.Behind the bond incubation, also has a washing step.Under the ELISA situation, also need be then with the substrate incubation of enzyme, typical incubation conditions also be 37 ℃ following 30 minutes.When finishing, incubation adds reacting terminating solution.Under the ELISA situation, the result obtains by the light absorption value of reading each unit in the spectrophotometer.If use another kind of Mk system, method will correspondingly be revised, and for example, in radioimmunoassay or fluorometric assay, just can obtain the result behind radioactive label or fluorescent labeled antigen bond incubation.For color grains, just can determine the positive and negative findings by eyes.

Under the situation of experiment outdoor application pick-up unit, the color grains thing that serves as a mark is particularly useful, because the positive or negative result can be by eye determining, reactions steps is minimum.The ELISA system can be used as more sensitive detection.

The present invention also provides complete equipment, and this device comprises:

(a) a kind of solid phase, especially plastic beads or microtiter plate, it carries a fixing class or the antibody of multiclass immunoglobulin (Ig), the mixtures of antibodies of particularly anti--IgG and anti--IgM,

(b) two or more antigen reagent, each can optionally combine with the specific antibody of a kind of pathogen of being studied, and each antigen is provided so that the mode of recognizable mark can directly or indirectly be provided, selectively also comprise following one or more:

(c) positive and negative control reagent, cleansing solution and dilution.

Complete equipment can provide like this, and perhaps, antibody cladding parts and antigen reagent parts are provided separately separately.

The present invention also provides the solid phase that is suitable for using in the immunoassay, has fixed the mixtures of antibodies of anti--IgG and anti--IgM on it.Can also comprise anti--IgA antibody in the mixtures of antibodies.These antibody are special anti-human antibodies.Solid phase is any in the above-mentioned solid phase for example, as microtiter plate and globule, also comprises the solid phase that those are applicable to pick-up unit.

" detection " and " mensuration " these two speech are with all showing qualitative, the quantitative and semiquantitative check of pathogen here.

Utilize the immunoglobulin (Ig) acquisition mode to measure the antibody of most of pathogen in the different samples (" joint detection method ") or simultaneously but measure respectively that most of different antibodies (" determination method simultaneously ") not only save time but also save money in the aliquot of same sample.The potential versatility of this mode is only to need to select antigen reagent, and multiple pathogen just can be measured in a unit or series of parallel unit, but this versatility was not recognized in the past.Can utilize general antibody to coat mode,, carry out any antibody test through fixing antibody in globule that antibody coats and the verifying attachment and should do not underestimated in the very tangible economically advantage of practical operation neutralization as the microtiter plate that antibody coats.

Certainly, recognize that the present invention is not limited to the detection pathogen, also can be applicable to detect interested any antibody, regardless of the character of the antigen that causes the antibody generation, for example with the irrelevant antibody of pathogen.The example that causes the condition that produces this antibody comprises autoimmune disease and allergic reaction: as non-organ specificity autoimmune disease, as rheumatic arthritis, lupus erythematosus and rheumatic fever, with the organ specificity autoimmune disease and be considered to and the autoimmunity diseases associated, as the thyroid gland autoimmune disease, myasthenia gravis, the colitis of autoimmune hemolytic anemia, multiple sclerosis, canker sore, pernicious anaemia and ulcer.Needed all be one can be specifically with the entity of interested any antibodies.

Therefore, antibody that the theory that should be appreciated that this instructions and detection and pathogen are irrelevant and the used theory of antibody that detects pathogen are relevant, and the present invention includes all these schemes.

The present invention can be used for discerning the mensuration of human or animal's pathogen, both can be used for the mankind, also can be used for animal doctor's application, is included in the application in the meat trade.

Following non-limiting example has been set forth the present invention.

Embodiment

Reagent

Following reagent is used for following detection:

1, solid phase: 96 hole microtiter plates (Nunc) are coated with the potpourri that anti-IgG of polyclone (DAKO) and polyclone resist human IgM (DAKO).(the Nunc product can be from LifeTechnologies, PO BOX 35, and Washington Road, Abbotts InchIndustrial Estate, Paisley, Renfrewshire, TA3 4EF, Scotla-nt obtains; The DAKO product is from DAKO, 16 Manor Courtyard, and HughendenAvenue, High Wycombe, Bucks HP3 5RE, England).

2, the HIV-1 recombinant protein that combines with HRP (horseradish peroxidase), it comprises core and periphery antigen (people (1986) Science such as Sattentau Q.J. of the separator CBL-1 that derives from HIV-1 2341120) (" HIV-1 bond ").

3, the HIV-2 peptide that combines with HRP, it comprises gp36 periphery antigen (" HIV-2 bond ").

4, the hepatitis core antigen that combines with HRP.

5, positive:

A) HIV-1, internal reference numbers 4034 is diluted in the HIV-1 negative serum.

B) HIV-2, internal reference number 91/174 is diluted in the HIV-2 negative serum.

C) positive of anti--HBc cAg is diluted in the HBc negative serum.

6, negative sample: the normal person's who from donated blood, obtains serum.

Method

The sample (10 μ l samples and 90 μ l sample diluting liquids) of 100 μ l dilution is joined in the aperture of microtiter plate, and incubation is 60 minutes under 37 ℃ wet condition.With cleansing solution aperture is thoroughly washed 5 times then, each washing all will be removed the content sucking-off in each hole, and aperture is full of ((the glycocoll borate buffer solution that contains tween)) with cleansing solution, soaks 30 seconds.After the last washing, remove the content in the aperture, aperture is inverted, tapping is to doing on facial tissue or thin paper.To be dissolved in the HEPES damping fluid that contains bovine serum albumin(BSA) and detergent, the working strength solution of 50 μ l related conjugates adds in the aperture, both can add separately or the adding that combines by following, then with titer plate incubation 30 minutes under 37 ℃ wet condition, after above-mentioned further washing, to contain TMB (3,3 ', 5,5 '-tetramethyl benzidine) and 100 μ l substrate solutions of hydrogen peroxide join in each hole, with titer plate incubation 30 minutes under 37 ℃ wet condition, use 50 μ l 2M sulfuric acid cessation reactions then, the absorptance in the aperture goes on record at the 450nm place, and reference wavelength is 690nm.

Embodiment 1

(a series of positive diluted and check according to the record of above-mentioned use HIV-1, HIV-2 and HBc bond of anti-HIV-1, anti-HIV-2 and anti--HBc) can be carried out as listed independent of following table or with different combinations respectively to contain a kind of interested antibody.The HIV-1 sample is 1/40,1/80,1/160 and 1/320 by serial dilution.The HIV-2 sample is diluted as 1/32,1/64,1/128 and 1/256.Many different HBc positive are with 1/2 dilution.

(i) with following bond in the ever-increasing HIV-1 positive of dilutability each is tested, this bond is: HIV-1:HIV-1+HIV-2; HIV-1+HBc; HIV-1+HIV-2+HBc.The result lists table 1 in.

Table 1

Sample-＞	????HIV-1	????HIV-1	????HIV-1	????HIV-1
					The dilutability that increases	????＞3	????2.897	????＞3	????＞3
	????2.015	????1.905	????2.011	????1.847
						????1.282	????1.148	????1.214	????1.129
	????0.694	????0.663	????0.695	????0.672
						????0.41	????0.378	????0.412	????0.378
	????0.232	????0.230	????0.282	????0.235
					Negative control	????0.058	????0.061	????0.068	????0.076
Negative control	????0.060	????0.064	????0.075	????0.075
					Bond	????HIV-1	????HIV-1+ ????HIV-2	????HIV-1+ ????HIV-2	????HIV-1+ ????HIV-2+ ????HBc

(ii) test with following bond each to the ever-increasing HIV-2 positive of dilutability, this bond is: HIV-2; HIV-2+HIV-1; HIV-2+HBc; HIV-1+HIV-2+HBc.The result lists table 2 in.

Table 2

Sample	????HIV-2	????HIV-2	????HIV-2	????HIV-2
					Constantly	????0.099	????1.045	????1.009	????1.002
The dilutability that increases	????0.588	????0.635	????0.619	????0.619
						????0.386	????0.397	????0.386	????0.383
	????0.232	????0.252	????0.249	????0.251
						????0.158	????0.167	????0.171	????0.183
	????0.111	????0.124	????0.126	????0.135
					Negative control	????0.053	????0.060	????0.061	????0.072
Negative control	????0.053	????0.062	????0.063	????0.075
					Bond	????HIV-2	????HIV-2+ ????HIV-1	????HIV-2+ ????HIV-1	????HIV-2+ ????HIV-1+ ????HBc

(iii) with following bond in six kinds of different HBc positive each is tested, this bond is: HBc; HBc+HIV-1; HBc+HIV-2; HBc+HIV-1+HIV-2.The result lists table 3 in.

Table 3

Sample	????HBc	????HBc	????HBc	????HBc
						????0.956	????1.072	????0.970	????0.926
	????0.980	????1.073	????0.986	????0.913
						????0.994	????1.053	????0.966	????0.032
	????1.072	????1.189	????1.058	????1.030
						????1.062	????1.177	????1.066	????1.025
	????1.058	????1.156	????1.062	????1.005
					Negative control	????0.056	????0.070	????0.061	????0.074
Negative control	????0.060	????0.069	????0.063	????0.073
					Bond	????HBc	????HBc+ ????HIV-1	????HBc+ ????HIV-2	????HBc+ ????HIV-1+ ????HIV-2

Discuss

Above-mentioned table 1 clearly illustrates that to the listed result of table 3 different antigen bonds can discern antibody separately effectively in association response or when being used separately.Almost do not have interference when two kinds of uses even three kinds of bonds, just when using two or more bonds, the background level of viewed negative sample has some slight risings.

Embodiment 2

The series of samples A that contains HIV-1, HIV-2 and HBc antibody with the preparation of original single sample dilution is to D, and is listed as table 4.

Table 4

	????HIV-1	????HIV-2	????HBc
				????A	????1/40	????1/32	????1/2
????B	????1/80	????1/64	????1/2
				????C	????1/160	????1/128	????1/2
????D	????1/320	????1/256	????1/2

Test with following antigen bond each to sample A to D then, this bond is: HIV-1; HIV-2; HBc; HIV-1+HIV-2; HIV-1+HBc; HIV-2+HBc; HIV-1+HIV-2+HBc.The results are shown in Table 5.

Table 5

A	??1.314	??0.961	??0.361	??1.602	??1.415	??1.185	??1.689
								B	??0.840	??0.619	??0.386	??1.232	??1.075	??0.848	??1.482
C	??0.519	??0.368	??0.401	??0.804	??0.803	??0.650	??1.102
								D	??0.298	??0.216	??0.402	??0.465	??0.614	??0.526	??0.828
Negative control	??0.062	??0.049	??0.052	??0.059	??0.059	??0.054	??0.068
								Negative control	??0.055	??0.049	??0.050	??0.054	??0.057	??0.052	??0.064
Negative control	??0.054	??0.047	??0.049	??0.054	??0.057	??0.051	??0.062
								Negative control	??0.053	??0.046	??0.050	??0.052	??0.057	??0.052	??0.063
Bond	??HIV-1	??HIV-2	??HBc	??HIV-1 ??HIV-2	??HIV-1 ??+HBc	??HIV-2 ??+HBc	??HIV-1 ??HIV-2 ??HBc

Discuss

These results clearly illustrate that when other antibody exists, each antibody still can be measured by selectivity, in other words, the existence of non-correlation antibody does not influence the mensuration of associated antibodies, and hang down the existence that the result who records under the dilutability shows three strain specific antibodies qualitatively, come down to add than the result who records under the high dilution.

When the gained result showed the antibody of measuring two kinds of different pathogens, specificity did not have undermined, showed that the sensitivity of simultaneous determination is very high simultaneously yet.

Claims (25)

1, a kind of method of measuring the specific antibody of two or more different pathogens in the liquid check sample, this method comprises:

(i) sample is contacted with solid phase, be fixed with the antibody of a class or multiclass immunoglobulin (Ig) on the solid phase, all kinds of or monoid immunoglobulin (Ig) that exists in the sample so just can be trapped on the solid phase,

(ii) simultaneously or contact solid phase successively with two or more different antigens, the immunoglobulin (Ig) that comes from sample on this solid phase is hunted down, each antigen can optionally combine with the specific antibody of a kind of pathogen of being studied, each antigen be provided in the mode that a kind of discernible sign can directly or indirectly be provided and

(iii) measure the final any immunoglobulin (Ig)-antigenic compound that forms on the solid phase.

2, the method described in the claim 1, wherein Gu Ding antibody is anti--IgG, anti--IgM or anti--IgA antibody, or the potpourri of two or more these antibody-likes.

3, the method described in the claim 1, wherein the mixtures of antibodies of anti--IgG and anti--IgM is fixed on the solid phase.

4, any one described method in the claim 1 to 3, wherein Gu Ding antibody is the polyclonal antibody or the monoclonal antibody of affinity purification.

5, any one described method in the claim 1 to 4, wherein each antigen directly is labeled in the mode that a recognizable mark can be provided.

6, any one described method in the claim 1 to 4, wherein can be antigen provides the method for a recognizable mark to comprise:

(i) antibody A b1, it can combine with this antigen, antibody A b1 self be provided in the mode that a recognizable mark can be provided or

(ii) comprise antibody A b1 and second antibody A b2 that energy combines with antibody A b1 that can combine with this antigen, this antibody A b2 is provided in the mode that a recognizable mark can be provided.

7, any one described method in the claim 1 to 6, wherein the sample of being studied is the sample of donated blood.

8, any one described method in the claim 1 to 7, two or more pathogen of wherein being studied are selected from HIV, hepatitis type B virus, hepatitis C virus, HTLV, cytomegalovirus, Epstein-Barr virus and syphilis.

9, any one described method in the claim 1 to 7, two or more pathogen of wherein being studied are selected from rubella, measles, bleb (simple with reproduction), chlamydiaceae, gonorrhoea, hepatitis A, varicella, mumps, the human parvovirus, Much's bacillus, Mycobacterium leprae, mycobacterium avium, staphylococcus aureus, monocyte Listeria monocytogenes, Bacillus anthracis (antigen/toxin), actinomyces, salmonella typhi, yersinia enterocolitica, pylorus convolution bacillus, campylobacter jejuni, pseudomonas mallei and pseudomonas pseudomallei, pseudomonas aeruginosa, legionella pneumophilia (Legionella pneumophila) and subspecies thereof, soil draws hot francis fungus, Bacterium melitense, Mycoplasma pneumoniae, question mark shape Leptospira, the subspecies of Borellia, Spirochaeta pallida, Candida albicans and by the caused disease of protozoan pathogen.

10, any one described method in the claim 1 to 8, two or more pathogen of wherein being studied are selected from rubella, toxoplasmosis, cytomegalovirus and kitchen exanthema virus.

11, a kind of while but independently measure the method for the single specific antibody of two or more different pathogens in the liquor sample separately, comprising:

(i) one in a large amount of unit in each sample and the single component is contacted, each unit comprises a kind of solid phase, be fixed with the antibody of a class or multiclass immunoglobulin (Ig) on it, all kinds of or the monoid immunoglobulin (Ig) that exists in each sample so just can be by solid-phase capture, in all unit of assembly, the antibody that is fixed belongs to same class or monoid.

(ii) will be with assembly that sample contacts in each unit contact with antigen again, this antigen can optionally combine with the specific antibody of a kind of pathogen of being studied, every kind of antigen be provided in the mode that a kind of recognizable mark can directly or indirectly be provided and

(iii) measure the final any immunoglobulin (Ig)-antigenic compound that forms on the solid phase.

12, the described method of claim 11, wherein at least one unit comprises the solid phase that is fixed with anti--IgG antibody, at least one unit comprise the solid phase that is fixed with anti--IgM antibody and, randomly, at least one unit comprises the solid phase that is fixed with anti--IgA antibody.

13, claim 11 or 12 described methods, wherein the sample of being studied is the aliquot of single sample.

14, the described method of any one of claim 1 to 13, wherein solid phase comprises hole or the cup on globule or the microtiter plate, or solid or hollow bar or transfer pipet, or particle; Or comprise film, thin slice, band, film or coating porose, that material suction or fiber is made, randomly be placed in the verifying attachment.

15, be used for different pathogens simultaneously but a kind of assembly that comprises two or more unit that independently detects separately, this unit comprises fixing AIA, at least one unit has fixing antibody, the fixing inhomogeneous immunoglobulin (Ig) of immunoglobulin (Ig) in the directly anti-a kind of and other unit of this antibody.

16, the described a kind of assembly of claim 15, this assembly comprise the unit and the one or more unit that has fixing IgG that have fixing anti--IgM.

17, the described assembly of claim 15, this assembly comprises three unit, and unit has fixing anti--IgG, and one has fixing anti--IgM and the 3rd unit has fixing anti--IgA.

18, the solid phase that is suitable in the immunoassay is fixed with the mixtures of antibodies of anti--IgG and anti--IgM on it.

19, the solid phase described in the claim 18 also is fixed with anti--IgA antibody on it.

20, the solid phase described in claim 18 or the claim 19, this solid phase comprises: globule, or the hole of microtiter plate or cup, or solid or hollow bar or transfer pipet, or particle; Or comprise by film, thin slice, bar, film or coating porose, that material suction or fiber is made, randomly be placed in the pick-up unit.

21, a kind of method of measuring the specific antibody of two or more different pathogens in the liquor sample, this method comprises:

(i) sample is contacted with solid phase, be fixed with the antibody of a class or multiclass immunoglobulin (Ig) on the solid phase, all kinds of or monoid immunoglobulin (Ig) that exists in the sample so just can be hunted down on solid phase.

(ii) that two or more are different antigen simultaneously or contact with solid phase successively, caught the immunoglobulin (Ig) in the sample on this solid phase, each antigen can optionally combine with the specific antibody of a kind of pathogen of being studied, every kind of antigen be provided in the mode that recognizable mark can directly or indirectly be provided and

(iii) measure the final any immunoglobulin (Ig)-antigenic compound that forms on the solid phase.

22, a kind of while but measure in the liquor sample two or more not methods of the specific antibody of synantigen independently of one another, this method comprises:

(i) one in a large amount of unit in each sample and the single component is contacted, each unit comprises the solid phase that is fixed with a class or multiclass immune globulin antibody, like this, all kinds of or the monoid immunoglobulin (Ig) that exists in each sample just can be hunted down on solid phase, in all unit of assembly, fixing antibody belongs to same class or monoid

Each unit of assembly that (ii) will contact with sample contacts with antigen, and this antigen can optionally combine with the specific antibody of a kind of pathogen of being studied, every kind of antigen be provided in the mode that a kind of recognizable mark can directly or indirectly be provided and

(iii) measure the final any immunoglobulin (Ig)-antigenic compound that forms on the solid phase.

23, the described method of claim 21 or claim 22, wherein the antibody of being studied is non-pathogen correlativity antibody.

24, the described method of claim 21 or claim 22, wherein the antibody of being studied is autoimmune antibody or the antibody relevant with allergic reaction.

25, the method for any one described in the claim 21 to 24, this method have the parameter that any one limited in the claim 2 to 6,11 and 12.