CN1316249C - Enzyme-linked assay kit and producing process thereof - Google Patents

Abstract

An enzyme-linked detection kit for detecting human vascular endothelial growth factor (VEGF), the kit box body includes: an enzyme-linked reaction plate coated with VEGF receptors expressed and purified from insect cells, horseradish peroxidase (HRP ) labeled antibody plus protective agent, lyophilized powder of VEGF protein as positive control, lyophilized powder of protein as negative control, sample diluent, PBST concentrated washing solution, 3, 3', 5, 5' - Chromogenic substrate prepared with tetramethylbenzidine, 2M H ₂ SO ₄ as stop solution, sealing tape. The kit can be used for the diagnosis and prognosis of patients with cancer, cardiovascular disease, diabetes or certain other diseases, and has the advantages of accuracy, specificity, sensitivity, stability and convenience. At the same time, the invention also provides a method for highly expressing foreign proteins using insect cells.

Description

A kind of enzyme-linked detection kit and preparation method

technical field

The invention belongs to the technical field of biomedicine, in particular to an enzyme-linked detection kit for detecting human vascular endothelial cell growth factor (VEGF).

The present invention also relates to the preparation method of the above kit.

Background technique

Tumor development and metastasis are multi-stage, complex and highly selective processes involving the intricate relationship between tumor cells and hosts, and the mechanism is still unclear. In recent years, research on the relationship between new angiogenesis and tumor development, metastasis and prognosis has progressed rapidly, proving that new angiogenesis (Angiogenesis) is a necessary condition for tumor growth, metastasis and metastatic focus growth, and vascular endothelial cell growth factor (vascular endothelial cell growth factor). Growth Factor (VEGF) is the most important cytokine regulating neovascularization.

A heparin-binding factor was first isolated and purified from the in vitro culture medium of bovine pituitary follicular stellate cells (Ferrara et al., Biochem Biophys Res Commun, 1989, 161(2): 851-855), because it can specifically act on the vascular endothelium Cells, causing vascular endothelial cell proliferation, and inducing angiogenesis in vivo, so it is called vascular endothelial growth factor (VEGF), and because it can increase microvascular permeability, it is also called vascular permeability factor , VPF). It is the most powerful vascular permeability factor found so far, and its effect is 50,000 times stronger than that of histamine.

Subsequently, VEGF protein was also purified in the cell culture medium of mouse anterior pituitary tumor cell line AtT20, human mononuclear cells, guinea pig tumor, and mouse glioma cell lines. It has been confirmed that VEGF is the only important growth factor specific to angiogenesis. Other growth factors, such as fibroblast growth factor and platelet-derived growth factor, can act on a variety of cells including vascular endothelial cells and are not specific. of. Studies have shown that VEGF plays an irreplaceable role in the development and differentiation of the vascular system (Ferrara et al. Endocr. Rev. 1997, 18: 4-25).

VEGF is a highly glycosylated basic protein with a molecular weight of 45KD. It is formed by combining two identical subunits through a disulfide bond. The isoelectric point is 8.5. It has strong heat and acid resistance. It is a member of the platelet-derived growth factor (PDGF) family, which can be produced and secreted by some normal cells, and can also be produced by a variety of tumor cells. The human VEGF gene is located at chromosome 6p21.3 (Vincenti, V. et al, Circulation 93, 1493-1495, 1996), with a full length of 28 kb. The gene encoding VEGF is about 14 kb long, consisting of 8 exons and 7 introns. Alternately formed. Different isoforms of VEGF are formed by different splicing patterns of this gene. After the human VEGF gene is encoded, it is spliced into five different transcripts (isomers) at the transcriptional level, namely VEGF206, VEGF189, VEGF165, VEGF145, and VEGF121. The functional difference between the isoforms is manifested by their different binding activities to heparin on the cell surface and in the extracellular matrix. Placental growth factor (PLGF) is cloned from human placenta, has 53% homology with VEGF at the amino acid level, and is also considered to be a member of the VEGF growth factor family, but PLGF is limited to expression in the placenta (Maglione D et al. PNAS USA, 1991, 88:9267).

VEGF high-affinity binding sites are only located on three VEGF receptors on vascular endothelial cells, namely VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR) and VEGFR-3 (Flt-4). The three receptors are mainly distributed on the surface of vascular endothelial cells, and consist of an extracellular region containing seven immunoglobulin-like structures, a membrane region and a tyrosine kinase region, all of which are transmembrane receptors and belong to RTK (receptor tyrosinekinase) III The common feature is that there is a tyrosine kinase insertion region in the catalytic domain. The activity of the tyrosine kinase is activated by the binding of the receptor and the ligand, and the phosphorylation of the receptor causes many enzymes and other reactions in the cell. important role in growth and differentiation. Studies have shown that VEGF receptors have a high affinity with VEGF in vitro (Kendall et al. PNAS USA, 1993, 90: 10705-9).

VEGF is a factor necessary for cancer growth and metastasis. Current research shows that in various human malignant tumors (i.e. cancer) diseases, the increase of VEGF secretion is universal and extensive, that is, when a malignant tumor grows in the human body, the secretion of VEGF will greatly increase and be released into the blood circulation . There are a large number of literature reports that the concentration of VEGF in normal people and cancer patients is significantly different, the concentration of VEGF in normal people is very low or undetectable, while the concentration of VEGF in cancer patients is very high. Therefore, detection of VEGF concentration in human blood can be used for early screening and diagnosis of cancer. On the other hand, the concentration of VEGF in human blood varies with the growth and decline of tumors. Blood levels of VEGF are high when the tumor is large and growing fast; it decreases when the tumor is "clinically cured" by chemotherapy or surgery. Therefore, the detection of VEGF can be used as an indicator of curative effect observation and prognosis. VEGF is an important component in the diagnosis of malignant tumors. In addition, increased blood VEGF levels are also seen in diabetes, vascular inflammatory diseases, certain immune diseases and pregnancy.

Yeo et al. (Clin.Chem.1992,38:71) reported an ELISA method for detecting VEGF based on immunofluorescence, but its detection sensitivity was low. Houck et al. (J Biol.Chem.1992,267:26031) reported an ELISA method for detecting VEGF based on colorimetry, but its detection sensitivity was only ng/ml. Hanatani et al. (Biosci. Biotechnol. Biochem. 1995, 59: 1985) reported a chemiluminescent ELISA for the determination of VEGF, and the serum VEGF level in normal people was determined to be 8-36 ng/l.

Contents of the invention

The purpose of the present invention is to provide a kit for enzyme-linked detection of human vascular endothelial growth factor (VEGF). The kit has the advantages of accurate and stable results, strong specificity, high sensitivity, convenient use and popularization.

Another object of the present invention is to provide the production, preparation and use methods of the above-mentioned enzyme-linked detection kit.

In order to achieve the above object, the kit provided by the invention consists of:

1. Enzyme-linked plate; 2. Positive control (lyophilized); 3. Negative control (lyophilized); 4. Sample diluent; 5. Enzyme-linked product; 6. Concentrated washing solution; , double distilled water; 9, stop solution; 10, sealing tape.

The preparation method and operation steps of the above-mentioned kit provided by the present invention are as follows:

a) using the enzyme-linked reaction plate as a solid support, coating the VEGF receptor expressed by genetic engineering on the solid support;

b) Block with blocking solution

c) contacting and incubating the biological sample with the VEGF receptor on the immobilized and enzyme-linked reaction plate, and the VEGF in the biological sample specifically binds to the VEGF receptor;

d) isolating biological samples from VEGF receptors;

e) VEGF was detected with a horseradish peroxidase (HRP)-labeled VEGF polyclonal antibody, and the chromogenic substrate used was 3,3',5,5'-tetramethylbenzidine (TMB).

The uniqueness of the kit of the present invention lies in that it uses VEGF receptors to coat the enzyme-linked plate, which can ensure that the lower level of VEGF can be detected by the kit, and can more accurately measure samples.

The kit detects VEGF in biological samples, preferably samples from cancer, vascular disease patients or other disease patients.

Description of drawings

Figure 1: The body and components of the kit of the present invention.

Figure 2: Polyacrylamide gel electrophoresis of VEGF protein expressed and purified from insect cells.

Figure 3: Polyacrylamide gel electrophoresis of VEGF receptor protein expressed and purified from insect cells.

Detailed ways

The following examples are for explaining the present invention in more detail, but should not be construed as limiting the present invention thereto.

Example 1:

The kit of the present invention can be exemplified by the following substances:

1) VEGF receptor coated enzyme-linked plate, and

2) One positive control (lyophilized powder of VEGF protein);

3) Negative control (animal serum freeze-dried powder) 1 tube;

4) Sample diluent: 0.05% Tween-20 (Tween-20), 1% bovine serum albumin (BSA), 10mM disodium ethylenediaminetetraacetic acid (EDTA), 0.05% thimerosal, 0.4M NaCl, pH6. 5, 1 bottle;

5) 2 bottles of enzyme conjugate (horseradish peroxidase labeled VEGF polyclonal antibody);

6) One bottle of PBST concentrated washing solution (137mM NaCl, 2.7mM KCl, 10mM Na ₂ HPO ₄ , 2mM KH ₂ PO ₄ , 0.05% Tween-20, pH 7.4);

7) Chromogens A & B, wherein liquid A is H ₂ O ₂ solution, and liquid B is 3,3',5,5'-tetramethylbenzidine (TMB) solution, 1 bottle each;

8) 1 bottle of double distilled water;

9) 1 bottle of stop solution (2M H ₂ SO ₄ );

10) 2 sheets of sealing tape;

The amounts of various substances described in this example are calculated based on the example of 96-well enzyme-linked plate (12 strips of 8-well or 8 strips of 12-well). If the number of wells of the enzyme-linked plate is more or less than 96 wells, each The amount of matter is correspondingly more or less.

The appearance of the kit can be seen in Figure 1.

Its preparation method:

1) Cloning of VEGF gene and VEGF receptor gene

The human placenta cDNA library was used as a template for polymerase chain reaction (polymerase chain reaction, PCR) reaction. Primers were designed according to the reporter sequence of the VEGF gene. PCR reaction conditions: denaturation at 94°C for 40 seconds, annealing at 52°C for 1 minute, extension at 72°C for 1 minute, and incubation at 72°C for 10 minutes after 30 cycles. Primers were designed according to the reporter sequence of the VEGF receptor gene. PCR reaction conditions: denaturation at 94°C for 40 seconds, annealing at 52°C for 1 minute, extension at 72°C for 2 minutes, and incubation at 72°C for 10 minutes after 30 cycles.

Reaction system: ddH ₂ O 20.0μl

10×Buffer 2.5μl

dNTP(10mM) 0.5μl

3'primer(100ng/μl) 0.5μl

5'primer (100ng/μl) 0.5μl

Taq enzyme (5U/μl) 0.5μl

Template DNA 0.5μl

Total volume 25μl

The fragment amplified by PCR was recovered by gel electrophoresis, cloned into the vector pMT18-T, transformed into Escherichia coli DH5α, and the recombinant plasmid was extracted for identification by enzyme digestion. The recombinant plasmid of VEGF gene was named pMT18-1, and the recombinant plasmid of VEGF receptor gene was named pMT18-2. DNA sequence analysis was completed by Shanghai Boya Bioengineering Co., Ltd.

2) Construction of insect expression vector

Plasmids pMT18-1 and pMT18-2 were digested with SalI and BamHI respectively, and VEGF gene fragments and VEGF receptor gene fragments were recovered respectively. At the same time, the plasmid pFastBac-HT was digested with SalI and BamHI, and the 4.7kb fragment was recovered. The VEGF gene fragment and the VEGF receptor gene fragment were respectively connected with the 4.7kb fragment to construct vectors pFastBac-1 and pFastBac-2. Different restriction endonucleases were used for enzyme digestion identification, which indicated that the constructed vector was correct.

The vectors pFastBac-1 and pFastBac-2 were respectively transformed into DH10Bac competent cells. Use blue and white spot screening, and after 24 hours, scratch the white spot on a new plate to confirm whether it is white spot. Pick the colony into 2ml LB medium, shake and culture at 250rpm for 12 hours at 37°C.

The method of extracting the plasmid in the cultured bacterial liquid is as follows:

a1) Transfer 1.5ml of bacterial solution into a centrifuge tube, centrifuge at 10,000rpm for 30s, discard the supernatant, and place the centrifuge tube upside down on a paper towel to make the bacterial pellet as dry as possible;

b1) Add 300 μl solution I (50mM glucose, 25mM Tris-HCl, 10mM EDTA, pH8.0), shake and mix on the shaker;

c1) Add 300 μl of the prepared solution II (0.2N NaOH, 1% SDS), mix well, and place at room temperature for 5 minutes;

d1) Add 300 μl of pre-cooled solution III (5M KAc, pH 5.5), and pre-ice for 5 minutes;

e1) Centrifuge at 12,000rpm for 15min;

f1) Transfer the supernatant to a new centrifuge tube, add 2 times the volume of absolute ethanol, and mix well;

g1) Centrifuge at 10,000～12,000rpm for 10min;

h1) Wash the precipitate with 70% and absolute ethanol;

i1) Blow dry, dissolve in 50 μl of RNase-added TE (10 mM Tris-HCl; 1 mM EDTA, pH 8.0), place at 37° C. for one hour, and store at -20° C. for use.

The extracted plasmid was identified by PCR, and the PCR reaction was carried out according to the aforementioned method.

3) Transfection of insect cells (taking sf9 cells as an example, but not limited to sf9 cells, the same effect can be obtained by using sf21, sf158 or c127 cell lines, which will not be described here).

The sf9 cells were cultured in Grace medium, supplemented with 15% fetal bovine serum, penicillin 50U/ml, and streptomycin 50ug/ml, and the transfection method was as follows:

a2) Divide cells into 24-well plates one day before transfection;

b2) 2 hours before transfection, replace with fresh serum-free and double-antibody-free culture medium, wash once, and then add 500ul of culture medium;

c2) Add 2ul of liposomes and 5ul of the above-mentioned recombinant plasmid to 50ul of serum-free and double-antibody-free culture medium, place at room temperature for 5 minutes, add the plasmid into the liposome, and place at room temperature for 20 minutes;

e2) Add the mixture from the previous step to the cells and incubate at 27°C;

f2) After 4 hours of transfection, suck off the culture medium, add 1ml of fresh serum-containing, double-antibody Grace culture medium, and incubate at 27°C for 72 hours;

g2) After 72 hours of transfection, observe whether there is any sign of toxicity;

h2) New sf9 cells will be infected with baculovirus supernatant.

4) Purification of insect expressed protein (Ni column adsorption method)

a3)) Preparation of Ni column

i) Take 1ml Ni-NTA into a centrifuge tube and centrifuge at 1500g for 5 minutes;

ii) Remove the supernatant, and resuspend the Ni-NTA with 1 volume of washing buffer (20mM Tris-HCl, 500mM KCl, 20mM diacid, 2mM mercaptoethanol, 10% glycerol, pH8.5);

iii) Put the resuspension into the column and equilibrate with 5-10 times the volume of wash buffer.

b3) Preparation of cell extract

i) Collect 50ml of cells by centrifugation at 500g for 5 minutes;

ii) resuspend the cells with lysis buffer (50mM Tris-HCl, 100mM KCl, 20mM methazine, 5mM mercaptoethanol, 1mM benzylsulfonyl fluoride, pH8.5);

iii) Centrifuge at 10,000 g for 10 minutes, and transfer the supernatant to a new tube.

c3) Protein purification

i) adding the supernatant of the previous step to a well-balanced Ni column;

ii) wash the column with 10 times the volume of buffer A (20mM Tris-HCl, 500mM KCl, 20mM methazine, 5mM mercaptoethanol, 10% glycerol, pH8.5);

iii) Wash the column with 2 times the volume of buffer B (20mM Tris-HCl, 1M KCl, 20mM imidazole, 5mM mercaptoethanol, 10% glycerol, pH8.5);

iv) Wash the column with 2 times the volume of buffer A;

v) Elute with 5-10 times buffer C (20mM Tris-HCl, 100mM KCl, 100mM methazine, 5mM mercaptoethanol, 10% glycerol, pH8.5), and collect the eluate.

vi) SDS-PAGE gel was used to detect the concentration and purity of the purified protein; the detection results are shown in Fig. 2 and Fig. 3 .

5) Preparation, purification and labeling of VEGF polyclonal antibody

Rabbits are used as immunized animals, and the animals are first stimulated by injecting 5-10 mg of BCG. One week later, the VEGF protein was made into Freund's complete adjuvant, and the first immunization was performed by subcutaneous multi-point injection. After 2 weeks, a second immunization was performed. After 2 weeks, blood was tested and the potency was measured. If the titer is low, booster immunization is carried out.

Rabbit whole blood was collected and centrifuged to obtain serum. Saturated ammonium sulfate is added to the serum, and the volume ratio of serum to ammonium sulfate is 2:1, so that the antibody is precipitated, centrifuged, and the purity of the antibody reaches more than 98%.

Horseradish peroxidase (HRP) was labeled with polyclonal antibody to VEGF by glutaraldehyde method.

6) Preparation of the kit

The coating of the enzyme-linked plate adopts the following steps: the antigen is diluted by a certain number of times with the coating buffer, and 100ul is added to each well. Coated in a water bath or incubator at 37°C for 2 hours, then left at room temperature overnight. Fill each well with PBST washing solution, leave it for about 30 seconds, and spin dry. Add 120ul of blocking solution to each well and place at room temperature for 2 hours. The blocking solution formula mainly contains BSA, some salts, etc. Shake off blocking solution and tap to remove residual blocking solution. Blow dry on a clean bench. Or blow it on a clean bench for several hours, and then leave it at room temperature overnight. Pack with a vacuum packaging machine.

Embodiment 2: Kit use method

1. Take out the required strips from the sealed bag that has been equilibrated to room temperature, and seal the other strips and put them back at 4°C.

2. Leave one well as a blank well, and add 100 μl (or 2 drops) of sample diluent to the other wells.

3. In addition to the blank well, add 100 μl each of the negative control well, the positive control well 2, and the serum sample to the corresponding well, and mix well by flicking. Seal the reaction well with sealing tape and keep at 37°C for 60 minutes.

4. Wash the plate 3 times: (1) Automatic plate washer: Shake off the liquid in the wells, the washing liquid required to be injected is 350μl, the interval between injection and aspiration is 15-30 seconds, and the plate is washed 3 times. (2) Manual plate washing: Shake off the liquid in the wells, add 350 μl of washing solution to each well, let it stand for 30 seconds, shake off the liquid, pat dry on thick absorbent paper, and wash 3 times.

5. Add 200 μl (or 4 drops) of enzyme conjugate to each well except the blank well, seal the plate well, and keep at 37°C for 60 minutes.

6. Wash the plate 3 times.

7. Color reaction: Add 100 μl (or 2 drops) of each of the color reagents A and B to the reaction wells (including blank wells), and develop the color at room temperature for 25 minutes in the dark.

8. Stop reaction: Add 50 μl (or 1 drop) of stop solution to each well (including blank wells), mix well, and measure the 450nmOD value.

The advantages that the present invention has:

1. The time is short, the cost is low, and the preparation is simple. Generally, monoclonal antibodies are used for coatings, and the preparation of monoclonal antibodies takes a long time, generally takes 9-12 months, and the fastest takes 6-8 months, the cost is high, and the preparation of monoclonal antibodies is relatively cumbersome; In the present invention, the receptor is used as a coating, and the preparation of the genetically engineered receptor expressed by insect cells only takes about 3 months, and the cost is low, and the preparation is relatively simple.

2. The results are highly specific. The combination of receptor and ligand has high specificity. In the present invention, the coating is the receptor of VEGF, and the test object (ligand) is VEGF. However, due to incomplete screening of a protein monoclonal antibody against a certain antigenic determinant, the prepared monoclonal antibody is likely to cross-react with other proteins, and has low specificity compared to receptor-ligand binding.

3. High sensitivity. The detection sensitivity can reach ng/L level.

4. Stable. The present invention is stored at 4°C, and the validity period is 9-12 months

5. Convenience. The invention is easy to operate, and the required detection instrument (microplate reader) is generally used in major hospitals and detection centers.

Claims (11)

1. elisa kit for detecting, it consists of:

Elisa plate, positive control, negative control, sample diluting liquid, enzyme connection thing, concentrated cleaning solution, developer A, developer B, distilled water, stop buffer and shrouding gummed paper;

Wherein:

Elisa plate be vascular endothelial growth factor can specificity the vegf receptor bag of combination by elisa plate;

Positive control is a vascular endothelial growth factor albumen freeze-dried powder;

Negative control is the animal blood serum freeze-dried powder;

Sample diluting liquid: 0.05% Tween-20,1% bovine serum albumin(BSA), 10mM disodium ethylene diamine tetraacetate, 0.05% thimerosal, 0.4M NaCl, pH6.5;

Enzyme connection thing is a horseradish peroxidase-labeled vascular endothelial growth factor polyclonal antibody;

Concentrated cleaning solution: 137mM NaCl, 2.7mM KCl, 10mM Na ₂HPO ₄, 2mMKH ₂PO ₄, 0.05% Tween-20, pH 7.4;

Developer A, developer B, wherein developer A is H ₂O ₂Solution, developer B are 3,3 ', 5,5 '-tetramethyl biphenyl amine aqueous solution;

Distilled water

Stop buffer is 2M H ₂SO ₄

2. according to the kit of claim 1, it is characterized in that each amount of substance is a standard with the hole count of elisa plate.

3. according to the kit of claim 1, it is characterized in that described elisa plate is 96 holes.

4. according to the preparation method of the described kit of claim 1, key step is:

(a) with the vascular endothelial growth factor receptor bag of gene engineering expression by on the solid support enzyme-linked reaction plate;

(b) seal with confining liquid;

(c) biological sample is joined in the enzyme-linked reaction plate reacting hole of pre-bag quilt, the vascular endothelial growth factor in the biological sample combines with vascular endothelial growth factor receptor specifically;

(d) detect vascular endothelial growth factor with the vascular endothelial growth factor polyclonal antibody:

(e) chromogenic substrate is 3,3 ', 5,5 '-tetramethyl benzidine.

5. according to the method for claim 4, it is characterized in that the described genetic engineering acceptor of step a insect cell expression.

6. according to the method for claim 5, it is characterized in that described insect cell is sf9, sf12, sf158 or c127 cell line.

7. according to the method for claim 5, it is characterized in that the genetic engineering acceptor of described insect cell expression Ni post purifying.

8. according to the method for claim 4, it is characterized in that the described biological sample of step c is cancer patient, angiosis patient's serum, blood plasma, cerebrospinal fluid, saliva, tissue extract or a urine.

9. according to the method for claim 4, it is characterized in that the vascular endothelial growth factor that the described vascular endothelial growth factor polyclonal antibody of steps d is used is with insect cell expression and use Ni post purifying.

10. according to the method for claim 4, it is characterized in that the polyclonal antibody described in the steps d is with vascular endothelial growth factor immunizing rabbit gained.

11. the method according to claim 4 is characterized in that, the horseradish peroxidase-labeled of polyclonal antibody described in the steps d.

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