CN117054648A - Kit for detecting HPV IgM antibody and preparation method thereof - Google Patents
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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Abstract
The invention relates to the technical field of detection, and particularly discloses a kit for detecting HPV IgM antibodies and a preparation method thereof. The kit comprises a goat anti-human IgM antibody marked by horseradish peroxidase, an ELISA plate, a sample diluent, a washing liquid, a color development liquid and a stop solution; the reaction hole of the ELISA plate is coated with HPV L1-VLP antigen; the HPV L1-VLP antigen is produced by deleting the N-terminal sequence of the L1 gene of HPV virus while modifying the L1 gene by point mutation and performing protein expression, protein purification and particle assembly. According to the invention, the goat anti-human IgM antibody is marked by horseradish peroxidase, and the HPV L1-VLP antigen is coated on the ELISA plate, so that the detection sample antibody and the coated and immobilized HPV L1-VLP antigen are combined with the goat anti-human IgM antibody marked by horseradish peroxidase to form an immune complex, thereby ensuring higher detection sensitivity and specificity and better result stability.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a kit for detecting HPV IgM antibodies and a preparation method thereof.
Background
Human papillomavirus (human papillomavirus, HPV) belongs to the genus papillomavirus of the family papillomaviridae, and is an epitheliophilic, non-enveloped, double-stranded circular DNA virus. The virus is about 50-55 nm in diameter and about 7-8 kb in genome size, encoding six early proteins (E1, E2, E4, E5, E6 and E7), two late proteins (L1 and L2) and one long regulatory region (long control region, LCR). HPV mainly causes proliferative lesions of human skin and mucous membrane, and is classified into three major types of high-risk type, medium-risk type and low-risk type. Vaccination with HPV prophylactic vaccines (hereinafter HPV vaccines) is the most effective, time-and cost-effective primary prophylactic measure for preventing HPV-related diseases and cancers. The main action mechanism of HPV vaccine is that the heterologous antigen carried by the vaccine enters the body, is captured by antigen presenting cells and presented to T lymphocytes, and assists B lymphocytes to produce protective antibodies, so as to prevent HPV infection. HPV vaccines currently licensed on the market are developed based on virus-like particles (VLPs) of the HPV major capsid protein L1. VLPs are therefore important antigen targets for immunogenicity evaluation after HPV vaccine immunization.
HPV vaccine immunogenicity is mainly assessed by humoral immune responses (including total and functional antibodies) as well as cellular immune responses, with evaluation indicators including antibody titer, serum positive turnover rate, and activated T-cytokines, among others. Among them, immunoglobulin M (IgM) is the immunoglobulin with the largest molecular weight, which is mainly secreted and synthesized by plasma cells in spleen and lymph nodes, and is divided into two subtypes of IgM1 and IgM 2. Mainly distributed in serum, exists in the form of pentamer and accounts for 5-10% of total immunoglobulin in serum. HPV vaccines require three vaccinations, three times within six months, and generally IgM antibodies can be produced one week after the primary immunization, and serum IgM antibody titres can fully reflect the early immune response level of the vaccinators.
However, the detection of gM antibodies after immunization against HPV vaccines still belongs to the technical blank field. The invention develops an HPV IgM antibody kit based on HPV L1-VLP, which can detect IgM antibodies of different HPV types, has good specificity and high sensitivity, is easy to obtain and store required consumables, can effectively reduce the transportation and use costs of the kit, is simple and convenient to operate, has objective result judgment, and can achieve the aim of rapidly evaluating the antibody level.
Disclosure of Invention
In order to make up the blank of the prior art, the invention provides a kit for detecting IgM antibodies of different types of HPV and a preparation method thereof, and the detection sensitivity and specificity are improved, so that the level of IgM antibodies can be rapidly estimated.
In a first aspect the invention provides a kit for detecting HPV IgM antibodies.
Specifically, the kit comprises a goat anti-human IgM antibody marked by horseradish peroxidase, an ELISA plate, a sample diluent, a washing liquid, a color development liquid and a stop solution;
the reaction hole of the ELISA plate is coated with HPV L1-VLP antigen;
the HPV L1-VLP antigen is prepared by deleting an N-terminal sequence of an L1 gene of HPV virus, modifying the L1 gene by point mutation, and performing protein expression, protein purification and particle assembly.
Preferably, the L1 gene of the HPV virus is at least one of HPV 6L1 gene, HPV 11L 1 gene, HPV16L1 gene, HPV 18L 1 gene, HPV 31L 1 gene, HPV 26L 1 gene, HPV 33L 1 gene, HPV 35L 1 gene, HPV45L1 gene, HPV 39L 1 gene, HPV45L1 gene, HPV 51L 1 gene, HPV 52L 1 gene, HPV 53L 1 gene, HPV 56L 1 gene, HPV 58L 1 gene and HPV59L1 gene.
Further preferably, the L1 gene of HPV virus is HPV16L1 gene.
In a second aspect, the invention provides a method for preparing a kit for detecting HPV IgM antibodies.
Specifically, the method comprises the following steps: diluting HPV L1-VLP antigen to coating concentration, adding the HPV L1-VLP antigen into a reaction hole of an ELISA plate, washing by using a washing solution, adding 5% Bovine Serum Albumin (BSA) for blocking, and washing and sealing after blocking is finished to coat the reaction hole of the ELISA plate with HPV L1-VLP antigen;
mixing the goat anti-human IgM antibody with horseradish peroxidase and dialyzing to obtain the goat anti-human IgM antibody marked by horseradish peroxidase.
Preferably, the coating concentration is 0.5-4. Mu.g/mL.
Further preferably, the coating concentration is 4.0. Mu.g/mL.
Preferably, the blocking temperature is 4 ℃ or 37 ℃.
Further preferably, the temperature of the enclosure is 37 ℃.
Preferably, the blocking time is 1h, 2h or overnight.
Preferably, the blocking solution used for the blocking is 5% BSA.
Further preferably, the closing time is 1h.
In a third aspect, the invention provides a detection method for detecting an HPV IgM antibody kit.
Specifically, the method comprises the following steps: diluting a sample to be detected by using a sample diluent, and then adding the diluted sample into an ELISA plate coated with HPV L1-VLP antigen for mixed incubation; discarding the mixed liquid and washing; adding horseradish peroxidase-labeled goat anti-human IgM antibody into an ELISA plate for incubation, discarding mixed liquid, and washing; and adding a color developing solution for incubation, adding a stopping solution to stop the reaction after the incubation is finished, and judging the result.
Preferably, the method for diluting the sample to be tested with the sample diluent is that the sample to be tested is diluted with the sample diluent according to 1:100. 1: 200. 1: 400. 1:800 volume ratio.
Further preferably, the method for diluting the sample to be tested with the sample diluent is to dilute the sample to be tested with the PBS buffer according to the following ratio of 1: dilution was performed at a volume ratio of 100.
Preferably, the incubation conditions are 60min at 37 ℃.
Preferably, the horseradish peroxidase-labeled goat anti-human IgM antibody dilution is 1:1000-2500.
Further preferred, the horseradish peroxidase-labeled goat anti-human IgM antibody dilution is 1:2000.
preferably, the color developing solution is TMB color developing solution.
Preferably, the optimal color development time of the TMB color development liquid is 15min.
Preferably, the stop solution is a sulfuric acid solution.
Further preferably, the sulfuric acid solution is 2mol/L H 2 SO 4 A solution.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the goat anti-human IgM antibody is marked by horseradish peroxidase, and the HPV L1-VLP antigen is coated on the ELISA plate, so that the detection sample antibody and the coated and immobilized HPV L1-VLP antigen are combined with the goat anti-human IgM antibody marked by horseradish peroxidase to form an immune complex, thereby ensuring that the detection has higher sensitivity and stronger specificity, and the repeatability and stability of the detection result are better.
Drawings
FIG. 1 is a roadmap of a detection technique;
FIG. 2 is a graph showing the judgment of the yin-yang threshold of IgM antibody.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
Example 1
A kit for detecting HPV IgM antibodies.
The HPV IgM antibody detection kit comprises goat anti-human IgM antibodies marked by horseradish peroxidase, an ELISA plate, a sample diluent, a washing liquid, a color development liquid and a termination liquid; the reaction well of the ELISA plate is coated with HPV L1-VLP antigen.
Example 2
A preparation method of a kit for detecting HPV IgM antibody.
A method for preparing hpv L1-VLP antigen:
(1) One of HPV6 (GenBank: AAC 80442.1), HPV11 (AAA 46935.1), HPV16 (ANA 05496), HPV18 (AAQ 92369), HPV31 (P17388), HPV26 (NP 041787.1), HPV33 (AMY 16565), HPV35 (P27232), HPV39 (P24838), HPV45 (P36741), HPV51 (ACV 88631.1), HPV52 (AML 80965), HPV53 (NP 041848), HPV56 (P36743), HPV58 (AFS 33402), HPV59 (CAA 54856) is selected for L1 expression and pseudovirus preparation. The N-terminal sequence of the selected HPV L1 gene was deleted and cloned into the pTO-T7 vector. Site-directed mutagenesis was performed at the C175 and C428 sites of the selected HPV L1 gene using a rapid mutagenesis kit (Vazyme, nanjing, china). The mutated gene was cloned into a pTO-T7 expression vector and protein expression was performed using E.coli ER2566 strain.
(2) Protein purification and particle assembly. Wild-type HPV (HPV WT) and mutant L1 proteins were produced in ER2566 e.coli strain. The strain was grown in LB medium at 37℃until OD600 reached 0.6. L1 protein expression was induced by the addition of isopropyl- β -D-thiogalactoside (IPTG, final concentration 10. Mu.M) and the strain was further incubated at 25℃for 8h. The strains were collected by centrifugation and resuspended with lysis solution (20 mM Tris, pH 7.4, 300mM NaCl and 5mM EDTA). HPV16L1 protein and HPV 18L 1 protein were released from the cells and bound to 20mM DTT, and then further purified by SP agarose (GE Healthcare, USA) and CHT-II resin (Bio-Rad, USA). After purification, the proteins were analyzed using SDS-PAGE and stored at a final concentration of 1mg/mL in a solution containing 20mM phosphate buffer (pH 8.0), 500mM NaCl and 20mM DTT.
The preparation method of the HPV L1-VLP antigen coated ELISA plate comprises the following steps:
HPV L1-VLP antigen was diluted to 2. Mu.g/mL using coating buffer (pH 9.6.05M carbonate buffer) and added at 0.1mL per reaction well in the ELISA plate overnight at 4 ℃. The mixture was discarded and wash solution (0.15M KH) was added to each well 2 PO 4 0.2g、Na 2 HPO 4 ·12H 2 Mixing 2.9g of O, 8.0g of NaCl, 0.2g of KCl and 0.05% Tween-200.5mL, adding distilled water to 1000mL, cleaning with a horizontal oscillator for 1min, discarding antigen washing buffer solution after finishing, taking excessive residual liquid on absorbent paper, and repeating for 3 times; adding 100 mu L of blocking solution into each reaction hole, and blocking for 1h at 37 ℃; discardingRemoving the sealing liquid, adding 100 mu L of washing liquid into each hole, washing for 1min by a horizontal oscillator, discarding the washing liquid after the washing liquid is finished, taking excessive residual liquid on the absorbent paper, and repeating for 3 times; the HPV L1-VLP antigen coating ELISA plate is completed.
3. The preparation method of the horseradish peroxidase-labeled goat anti-human IgM antibody comprises the following steps:
(1) Horseradish peroxidase activation: 5mg of horseradish peroxidase was weighed and dissolved in 500. Mu.l of double distilled water, then 0.5mL of 0.06M sodium periodate solution was added, and the mixture was left to stand at 4℃in the dark for 30 minutes. 160mM ethylene glycol solution (0.5 mL) was added thereto, and the mixture was left at room temperature for 30 minutes. The final concentration of horseradish peroxidase after activation was 3.33mg/mL.
(2) Mixing goat anti-human IgM antibody with activated horseradish peroxidase solution according to a mass ratio of 1:1, placing the mixture into a dialysis bag, and placing the dialysis bag into 3L of 0.05mM CB Buffer for dialysis at 4 ℃ overnight.
(3) The dialyzed liquid was aspirated, the volume was recorded, and 20. Mu.L of a 5mg/mL sodium borohydride solution (NaBH) 4 ) Mixing and placing at 4 ℃ for 2h.
(4) Adding an equivalent amount of saturated ammonium sulfate solution, mixing, incubating at 4 ℃ for 1h, centrifuging at 12000r at 4 ℃ for 20min after incubation is completed, and discarding the supernatant.
(5) The precipitate was dissolved in 500. Mu.L of PBS solution, filled into dialysis bags, placed into 3L 0.05mM CB Buffer dialysate, and stirred overnight at 4 ℃. The dialysate was changed once in the middle.
(6) After dialysis, the liquid was centrifuged at 4℃for 12000r for 20min in an EP tube to remove the precipitate, and the supernatant was horseradish peroxidase-labeled goat anti-human IgM antibody.
4. Preparation of sample dilutions: 5% BSA (5 g Bovine Serum Albumin (BSA) was added to PBS to volume 100 mL).
5. The preparation of the lotion comprises the following steps:
(1) 10X wash: KH (KH) 2 PO 4 4g，Na 2 HPO 4 ·12H 2 O58g,NaCl 160g,KCl 4g,Tween-20 mL water is added to a constant volume of 2L.
(2) 1X washing liquid: the 10X washing liquid and the single distilled water are diluted and configured according to the volume ratio of 1:9.
6. Preparation of a color development liquid:
the reagents required for preparing the color development liquid are as follows, and all three solutions need to be prepared on site.
(1) And (3) solution A: 0.1M citric acid (1.05 g citric acid added double distilled water constant volume to 50 mL)
(2) And (2) liquid B: 0.2M Na 2 HPO 4 .12H 2 O(7.1628g Na 2 HPO 4 .12H 2 O adding double distilled water to constant volume to 100 mL)
(3) And C, liquid: 1% TMB (10 mg3,3', 5' -Tetramethylbenzidine (TMB) with 200uL absolute ethanol, 800uL double distilled water)
24.3mL of solution A and 25.7mL of solution B are taken and mixed, and 50mL of double distilled water is added. Then 9.9mL was removed therefrom and transferred into a new tube, 100uL of C solution was added, and finally 10uL of 30% H was added 2 O 2 。
7. Preparation of a stop solution: 2mol/L H 2 SO 4 。
Example 3
A detection method for detecting HPV IgM antibody kit (taking HPV16 type IgM antibody detection kit as an example). The detection technology roadmap is shown in fig. 1.
1. Determination of the concentration of HPV16L 1-VLP antigen coated ELISA plate and selection of the optimal dilution of serum to be tested
Optimal coating concentration and optimal serum dilution of HPV16L 1-VLP antigen were optimized by a square method.
(1) HPV L1-VLP antigen was diluted to 0.5, 1.0, 2.0, 4.0 μg/mL using coating buffer (ph 9.6.05M carbonate buffer), each concentration in 96-well elisa plate was coated 2-way, 100 μl was added per reaction well and incubated for 1h at 37 ℃.
(2) Removing the mixed solution, adding 100 μl of 1×lotion into each well, washing with a horizontal shaker for 5min, removing 0.05% PBST lotion after the washing, taking excessive residual liquid on absorbent paper, and repeating for 3 times;
(3) After the ELISA plate is coated, the ELISA plate can be directly used or packaged by a self-sealing bag, and then the ELISA plate is inverted to a refrigerator with the temperature of-20 ℃ for standby; positive serum to be tested and negative serum were diluted with 1:100. diluting at the ratio of 1:200, 1:400 and 1:800, adding 3 columns of 100 mu L of each dilution in the 96-well ELISA plate, and incubating for 1h at 37 ℃;
(4) Discarding the solution in the ELISA plate, adding 250 mu L of 1 Xwashing liquid into each reaction hole, cleaning for 1min by a horizontal oscillator, discarding the washing liquid after finishing, taking redundant residual liquid on the absorbent paper, and repeating for 4 times;
(5) Diluting goat anti-human IgM antibody with a diluent according to the proportion of 1:2000, and sealing a constant temperature box at 37 ℃ for 1h with 100 mu L of each reaction hole;
(6) Discarding the solution in the ELISA plate, adding 250 mu L of 1 Xwashing liquid into each reaction hole, cleaning for 1min by a horizontal oscillator, discarding the washing liquid after finishing, taking redundant residual liquid on the absorbent paper, and repeating for 4 times;
(7) Under the condition of avoiding light, adding 100 mu L of TMB color development liquid into each reaction hole, and incubating for 15min at room temperature; after incubation of the color development solution, 50 mu L of 2mol/L H is added into each reaction well 2 SO 4 Stopping the liquid, and then detecting on the machine;
(8) And adjusting the enzyme labeling instrument to OD450 nm reading, calculating P/N, and determining the protein concentration corresponding to the maximum value of P/N.
TABLE 1 OD values at different antigen concentration coatings and serum dilution
Note that: p is positive serum OD 450 A measured value; n is negative serum OD 450 Measurement value
The assay showed that the antigen was coated at a concentration of 4.0 μg/mL and serum at 1: 100-fold dilution was performed, at which point the P/N value was at most 18.06. Thus, the optimal coating concentration for antigen was determined to be 4.0. Mu.g/mL and the optimal dilution concentration for serum was determined to be 1:100.
2. Optimum sealing liquid composition and sealing time
In order to determine the optimal sealing liquid components and sealing time, based on the optimized coating antigen and serum dilution concentration, different sealing conditions and times are respectively set for detection, P/N values are compared, the highest P/N value group is selected as the optimal sealing parameter, and the detection results are shown in the following table 2. The detection result shows that when 5% BSA is selected as a blocking solution, the blocking solution is incubated for 1h at 37 ℃, and the maximum P/N value is 17.06. Therefore, the blocking effect of 5% BSA was determined to be best, and the optimal blocking time was 1h at 37 ℃.
TABLE 2 OD values of different confining liquid components
Note that: p is positive serum OD 450 A measured value; n is negative serum OD 450 Measurement value
3. Optimal incubation time of sample to be examined
In order to determine the optimal incubation time of the sample to be detected, based on the optimal conditions, the serum to be detected is incubated for 30min, 45min, 60min and 90min respectively and then detected, the P/N values are compared, the highest P/N value group is selected as the optimal condition, and the detection results are shown in the following table 3. The test result showed that the serum incubation time was 60min, at which the P/N value was 9.37 at maximum. Thus, the optimal serum incubation time was determined to be 60min.
TABLE 3 OD values for different serum incubation times
Note that: p is positive serum OD 450 A measured value; n is negative serum OD 450 Measurement value
4. Optimal dilution concentration and incubation time of enzyme-labeled IgM antibody
In order to determine the optimal dilution times and incubation time of the enzyme-labeled IgM antibodies, the conditions for setting the enzyme-labeled antibodies are shown in tables 4 and 5, the P/N values are compared, the highest P/N value group is selected as the optimal conditions, and the detection results are shown in tables 4 and 5. The results showed that the P/N value was maximum at 5.94 and 9.45 when the dilution of the enzyme-labeled antibody was 1:2000 and the incubation time was set to 60min.
TABLE 4 dilution OD values of different horseradish peroxidase-labeled goat anti-human IgM (HRP-IgM) antibodies
| Dilution factor of HRP-IgM | P | N | P/N |
| 1:1000 | 2.107 | 0.280 | 5.74 |
| 1:1500 | 1.948 | 0.308 | 5.36 |
| 1:2000 | 1.954 | 0.160 | 5.94 |
| 1:2500 | 1.538 | 0.261 | 5.89 |
Note that: p is positive serum OD 450 A measured value; n is negative serum OD 450 Measurement value
TABLE 5 OD values of incubation time of different horseradish peroxidase-labeled goat anti-human IgM (HRP-IgM) antibodies
| Incubation time of HRP-IgM antibody | P | N | P/N |
| 30min | 0.887 | 0.107 | 8.30 |
| 60min | 1.211 | 0.128 | 9.45 |
| 90min | 1.398 | 0.156 | 8.96 |
| 120min | 1.602 | 0.170 | 9.42 |
Note that: p is positive serum OD 450 A measured value; n is negative serum OD 450 Measurement value
5. Optimal color development time of TMB substrate
Based on the optimized conditions, TMB is detected after being developed for 5min, 10min, 15min and 20min in dark, the P/N values are compared, and the highest P/N value group is selected as the optimal optimized condition. As a result, TMB optimum development time was set to 15min as shown in the table.
TABLE 6 OD values at different TMB development times
Note that: p is positive serum OD 450 A measured value; n is negative serum OD 450 Measurement value
After exploring the above working conditions, the optimal working conditions of the type 16 HPV IgM ELISA kit were finally set as follows: 4.0. Mu.g/mL HPV L1-VLP coated ELISA plates, 100. Mu.L per well, incubated at 37℃for 60min. The coating was discarded, the plate was washed 3 times with 1 Xwashing solution, 200. Mu.L of 5% BSA blocking solution was added, and the plate was blocked at 37℃for 60 minutes. The plate was washed 3 times with 1X wash solution, added with serum to be tested and incubated at 37℃for 60min. Plates were washed 5 times with 1 Xwash solution, 100. Mu.L of 1:2000 pre-diluted enzyme-labeled IgM antibody was added and incubated at 37℃for 60min. Washing the plate with 1 Xwashing solution for 5 times, adding 100 μl TMB color development solution into each well, reacting at room temperature in dark place for 15min, adding 50 μl 2M H after color development 2 SO 4 The reaction was terminated.
6. Determination of yin-yang limit value of HPV IgM ELISA kit
By using the final optimized working conditions, a 16-type HPV IgM antibody detection method is established, 50 negative serum samples are detected, and the detection results are shown in the following table 7. The Cut-off value setting is calculated according to the following formula: cut-off = OD of negative samples 450 Mean +3 x standard deviation. OD of 50 negative serum samples 450 Average value 0.086,3X standard deviation of 0.078, i.e. Cut-off value of 0.042 for type 16 HPV IgM ELISA kit established by this study (see FIG2). The decision criteria are as follows: when the OD450 value of the sample to be detected is more than or equal to 0.127, judging that the sample is an HPV16 type positive sample; sample OD450 value<0.127, HPV type 16 negative samples were judged.
TABLE 7 ELISA assay results for 50 HPV negative serum
7. HPV IgM ELISA kit sensitivity detection result analysis
100 serum samples of the bivalent vaccine group without HPV infection (collected 7-14 days after the first or two-needle inoculation) are detected by using the kit, and the detection steps are as follows:
(1) Preparing a washing liquid: 10 Xthe wash was diluted with deionized or distilled water in a 1:9 ratio. For example, 10mL of 10 Xwashing solution is added to 90mL of deionized water to prepare 100mL of 1 Xwashing solution, and the solution is stored at 2-8deg.C; if crystallization occurs in the 10 Xwash solution, the 20 Xwash solution must be heated by a 50℃water bath until the crystallization is completely removed, and the dilution operation is performed after shaking thoroughly.
(2) Preparing an ELISA plate: the ELISA plate strips were counted and mounted according to the number of test samples. Ensure that the ELISA strips are firmly clamped into the plate frame, and the unused strips are left in an aluminum foil bag and stored at 2-8 ℃. The strips must be stored in a closed aluminum foil bag to avoid moisture.
(3) Sample and coating buffer preparation: the sample and coating buffer are diluted in the ratio of the optimal coating concentration.
(4) Adding diluted sample into ELISA plate for incubation, mixing with microplate constant temperature shaker for 5-10s, sealing, and incubating at 37deg.C for 60min.
(5) Washing: the cover plate membrane was removed, the wells were discarded, and the microplate was washed four times with 260. Mu.L of 1 Xwash. After washing, the water in the wells was completely tapped off on the dried absorbent paper.
(6) Horseradish peroxidase-labeled goat anti-human IgM antibody was added to the ELISA plate for immobilization of HPV L1-VLP antigen, mixed, sealed, and incubated at 37℃for 60 minutes.
(7) Washing: the cover plate membrane was removed, the wells were discarded, and the ELISA plate was washed four times with 250. Mu.L of 1 Xwash solution. After washing, the water in the wells was completely tapped off on the dried absorbent paper.
(8) The chromogenic solution was added and reacted with HRP to yield a blue product. Incubating at 37℃for 15 minutes.
(9) Stop solution was added to stop the reaction and turn the blue product yellow.
(10) Detection was performed using an enzyme-labeled instrument at a wavelength of 450 nm.
The decision criteria are as follows: when the sample to be measured OD 450 When the value is more than or equal to 0.127, judging that the sample is an HPV16 type positive sample; sample OD450 value<0.127, HPV type 16 negative samples were judged. The results are shown in Table 8 below:
TABLE 8 OD value measurement results of 100 Positive serum
The serum samples after 100 HPV bivalent vaccine immunizations are detected by using the optimized HPV IgM ELISA kit, the results are shown in Table 8, and the kit detects 98 positive samples in total in the 100 positive samples after HPV bivalent vaccine immunizations, and the sensitivity is 98%.
8. Within-batch and inter-batch repeatability test
(1) In-batch reproducibility test was performed by taking three ELISA plates of the same batch, performing ELISA assay on 4 positive serum and 4 negative serum, and performing 3 wells in parallel each.
(2) Batch-to-batch repeat test: three batches of antigen-coated ELISA plates prepared at different times were used to perform ELISA assays on 4 positive and 4 negative sera, 3 wells were made in parallel each, and batch-to-batch reproducibility assays were performed. The results are shown in Table 9 below.
TABLE 9 results of within-and inter-lot reproducibility test OD values
The results show that CV ranges of the intra-batch and inter-batch repeatability tests are 0.6% -5.7%, and CV ranges are 0.5% -6.0%, respectively, and CV is less than 10%, so that the established HPV IgM antibody kit has good repeatability and stability.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, any modification, equivalent replacement, improvement or the like of the prior art through logic analysis, reasoning or limited experiments according to the present invention will be within the scope of protection defined by the claims.
Claims (10)
1. The kit for detecting the HPV IgM antibody is characterized by comprising a goat anti-human IgM antibody marked by horseradish peroxidase, an ELISA plate, a sample diluent, a washing liquid, a chromogenic liquid and a stop solution;
the reaction hole of the ELISA plate is coated with HPV L1-VLP antigen;
the HPV L1-VLP antigen is prepared by deleting an N-terminal sequence of an L1 gene of HPV virus, modifying the L1 gene by point mutation, and performing protein expression, protein purification and particle assembly.
2. The kit for detecting an HPV IgM antibody of claim 1, wherein the L1 gene of HPV virus is at least one of HPV 6L1 gene, HPV 11L 1 gene, HPV16L1 gene, HPV 18L 1 gene, HPV 31L 1 gene, HPV 26L 1 gene, HPV 33L 1 gene, HPV 35L 1 gene, HPV45L1 gene, HPV 39L 1 gene, HPV45L1 gene, HPV 51L 1 gene, HPV 52L 1 gene, HPV 53L 1 gene, HPV 56L 1 gene, HPV 58L 1 gene, HPV59L1 gene.
3. The method for preparing a kit for detecting HPV IgM antibodies according to any one of claims 1 to 2, characterized by comprising the steps of:
diluting HPV L1-VLP antigen to coating concentration, adding the HPV L1-VLP antigen into a reaction hole of an ELISA plate, washing by using an antigen washing buffer solution, adding 5% bovine serum albumin for sealing, and washing and sealing after sealing is finished to coat the reaction hole of the ELISA plate with HPV L1-VLP antigen;
mixing the goat anti-human IgM antibody with horseradish peroxidase and dialyzing to obtain the goat anti-human IgM antibody marked by horseradish peroxidase.
4. A method of preparation according to claim 3, wherein the coating concentration is 0.5-4 μg/mL.
5. A method of preparation according to claim 3, wherein the blocking temperature is 4 ℃ or 37 ℃.
6. A method of preparation according to claim 3, wherein the blocking time is 0.5-2 hours.
7. The detection method of the HPV IgM antibody detection kit of any one of claims 1 to 2, characterized by comprising the steps of:
diluting a sample to be detected by using a sample diluent, and then adding the diluted sample into an ELISA plate coated with HPV L1-VLP antigen for mixed incubation; discarding the mixed liquid and washing; adding horseradish peroxidase-labeled goat anti-human IgM antibody into an ELISA plate for incubation, discarding mixed liquid, and washing; and adding a color developing solution for incubation, adding a stopping solution to stop the reaction after the incubation is finished, and judging the result.
8. The method according to claim 7, wherein the method of diluting the sample to be measured with the PBS buffer solution comprises the steps of diluting the sample to be measured with the sample diluent according to 1: the volume ratio of 100-800.
9. The method according to claim 7, wherein the color developing solution is TMB color developing solution.
10. The method according to claim 7, wherein the stop solution is a sulfuric acid solution.
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| US5629146A (en) * | 1988-10-28 | 1997-05-13 | Ferring Ab | Method for detection of human papillomavirus (HPV) for diagnostic purposes |
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