CN117110606A - Method for detecting neutralizing antibodies of different types of HPV (human papilloma Virus) based on competition method - Google Patents
Method for detecting neutralizing antibodies of different types of HPV (human papilloma Virus) based on competition method Download PDFInfo
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- CN117110606A CN117110606A CN202311057183.6A CN202311057183A CN117110606A CN 117110606 A CN117110606 A CN 117110606A CN 202311057183 A CN202311057183 A CN 202311057183A CN 117110606 A CN117110606 A CN 117110606A
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention relates to the technical field of detection, and particularly discloses a method for detecting neutralizing antibodies of different types of HPV based on a competition method. The kit used in the method comprises an HPV neutralizing antibody standard, an HPV L1 (E7) VLP antigen; HPV L1 (E7) VLP antigens are prepared by deleting the N-terminal sequence of the L1 gene of HPV virus while encapsulating the mutant HPV E7 gene inside the VLP, and performing protein expression, protein purification and particle assembly; the nucleotide sequence of the mutant HPV E7 gene is shown in SEQ ID NO. 1. The invention develops a quantitative detection method special for neutralizing antibody serum after HPV vaccination based on a competition method, can detect the levels of neutralizing antibodies of different types of HPVs in the same sample, and has the advantages of simple operation, high flux, short detection period, high accuracy, high sensitivity and good specificity.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a method for detecting neutralizing antibodies of different types of HPV based on a competition method.
Background
Human papillomavirus (Human Papillomavirus, HPV) infection has become one of the most common sexually transmitted infections worldwide. HPV infection is not only associated with various cancers (such as cervical cancer, penile cancer, anal cancer, etc.), but also can lead to undesirable lesions such as genital warts. In order to prevent HPV infection and its related diseases, injection of HPV vaccine has been widely recommended and adopted.
HPV vaccine is a highly safe and effective prophylactic measure that reduces the risk of infection with HPV by activating the immune system to produce antibodies. The injection of such vaccines typically occurs during childhood and adolescence to establish immune protection prior to the onset of sexual activity. According to the recommendations of the world health organization (World Health Organization, WHO for short), the worldwide popularization of HPV vaccination is of great importance for the prevention of the occurrence of related diseases. In developing countries, HPV vaccine promotion faces challenges, where the safety and effectiveness of HPV vaccines are known to be limited. Meanwhile, the cost of HPV vaccines is still high for some developing world population, and is burdensome. If no exact vaccine effectiveness evaluation means is available, the protection effect of the vaccine after injection on the diseases is intuitively reflected, and the popularization of the vaccine is not facilitated.
The WHO vaccine evaluation system includes four major indicators of Immunogenicity (Immunogenicity), efficacy and Safety (Safety), and can be double-validated by clinical control studies and real-world studies. Immunogenicity is primarily 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, and activated T-cytokines, among others. HPV vaccines mainly induce a humoral immune response in the body, and neutralizing antibodies produced by them bind to HPV antigens when HPV enters the body, thereby preventing primary or persistent infection of HPV. WHO indicates in recommendations (WHO/BS/2015.2252) on ensuring safety, efficacy and quality of recombinant human papillomavirus-like particle (VLP) vaccines that neutralizing antibodies are the gold standard for evaluating whether HPV vaccine-induced immune responses are protective. Thus, the immunogenicity evaluation of HPV vaccines has focused on evaluating the anti-neutralizing response of the vaccine.
The serological detection methods of antibodies after HPV vaccine immunization mainly comprise three methods: VLP-based enzyme linked immunosorbent assay (VLP-ELISA), competitive Luminol Immunoassay (CLIA), pseudovirus neutralization assay (PBNA). The enzyme-linked immunosorbent assay based on VLP is widely used due to simple operation, short period and low cost, but the method detects the class total antibodies (such as total IgG) of the anti-L1 VLP, namely the antibodies containing neutralizing antibodies and non-neutralizing binding antibodies, and overestimates the effective immune response level of HPV vaccine. The competitive Luminex immunoassay can achieve high throughput detection and has good type specificity, but the method can only detect neutralizing antibodies aiming at single neutralizing epitopes in principle, and underestimate the effective immune response level of HPV vaccine. The pseudovirus neutralization test is closest to the process of blocking virus infected cells by neutralizing antibodies in vivo, can intuitively reflect the immunogenicity and the protective effect of the vaccine, and is also recommended by WHO as a gold standard for evaluating the effectiveness of HPV vaccines. However, this method requires cell culture and is time-consuming and labor-consuming to operate.
At present, HPV vaccination is in great demand, however, at present, no detection method for rapidly detecting the effectiveness of HPV vaccines exists on the market, and related detection of HPV is more directed to clinical diagnosis. Aiming at the pain point, the patent provides a high-flux, stable and convenient HPV vaccine effectiveness detection method, which judges the concentration level of the antibody after vaccination by detecting neutralizing antibodies of different types of HPVs, and based on the detection method, the effectiveness of the vaccine is judged, the detection period is greatly shortened, and the specific and high-precision detection is realized.
Disclosure of Invention
The invention aims at least solving one of the technical problems in the prior art, and therefore, the invention provides a method for detecting neutralizing antibodies of different types of HPVs based on a competition method, which is used for detecting neutralizing antibody levels of HPVs of different types in the same serum sample, shortens the detection period and improves the detection precision.
The invention is characterized in that: the neutralizing antibody in the sample is specifically bound with the neutralizing epitope on the HPV L1VLP antigen, so that the neutralizing antibody standard with the signal molecule can be blocked against the binding of the HPV L1VLP antigen to a certain extent, and whether the neutralizing antibody with the signal molecule is present or not can be judged by detecting the binding condition of the neutralizing antibody standard with the signal molecule and the HPV L1VLP antigen.
In a first aspect, the invention provides a kit for detecting different types of neutralizing antibodies to HPV based on a competition method.
Specifically, HPV L1 (E7) VLP antigens, including HPV neutralizing antibody standards;
the HPV L1 (E7) VLP antigen is prepared by deleting the N-terminal sequence of the L1 gene of HPV virus while wrapping the mutant HPV E7 gene inside the VLP, and performing protein expression, protein purification, and particle assembly;
the nucleotide sequence of the mutant HPV E7 gene is shown as SEQ ID NO. 1.
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 33L 1 gene, HPV 35L 1 gene, HPV 45L 1 gene, HPV 52L 1 gene, HPV58L1 gene and HPV 59L 1 gene.
Preferably, the HPV neutralizing antibody standard contains a tag molecule.
Preferably, the label molecule comprises any one of horseradish peroxidase and acridinium ester.
Preferably, the deletion N-terminal sequence of the L1 gene of the HPV virus is amplified by PCR with the upstream and downstream primers shown as SEQ ID NO. 2 and SEQ ID NO. 3.
In a second aspect, the invention provides a detection method of a kit for detecting different types of neutralizing antibodies of HPV based on a competition method.
Specifically, the method comprises any one of the following steps:
(1) Diluting a sample to be detected, and then adding the diluted sample to an ELISA plate coated with HPV L1 (E7) VLP antigen for incubation; discarding the mixed liquid and washing; adding HPV neutralizing antibody standard substances into an ELISA plate for incubation, discarding mixed liquid, and washing; adding a color developing solution for incubation, adding a stopping solution to stop the reaction after the incubation is finished, and judging the result;
(2) Mixing the sample to be tested with HPV L1 (E7) VLP antigen and HPV neutralizing antibody standard, adding an external magnetic field to prepare a precipitation complex, discarding supernatant, cleaning the precipitation complex, and measuring by using a chemiluminescence immunoassay method.
Preferably, in step (1), the HPV neutralizing antibody standard is first mixed with horseradish peroxidase and dialyzed to form a horseradish peroxidase-labeled HPV neutralizing antibody standard.
Preferably, in step (1), the concentration of the coating is 1.5-2.5. Mu.g/mL.
Preferably, in step (2), the HPV L1 (E7) VLP antigen is first reacted with an activated magnetic particle to produce an HPV L1 (E7) VLP antigen-magnetic particle conjugate.
Preferably, in the step (2), the HPV neutralizing antibody standard is first mixed with acridinium ester, coupled, and dialyzed to prepare the HPV neutralizing antibody standard-acridinium ester luminescent reagent.
The third aspect of the invention provides an application of a detection method of a kit for detecting different types of neutralizing antibodies of HPV based on a competition method in a vaccine evaluation system.
Compared with the prior art, the invention has the following beneficial effects:
the invention develops a quantitative detection method special for neutralizing antibody serum after HPV vaccination based on a competition method. The method can detect the levels of HPV neutralizing antibodies of different types in the same sample, and has the advantages of simple operation, high flux, short detection period, high accuracy, high sensitivity and good specificity.
Drawings
Fig. 1 is a schematic diagram of the detection principle of the present invention.
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 different types of neutralizing antibodies of HPV based on a competition method.
The kit comprises an HPV neutralizing antibody standard substance marked by horseradish peroxidase, an ELISA plate, a sample diluent, a washing buffer solution, a color development solution and a termination solution; the reaction wells of the elisa plate were coated with HPV16L1 (E7) VLP antigen.
Example 2
A kit for detecting different types of neutralizing antibodies of HPV based on a competition method.
The kit comprises HPV16L1 (E7) VLP antigen-magnetic particle conjugate, HPV neutralizing antibody standard substance-acridine ester luminescent reagent, excitation solution and pre-excitation solution.
Example 3
Example 1 method of detection of kit.
A method for preparing hpv16L1 (E7) VLP antigen:
(1) PCR amplification and clone sequencing. The upstream and downstream primers used for amplifying HPV16L1 gene are respectively: CAGGTCGACATGTCCGTGTGGCGGCCTAGTGAG (SEQ ID NO: 2) and GGAGAGCTCTTACTT TCGTCCCAAAGGAAACTGATCTAGATC (SEQ ID NO: 3). Since the C-terminal deletion mutation of HPV16L1 protein does not affect VLP formation and bioactivity when it reaches 34 amino acids, the downstream primer is designed to have 33 amino acids deleted at C-terminal. The truncated HPV16L1 gene is amplified by using the upstream and downstream primers and the HPV16 whole genome as templates. Recombinant into a PUCMT cloning vector to construct pUC-T-HPV 16L1 plasmid, and carrying out sequencing identification.
(2) Construction of recombinant plasmid pFB-HPV 16L1 and acquisition of recombinant baculovirus. pUC-T-HPV 16L1 plasmid is digested with NcolI and XholI, truncated HPV16L1 gene is recovered, and the obtained product is connected with pFastBac-Htb shuttle vector to construct recombinant plasmid pFB-HPV 16L 1. DH10Ba competent cells containing baculovirus genome Bacmid and helper plasmid were transformed with pFB-HPV 16L 1. After plating, positive clones were picked and incubated at 37℃for 24h.
(3) Cell culture and transfection of Sf9 insect cells. Insect cells Sf9 were cultured at 27℃in Grace's medium containing 10% high quality foetal calf serum. Inoculation in 6 well plates of about 9X 10 5 Sf-9 cells/well were cultured at 27℃for 1 hour or more to allow complete adherence, followed by cell transfection.
(4) SDS-PAGE and Western blot analysis of expression products and purification of proteins. 10 mu L of transfected cells obtained by centrifugation are added with an equal amount of loading buffer solution, boiled for 3min and subjected to 12% SDS-PAGE, and then respectively subjected to Coomassie brilliant blue staining and Western blot detection. After electrotransfer, the transfer film was subjected to immunochemical reaction with HPV-16 monoclonal antibody and developed with DAB. HPV16L1 protein was purified using the ProBondTM purification system. HPV16L1 protein was purified using both denaturing and non-denaturing conditions. Sf9 cells expressing HPV16L1 protein were first suspended in an appropriate amount of 6mol/L guanidine hydrochloride (4 h at room temperature) and phosphate buffer (3 times repeated freeze thawing with liquid nitrogen) at ph7.2, respectively, cell lysate supernatant containing HPV16L1 protein was mixed with Ni column to bind L1 protein to Ni column, unbound and non-specific binding proteins were then washed off with wash solution, and finally L1 protein was eluted with eluent.
(5) Expression identification of HPV16L1 recombinant proteins and assembly of pseudoviruses. The sf9 insect cells are infected by the recombinant baculovirus, the cells are collected after 4 days, 0.5mg/dl NP-40 is used for cracking the cells, the nuclei are collected by centrifugation at 1000r/min for 10min, the nuclei are cracked by ultrasound, the supernatant is collected by centrifugation at 12000r/min, his-Ni affinity chromatography is added for purifying HPV16L 1VLP, and 100 mug HPV16L 1.Ps were dialyzed against dissociation buffer (pH 7.15Tris-HCl,0.115mmol/L NaCl,1mmol/L EDTA,20mmol/L DTT) at room temperature for 2h, and 100. Mu.g of mE7-pcDNA3.1 was added to the dissociated capsid particles + The plasmid was dialyzed overnight at room temperature against 5mmol/L CaCl2, and after ultracentrifugation, the component HPV16L1 (E7) VLP antigen gene was collected for PCR amplification.
The insect-baculovirus system is utilized to construct virus-like particles composed of HPV16L1 protein, the mutant HPV 16E 7 gene is wrapped in the VLP, and the transformation activity of the E7 protein is removed through mutation, but the antigenicity of the virus-like particles is maintained.
2. The preparation method of the solid phase antigen comprises the following steps:
preparation of an ELISA plate coated with HPV16L1 (E7) VLP antigen.
HPV16L1 (E7) VLP antigen was diluted to 2. Mu.g/mL using coating buffer (pH 9.6.0.05M carbonate buffer) and added at 37℃for 2h per reaction well in the ELISA plate. 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-20.5 mL, 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 2 hours at 37 ℃; discarding 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; coating the ELISA plate with HPV16L1 (E7) VLP antigen is completed.
3. A preparation method of a horseradish peroxidase-labeled HPV neutralizing antibody standard.
(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 the neutralizing antibody with the activated horseradish peroxidase solution according to the mass ratio of 1:1, filling 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 was completed, the liquid was centrifuged at 4℃for 12000r for 20min in an EP tube to remove the precipitate, and the supernatant was a horseradish peroxidase-labeled HPV neutralizing antibody standard.
4. Other preparation methods of the solution:
(1) Sample dilution: 5% BSA (5 g Bovine Serum Albumin (BSA) was added to PBS to volume 100 mL).
(2) Washing buffer: 10X washing liquid, 1X washing liquid;
10 x wash buffer: 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.
1 x wash buffer: the 10 Xwashing liquid and the single distilled water are diluted and configured according to the volume ratio of 1:9.
(3) 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.
And (3) solution A: 0.1M citric acid (1.05 g citric acid added to double distilled water to volume 50 mL);
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);
and C, liquid: 1% TMB (10 mg3,3', 5' -Tetramethylbenzidine (TMB) plus 200uL absolute ethanol, then 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 。
(4) Stop solution: 2mol/L H 2 SO 4 。
5. Determining the concentration of HPV16L1 (E7) VLP antigen coated ELISA plate and selecting the optimal dilution of serum to be detected.
(1) HPV16L1 (E7) VLP antigen was diluted to 0.5, 1.0, 2.0, 4.0 μg/mL using a coating buffer (ph 9.6.05M carbonate buffer), each concentration in a 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 Xwashing solution into each well, washing with a horizontal vibrator for 5min, removing 0.05% PBST washing buffer solution after finishing, 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) Neutralizing antibodies were diluted with a diluent according to 1:2000, each reaction well is 100 mu L and a 37 ℃ incubator is closed for 1h;
(6) Discarding the solution in the ELISA plate, adding 250 mu L of 1 Xwashing buffer solution into each reaction hole, washing for 1min by a horizontal oscillator, discarding the washing buffer solution after finishing, taking redundant residual liquid on water-absorbing 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 10min 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 label instrument to OD450 nm reading, calculating P/N, and determining the concentration corresponding to the maximum value of P/N.
TABLE 1 OD values at different antigen concentration coatings and serum dilution
Coating concentration μg/mL | Serum dilution concentration | P | N | P/N |
0.5 | 1:100 | 1.144 | 0.093 | 12.30 |
1:200 | 0.850 | 0.089 | 9.55 | |
1:400 | 0.601 | 0.082 | 7.33 | |
1:800 | 0.421 | 0.085 | 4.95 | |
1.0 | 1:100 | 1.328 | 0.098 | 13.55 |
1:200 | 1.105 | 0.092 | 12.01 | |
1:400 | 0.942 | 0.087 | 10.83 | |
1:800 | 0.790 | 0.082 | 9.63 | |
2.0 | 1:100 | 1.589 | 0.094 | 16.90 |
1:200 | 1.793 | 0.086 | 20.85 | |
1:400 | 1.432 | 0.085 | 16.85 | |
1:800 | 1.205 | 0.083 | 14.52 | |
4.0 | 1:100 | 1.358 | 0.103 | 13.18 |
1:200 | 1.285 | 0.094 | 13.67 | |
1:400 | 1.084 | 0.089 | 12.18 | |
1:800 | 0.943 | 0.087 | 10.84 |
Note that: p is a positive serum OD450 measurement; negative serum OD450 measurement
The assay showed that HPV16L1 (E7) VLP antigen was coated at a concentration of 2.0 μg/mL, serum at 1: 200-fold dilution was performed, at which point the P/N value was 20.85 at maximum. Thus, the optimal coating concentration for HPV16L1 (E7) VLP antigen was determined to be 2.0. Mu.g/mL and the optimal dilution concentration for serum was 1:200.
6. And determining the optimal sealing liquid component 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.
TABLE 2 OD values of different confining liquid compositions
Note that: p is a positive serum OD450 measurement; negative serum OD450 measurement
The detection result shows that when 5% BSA is selected as a blocking solution, the blocking solution is incubated for 2 hours at 37 ℃, and the maximum P/N value is 18.06. Therefore, the blocking effect of 5% BSA was determined to be best, and the optimal blocking time was 2h at 37 ℃.
7. The sample to be examined is incubated for an optimal time.
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.
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
The test result shows that the incubation time of the serum is 60min, and the maximum P/N value is 19.68. Thus, the optimal serum incubation time was determined to be 60min.
8. Optimal dilution concentration and incubation time of enzyme-labeled neutralizing antibody.
In order to determine the optimal dilution and incubation time of the enzyme-labeled neutralizing antibody, the conditions for setting the enzyme-labeled antibody 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.
TABLE 4 dilution OD values of different horseradish peroxidase-labeled HPV neutralizing antibody standards
Dilution factor | P | N | P/N |
1:1000 | 2.185 | 0.206 | 10.60 |
1:1500 | 1.948 | 0.308 | 6.32 |
1:2000 | 1.958 | 0.258 | 7.59 |
1:2500 | 1.628 | 0.261 | 6.24 |
Note that: p is positive serum OD 450 A measured value; n is negative serum OD 450 Measurement value
TABLE 5 OD values of different horseradish peroxidase-labeled HPV neutralizing antibody standards incubation times
Antibody incubation time | P | N | P/N |
30min | 0.833 | 0.129 | 6.46 |
60min | 1.383 | 0.135 | 10.24 |
90min | 1.470 | 0.153 | 9.61 |
120min | 1.701 | 0.168 | 10.13 |
Note that: p is positive serum OD 450 A measured value; n is negative serum OD 450 Measurement value
The results showed that the P/N value was maximum at 10.60 and 10.24 when the dilution of the enzyme-labeled antibody was 1:1000 and the incubation time was set to 60min.
Determination of the optimal 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.
TABLE 6 OD values at different TMB development times
Color development time (min) | P | N | P/N |
5min | 0.593 | 0.104 | 5.70 |
10min | 0.993 | 0.117 | 8.49 |
15min | 1.007 | 0.124 | 8.12 |
20min | 1.152 | 0.138 | 8.35 |
Note that: p is positive serum OD 450 A measured value; n is negative serum OD 450 Measurement value
As a result, as shown in Table 6, the TMB optimum development time should be set to 10min.
After the above working conditions were explored, the optimum working conditions of the kit of example 1 were finally set as follows: 2.0. Mu.g/mL HPV16L1 (E7) VLP antigen coated ELISA plates were incubated at 37℃for 2h at 100. Mu.L per well. 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 2 hours. The plate was washed 3 times with 1X wash solution, added with serum to be tested, and incubated at 37℃for 1h.1 Xwash plates were washed 5 times and 100. Mu.L 1 was added: 1000 pre-diluted enzyme-labeled neutralizing antibody, incubated at 37℃for 1h. 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 10min, adding 50 μl 2M H after color development 2 SO 4 The reaction was terminated.
10. Example 1 kit yin-yang limit determination.
Using the final optimized working conditions described above, 50 negative serum samples were tested and the test results are shown in Table 7 below. 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 The average value was 0.069,3X standard deviation was 0.067, i.e., the Cut-off value of the example 1 kit established by this study was 0.137. Decision criteria such asThe following steps: when the OD450 value of the sample to be detected is more than or equal to 0.137, judging that the sample is an HPV16 type positive sample; sample OD450 value<0.137, HPV type 16 negative samples were judged.
TABLE 7 ELISA assay results for 50 HPV negative serum
11. Example 1 analysis of the sensitivity test results of the kit.
The kit is used for detecting 50 serum samples (collected 14-18 days after three-needle inoculation) of the non-infected HPV nine-vaccine population, and the detection steps are as follows:
(1) Preparing a washing liquid: 10 Xthe wash was diluted with deionized or distilled water in a 1:10 ratio. Adding 10mL of 10 Xwashing solution into 90mL of deionized water to prepare 100mL of 1 Xwashing solution, and preserving 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 the mixture 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 HPV neutralizing antibody standard was added to the elisa plate immobilized with HPV16L1 (E7) VLP antigen, mixed, sealed, and incubated at 37 ℃ for 60min.
(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 OD450 value of the sample to be detected is more than or equal to 0.137, judging that the sample is an HPV16 type positive sample; samples with OD450 <0.137 were judged to be HPV type 16 negative samples. The results are shown in Table 8 below.
TABLE 8 OD value measurement results of 50 Positive serum
The results show that the kit of the example 1 is used for respectively detecting serum samples after 50 HPV nine-valent vaccine immunization, the results are shown in table 8, and the kit can be used for detecting 49 positive samples in total among the 50 positive samples after HPV nine-valent vaccine immunization, and the sensitivity is 98%.
Example 4
Example 2 method of detection of kit.
The preparation of the HPV16L1 (E7) VLP antigen was the same as in example 3.
A method for preparing a hpv16L1 (E7) VLP antigen-magnetic particle conjugate.
Washing carboxyl magnetic particles with 0.05mol/L morpholinesulfonic acid (pH 6.0) for several times, adding equal amounts of EDC and NHS, (EDC: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, NHS: N-hydroxysuccinimide) and activating at normal temperature for 40min; after 4 washes in wash buffer, VLP antigen according to HPV16L1 (E7) with magnetic particle 1:20, and carrying out oscillation reaction for 5h at room temperature. After blocking for 2h with blocking buffer (0.05 mol/L Tris) +2% bovine serum albumin+0.05% TritonX-100+0.5% Proclin 300), washing with washing buffer to obtain HPV16L1 (E7) VLP antigen-magnetic particle conjugate. Microsphere dilution (0.05 mol/LTris+3% bovine serum albumin+0.05% TritonX-100+5% trehalose+1% sodium chloride+0.2% Proclin 300) was used for dissolution and placed in a refrigerator at 4deg.C for use.
Preparation method of HPV neutralizing antibody standard.
HPV neutralizing antibody standard with acridine ester at 2: mixing the materials according to the mass ratio of 1, performing oscillation coupling for 0.5h at normal temperature, removing free acridinium ester by a dialysis method, purifying by a gel chromatographic column (SephadexG 50), and collecting effluent with high luminous intensity to prepare the HPV neutralizing antibody standard product-acridinium ester luminous reagent. Dilute with luminescent diluent (0.01 mol/L Phosphate Buffer (PBS) +0.05% polyethylene glycol octyl phenyl ether (Triton X-100) +5% trehalose+0.2% casein+0.5% Proclin 300), and place in the dark at 4deg.C for use.
4. Serum sample detection effect determination:
the HPV positive serum sample is a serum sample of a group of people which are not infected with HPV nine-valent vaccine (collected 28-60 days after the whole inoculation is completed); the HPV negative serum sample is a serum sample of healthy people which are not infected with HPV vaccine, 50 parts of HPV positive serum sample and 50 parts of HPV negative serum sample are used, and the test is carried out by the kit, wherein the detection steps are as follows:
positive serum sample test: taking HPV16L1 (E7) VLP antigen-magnetic particle conjugate, adding 60 mu L of HPV positive serum and 100 mu LHPV neutralizing antibody standard substance-acridinium ester luminescent reagent, mixing, incubating at 37 ℃ for 30min, adding magnetic particles and magnetic particle conjugate thereof to aggregate at the bottom of a test tube by an external magnetic field, and lightly washing the test tube by using a washing buffer (PBS+0.05% Tween 20) to remove free substances; after adding 100. Mu.L of excitation solution and 100. Mu.L of pre-excitation solution, respectively, the results were automatically detected by using a chemiluminescent apparatus.
Negative serum sample test: taking HPV16L1 (E7) VLP antigen-magnetic particle conjugate, adding 60 mu L of HPV negative serum and 100 mu L of HPV neutralizing antibody standard substance-acridinium ester luminescent reagent, performing incubation reaction for 30min at 37 ℃, adding an external magnetic field to promote the magnetic particles and the magnetic particle conjugate thereof to aggregate at the bottom of a test tube, and lightly washing the test tube by using a washing buffer (PBS+0.05% Tween 20) to remove free substances; after adding 100. Mu.L of excitation solution and 100. Mu.L of pre-excitation solution, respectively, the results were automatically detected by using a chemiluminescent apparatus.
Negative result judgment: counting the average value and standard deviation of the RLU value of the negative sample, namely judging positive when the RLU value of the sample is greater than the average value plus 3 times of standard deviation; sample RLU value < average value+2×standard deviation, and is judged as negative; the average value +2×standard deviation is less than or equal to the RLU value of the sample and less than or equal to the average value +3×standard deviation, and the sample is judged to be suspected.
TABLE 9 detection results of 50 HPV negative serum samples
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TABLE 10 detection results of 50 HPV Positive serum samples
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TABLE 11 statistics of 100 sets of serum sample test results
As can be seen from tables 9 and 10, the positive compliance rate of the test results was 96%, the negative compliance rate was 100%, and the total compliance rate was 98%. The sensitivity of the reagent is 100%, the specificity is 100%, and the accuracy is 98%. The method for detecting the HPV neutralizing antibodies in different types based on the competition method is high in sensitivity and good in specificity.
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. A kit for detecting different types of neutralizing antibodies of HPV based on a competition method, comprising an HPV neutralizing antibody standard, HPV L1 (E7) VLP antigen;
the HPV L1 (E7) VLP antigen is prepared by deleting the N-terminal sequence of the L1 gene of HPV virus while wrapping the mutant HPV E7 gene inside the VLP, and performing protein expression, protein purification, and particle assembly;
the nucleotide sequence of the mutant HPV E7 gene is shown as SEQ ID NO. 1.
2. The kit 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 33L 1 gene, HPV 35L 1 gene, HPV 45L 1 gene, HPV 52L 1 gene, HPV58L1 gene, HPV 59L 1 gene.
3. The kit of claim 1, wherein the HPV neutralizing antibody standard comprises a tag molecule; the label molecule comprises any one of horseradish peroxidase and acridine ester.
4. The kit according to claim 1, wherein the L1 gene of HPV virus is deleted for N-terminal sequence and PCR amplified with the upstream and downstream primers shown in SEQ ID NO. 2 and SEQ ID NO. 3.
5. The method for detecting the kit for detecting different types of neutralizing antibodies to HPV according to claim 1, comprising any one of the following steps:
(1) Diluting a sample to be detected, and then adding the diluted sample to an ELISA plate coated with HPV L1 (E7) VLP antigen for incubation; discarding the mixed liquid and washing; adding HPV neutralizing antibody standard substances into an ELISA plate for incubation, discarding mixed liquid, and washing; adding a color developing solution for incubation, adding a stopping solution to stop the reaction after the incubation is finished, and judging the result;
(2) Mixing the sample to be tested with HPV L1 (E7) VLP antigen and HPV neutralizing antibody standard, adding an external magnetic field to prepare a precipitation complex, discarding supernatant, cleaning the precipitation complex, and measuring by using a chemiluminescence immunoassay method.
6. The method for detecting different types of neutralizing antibodies against HPV according to claim 5, wherein in step (1), the HPV neutralizing antibody standard is mixed with horseradish peroxidase and dialyzed to form a horseradish peroxidase-labeled HPV neutralizing antibody standard.
7. The method according to claim 5, wherein the concentration of the coating in the step (1) is 1.5-2.5. Mu.g/mL.
8. The method of claim 5, wherein in step (2), the HPV L1 (E7) VLP antigen is reacted with activated magnetic particles to produce HPV L1 (E7) VLP antigen-magnetic particle conjugates.
9. The method for detecting a kit for detecting different types of neutralizing antibodies to HPV according to claim 5, wherein in step (2), the HPV neutralizing antibody standard is first mixed with acridinium ester, coupled and dialyzed to prepare an HPV neutralizing antibody standard-acridinium ester luminescent reagent.
10. Use of the detection method of the kit for detecting different types of neutralizing antibodies to HPV based on the competition method according to any one of claims 5 to 9 in a vaccine evaluation system.
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Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998014564A1 (en) * | 1996-10-04 | 1998-04-09 | Merck & Co., Inc. | Synthetic hpv16 virus-like particles |
CN101251537A (en) * | 2008-03-12 | 2008-08-27 | 中国人民解放军军事医学科学院军事兽医研究所 | Kit for testing neutralizing antibody racing ELISA in human and animal rabies |
CN101609097A (en) * | 2009-04-29 | 2009-12-23 | 唐山怡安生物工程有限公司 | The competitive enzyme-linked immune detection method of EV71 neutralizing antibody, kit or reagent and preparation method thereof |
US20110256182A1 (en) * | 2007-11-23 | 2011-10-20 | Gaoxia Zhang | Genes encoding major capsid protein l1 of human papilloma virus |
CN104880555A (en) * | 2015-06-19 | 2015-09-02 | 中国食品药品检定研究院 | High-throughput detection method for human papilloma virus (HPV) neutralizing antibodies |
CN111562369A (en) * | 2020-06-18 | 2020-08-21 | 威海威高生物科技有限公司 | SARS-CoV-2 neutralizing antibody detection kit |
CN113009153A (en) * | 2021-02-25 | 2021-06-22 | 山东莱博生物科技有限公司 | Magnetic particle chemiluminescence-based detection kit for neutralizing antibody of novel coronavirus and application thereof |
CN113030483A (en) * | 2021-02-25 | 2021-06-25 | 山东省大健康精准医疗产业技术研究院 | Novel coronavirus neutralizing antibody enzyme-linked immunosorbent assay detection kit and application thereof |
CN114062677A (en) * | 2021-11-12 | 2022-02-18 | 郑州安图生物工程股份有限公司 | Kit for detecting neutralizing antibody of new coronavirus and preparation method thereof |
-
2023
- 2023-08-21 CN CN202311057183.6A patent/CN117110606A/en active Pending
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998014564A1 (en) * | 1996-10-04 | 1998-04-09 | Merck & Co., Inc. | Synthetic hpv16 virus-like particles |
US20110256182A1 (en) * | 2007-11-23 | 2011-10-20 | Gaoxia Zhang | Genes encoding major capsid protein l1 of human papilloma virus |
CN101251537A (en) * | 2008-03-12 | 2008-08-27 | 中国人民解放军军事医学科学院军事兽医研究所 | Kit for testing neutralizing antibody racing ELISA in human and animal rabies |
CN101609097A (en) * | 2009-04-29 | 2009-12-23 | 唐山怡安生物工程有限公司 | The competitive enzyme-linked immune detection method of EV71 neutralizing antibody, kit or reagent and preparation method thereof |
CN104880555A (en) * | 2015-06-19 | 2015-09-02 | 中国食品药品检定研究院 | High-throughput detection method for human papilloma virus (HPV) neutralizing antibodies |
CN111562369A (en) * | 2020-06-18 | 2020-08-21 | 威海威高生物科技有限公司 | SARS-CoV-2 neutralizing antibody detection kit |
CN113009153A (en) * | 2021-02-25 | 2021-06-22 | 山东莱博生物科技有限公司 | Magnetic particle chemiluminescence-based detection kit for neutralizing antibody of novel coronavirus and application thereof |
CN113030483A (en) * | 2021-02-25 | 2021-06-25 | 山东省大健康精准医疗产业技术研究院 | Novel coronavirus neutralizing antibody enzyme-linked immunosorbent assay detection kit and application thereof |
CN114062677A (en) * | 2021-11-12 | 2022-02-18 | 郑州安图生物工程股份有限公司 | Kit for detecting neutralizing antibody of new coronavirus and preparation method thereof |
Non-Patent Citations (6)
Title |
---|
"MK387723", GENBANK * |
"OQ659457", GENBANK * |
FRANCIS J. DESSY等: "Correlation between direct ELISA, single epitope-based inhibition ELISA and pseudovirion-based neutralization assay for measuring anti-HPV-16 and anti-HPV-18 antibody response after vaccination with the AS04-adjuvanted HPV-16/18 cervical cancer vaccine", HUMAN VACCINES, vol. 4, no. 6, pages 425 - 434 * |
王亚婷 等: "人乳头瘤病毒58型L1/E7"假病毒"疫苗的制备及免疫效果评价", 现代检验医学杂志, vol. 33, no. 3, pages 35 - 37 * |
秦焱 等: "人16型乳头瘤病毒"假病毒"免疫保护作用的研究", 免疫学杂志, vol. 22, no. 6, pages 623 - 625 * |
秦焱 等: "人16型乳头瘤病毒"假病毒"的制备", 北京大学学报(自然科学版), vol. 42, no. 2, pages 180 - 183 * |
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