CN110646612A

CN110646612A - Reagent and kit for detecting serum HPV antibody

Info

Publication number: CN110646612A
Application number: CN201910941601.5A
Authority: CN
Inventors: 吴稚伟; 吴喜林; 袁换云; 马晓花; 李彦磊
Original assignee: Source Daolong (suzhou) Medical Technology Co Ltd
Current assignee: Y Clone Medical Science Co ltd
Priority date: 2019-09-30
Filing date: 2019-09-30
Publication date: 2020-01-03

Abstract

The invention relates to a reagent for detecting serum HPV antibodies, which comprises nine types of VLPs of HPV6,11, 16, 18,31,33,45,52 and 58 subtypes, wherein each VLP of the HVP subtypes is packaged by L1 protein and L2 protein of the subtype. Also relates to a kit comprising the reagent.

Description

Reagent and kit for detecting serum HPV antibody

Technical Field

The invention relates to the field of HPV prevention and treatment, in particular to a reagent and a kit for detecting serum HPV antibodies.

Background

Cervical cancer is the second largest cancer of women worldwide, second only to breast cancer. In recent years, the incidence rate of cervical cancer in China is continuously high, and the average speed is increased by 8.7% every year, wherein the growth rate in rural areas is 10.3%, and the growth rate in urban areas is 5.6%; the mortality rate increases at a rate of 8.1%, and the prevention and treatment form is not optimistic. The allelochemicals of cervical cancer are persistent infections with Human Papilloma Virus (HPV). According to different pathogenicity of HPV, the HPV is divided into low-risk type HPV and high-risk type HPV; low risk types (HPV6, type 11, etc.), which can lead to warts and benign tumors of the reproductive organs; high-risk HPV types (HPV16, 18,31,33,45,52,58, and the like) can cause female cervical cancer and male penile cancer. Among them, about 54% of cervical cancer is caused by HPV16 infection, about 16% of cervical cancer is caused by HPV18 infection, so that the control of the persistent infection of high-risk HPV virus has great significance for the prevention and treatment of cervical cancer, and the main prevention and control mode at present is to largely popularize HPV screening and inoculation of HPV vaccine. Currently available HPV vaccines on the market are the siraitia bivalent vaccine against HPV16 and HPV18 from Glactin Stackers (GSK) and the hadamard tetravalent vaccine against HPV16, HPV18, HPV11 and HPV6 from visadon and the hadamard ninth vaccine against HPV (types 6,11, 16, 18,31,33,45,52, 58). These vaccines are virus-like particles (VLPs) that use different expression systems to produce the HPV1 protein, and inhibit Viral infection by vaccination with HPV VLPs to produce highly effective protective antibodies in vivo. Whether or not highly effective protective antibodies are produced is therefore an important indicator of success of HPV vaccination.

Research shows that antibodies induced by HPV VLPs of different subtypes mainly inhibit infection of self-type HPV viruses, and cross-inhibition effect on infection of other types of HPV is not strong. Since the immune response to vaccines varies from individual to individual, the level of production of corresponding protective antibodies varies, and the antigenicity of different types of VLPs in multivalent vaccines varies, it is necessary to detect whether HPV vaccines are successful following widespread vaccination. Research shows that when the antiserum titer induced by the virus-like particles reaches 1000 times of serum dilution, the virus-like particles have good protection effect on HPV infection. In addition, the recent outbreak of HPV pseudovaccine events and the failure of high-titer protective antibodies after HPV vaccination due to the fact that the HPV vaccine is ineffective in the transportation and storage process or a plurality of subjects are vaccinated with the single HPV vaccine.

In view of this, it is necessary to evaluate whether high titers of protective antibodies are produced after HPV vaccination.

Disclosure of Invention

To solve the above problems, the present invention provides a reagent for detecting serum HPV antibodies, characterized by comprising nine subtypes of HPV6,11, 16, 18,31,33,45,52, and 58 VLPs, wherein each of the HVP subtypes of VLP is packaged by L1 protein and L2 protein of the subtype.

In a preferred embodiment, the nine subtypes of VLPs are dissolved in CBS buffer solution at pH 8.5-10.

In a preferred embodiment, the concentration of the VLPs of each HVP subtype is 60-150 ng/mL.

The invention also provides a kit for detecting serum HPV antibodies, which comprises the reagent for detecting serum HPV antibodies.

In a preferred embodiment, the kit is an ELISA kit, and the reagents for detecting serum HPV antibodies serve as a coating solution.

In a preferred embodiment, the kit further comprises a blocking solution.

In a preferred embodiment, the blocking solution is a PBST solution containing 3-10g/100mL skim milk powder.

In a preferred embodiment, the kit further comprises a secondary human antibody having a recognizable marker.

Drawings

FIG. 1 is HPV vaccinator titer assay against HPV VLP. PV4001, PV4002 and PV4003 were sera from subjects vaccinated with HPV4 vaccine, PV9004 was serum from subjects vaccinated with HPV9 vaccine, and H001,002,003 was serum from subjects not vaccinated with HPV vaccine, as negative controls.

FIG. 2 HPV vaccinator titer determination against different subtypes of HPV VLPs. PV4001, PV4002 and PV4003 were sera from subjects vaccinated with HPV4 vaccine, PV9004 was serum from subjects vaccinated with HPV9 vaccine, and H001,002,003 was serum from subjects not vaccinated with HPV vaccine, as negative controls. 1:500, 1:1000, 1:10000 represent serum dilution times.

Detailed Description

1. Preparation of HPV VLPs

The sequences of L1 and L2 of HPV6,11, 16, 18,31,33,45,52 and 58 subtypes of HPV viruses are obtained from NCBI website and cloned to pCDNA3.1. The two ORF frames L1 and L2 are linked by IRES using CMV as promoter. And (4) carrying out bacterium shaking on the correctly constructed clone, and greatly extracting endotoxin-free plasmids for use.

Transfecting 293T cells with the constructed plasmids, after 44-52h, cracking the cells, adding ammonium sulfate solution with pH9.0, whirling, putting the cells into a 37 ℃ incubator for ripening overnight, and purifying by crude salt extraction Hertz ultracentrifugation to obtain high-purity HPV VLP proteins of different subtypes.

HPV antibody detection agent

The above 9 VLPs were mixed in equal proportions, followed by equal proportions of 9 subtypes of VLPs at a total concentration of 900ng/ml, followed by dilution into CBS coating solution at a total concentration of 900ng/ml, 100 ul/well, i.e. 10 ng/well of each VLP was coated in ELISA plates and incubated overnight at 4 ℃. The next day, wash 3 times with wash buffer PBST, 250ul PBST/well, spin dry the wash in the well and pat dry on dust free absorbent paper. And (3) sealing: PBST containing 5% bovine serum is prepared as a sealing solution, the sealing solution is added into an enzyme label plate at 250 ul/hole, the plate is placed at 37 ℃ for 2 hours, and the plate is washed for 3 times after sealing. Loading: serum samples of vaccinees and healthy volunteers were diluted 3-fold in blocking solution starting at 1:900 for a total of 11 gradients of 100 ul/well, loaded at 37 ℃ for 1.5 hours, and washed 3 times after loading. Secondary antibody: a goat anti-human-HRP secondary antibody was added at a rate of 1: diluting with 5000 solution in blocking solution, adding 100 ul/well of the blocking solution into an ELISA plate, placing at 4 ℃ for 45min, and washing for 4 times after the second antibody incubation is finished. Color development: mixing the color substrate solution TMB A and B at a ratio of 1:1, adding 100 ul/well into the ELISA plate, and keeping the temperature at 37 deg.C for 10 min. And (4) terminating: stop solution 1M H2SO4 was added to the well after the color development, 50ul per well. Reading: and (4) reading the OD value at the position of 450nm of the wavelength on the microplate reader, and storing the data. The results are shown in FIG. 1: in the sera of four HPV vaccinees (PV4001, PV4002, PV4003 and PV9004), PV4003 and PV9004 produced high titers of antiserum to HPV VLPs, with the serum titer of PV4003 being 729,000-fold dilution and the serum titer of PV9004 being 243,000-fold dilution. No high titers of antiserum to HPVVLP were observed in the unvaccinated subjects (H001, H002 and H003) by any of the other vaccinees. The above results suggest that some HPV vaccinees produced high titers of antisera against HPV VLPs in vivo, whereas some subjects did not succeed in producing high titers of antibodies against HPV VLPs after vaccination.

Since antibodies induced by VLP proteins of different subtypes mostly inhibit HPV infection of self-type and HPV16 and HPV18 induce 80% of cervical cancer, it is necessary to detect whether antibodies with high titer against different types, especially against HPV16 and HPV18, are generated after vaccination.

Coating: VLPs of different subtypes were diluted at 100ng/ml into CBS coating solution and 100 ul/well, i.e. 10 ng/well, were added to ELISA plates and incubated overnight at 4 ℃. Sealing as before. Loading: serum samples from vaccinees and healthy volunteers were diluted in blocking solution at 100 ul/well for 1.5h at 37 ℃ starting with three dilutions, 1:500, 1:1000, 1: 5000. The rest is the same as before. The results are shown in figure 2, the PV4001 subjects contained only high titer antiserum against HPV18 and HPV33 in their sera, and the PV4003 subjects vaccinated with the 4-valent HPV vaccine successfully produced specific antibodies against the four subtypes HPV6,11, 16 and 18, in addition to cross-binding HPV 33; in addition, the subject PV9004 vaccinated with the 9-valent vaccine successfully produced high titers of specific antibodies against the 8 subtypes HPV6,11,18,31,33,45,52,58, but failed to detect specific antibodies against the high-risk subtype HPV 16. It can be seen that although PV9004 vaccinees produced high titers of HPV VLP antibodies in vivo, they failed to produce high titers of antibodies against the high risk subtype HPV 16.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A reagent for detecting serum HPV antibodies, comprising nine subtypes of VLPs of HPV6,11, 16, 18,31,33,45,52 and 58, wherein each VLP of HVP subtype is packaged with the L1 protein and L2 protein of that subtype.

2. The reagent for detecting serum HPV antibodies of claim 1, wherein said nine subtypes of VLPs are dissolved in CBS buffer solution at pH 8.5-10.

3. The reagent for detecting serum HPV antibodies according to claim 2, wherein the concentration of VLPs per HVP subtype is 60-150 ng/mL.

4. A kit for detecting serum HPV antibodies, comprising the reagent for detecting serum HPV antibodies of any one of claims 1-3.

5. The kit according to claim 4, wherein the kit is an ELISA kit, and the reagent for detecting serum HPV antibodies is used as a coating solution.

6. The kit of claim 5, further comprising a blocking solution.

7. The kit according to claim 6, wherein the blocking solution is PBST solution containing 3-10g/100mL skimmed milk powder.

8. The kit of claim 5, further comprising a secondary human antibody having a recognizable marker.