CN109828109A

CN109828109A - For detecting the preparation method and kit of the antigen protein of rabies virus antibodies

Info

Publication number: CN109828109A
Application number: CN201811549633.2A
Authority: CN
Inventors: 华俊清; 曾强; 黄晶; 王诺; 项雅丽; 吴边
Original assignee: WUHAN LIFE TECHNOLOGY Co Ltd
Current assignee: WUHAN LIFE TECHNOLOGY Co Ltd
Priority date: 2018-12-18
Filing date: 2018-12-18
Publication date: 2019-05-31

Abstract

The present invention relates to technical field of in vitro diagnostic reagents, more particularly to a kind of for detecting the preparation method and kit of the antigen protein of rabies virus antibodies, the recombinate shape virus infection insect cell of present invention expression cassette containing rabies virus G protein, the insect cell infected；Metainfective Insect cellculture is subjected to culture proliferation；By the antigen protein for extracting rabies virus antibodies in insect cell; and kit has been made with antigen protein; it solves and uses conventional method rabies viruses whole virus particles as envelope antigen; because containing neutralizing antibody and nonneutralizing antibody in antibody test result; not the problem of not can accurately reflect internal effective protection antibody true horizon; the present invention is produced on a large scale; detect other infectious disease positive serums; no cross reaction, high specificity, high sensitivity; it is reproducible; the range of linearity is wide, avoids bio-safety risk, has very strong creativeness.

Description

Preparation method and kit of antigen protein for detecting rabies virus antibody

Technical Field

The invention relates to the technical field of medical supplies, in particular to a preparation method and a kit of an antigen protein for detecting a rabies virus antibody.

Background

Rabies (also called hydropathia) is a zoonotic infectious disease caused by Rabies Virus (RV), and once the Rabies is developed, the fatality rate is almost 100 percent, and the Rabies is an acute infectious disease with the highest fatality rate of human. The rabies virus is bullet in shape and is a single-strand negative-strand RNA virus. The virus genome is about 12kb in length, and N, P, M, G and L five rabies virus structural proteins are arranged from the 3 'end to the 5' end in sequence and respectively code nucleoprotein, phosphoprotein, matrix protein, glycoprotein and large transcriptase protein. The glycoprotein is related to the infection and immunity of rabies virus, and is the only protein in the five proteins which can stimulate the body to produce neutralizing antibodies.

Rabies is distributed globally and domestic animals are the main source of infection. The world health organization estimates that more than 5.5 million people worldwide die from rabies annually, with over 99% occurring in developing countries. Children are the most vulnerable group to rabies. Rabies virus poses a serious threat to human health. At present, rabies vaccines are the most effective means for resisting rabies viruses, and with the continuous development of biotechnology in recent years, human beings have achieved remarkable achievements on the development of novel vaccines, and safe and effective rabies vaccines for human beings are provided, so that more effective protection is provided for the human beings for resisting rabies viruses. In order to prevent rabies, after the dangerous animals are injured, the rabies can be prevented by injecting a passive immune preparation and inoculating a rabies vaccine, but sufficient and effective antibodies can be obtained to resist virus infection and prevent the rabies without completing the inoculation of the rabies vaccine. Due to the reasons of individual immunity level difference, vaccine quality, normalization of vaccination program, gradual reduction of antibody level along with time and the like, most of people obtain immune protection after vaccination, but some people can still have low antibody level due to the reasons and cannot resist the attack of rabies viruses.

The antibody detection is carried out on the corresponding antibody of the rabies virus, the serum neutralizing antibody level of a patient is tracked, the effect of the immunity before exposure and the vaccine immunity in the treatment process after exposure is evaluated, the targeted reseeding is carried out on the crowd with insufficient antibody level, and the method has very important significance for reducing the psychological burden of the patient, preventing the rabies and protecting the health of the related crowd.

At present, 4 methods for detecting rabies virus antibodies are available, including a fluorescent antibody virus neutralization test (FAVN), a Rapid Fluorescent Focus Inhibition Test (RFFIT), a Mouse Neutralization Test (MNT) and an enzyme-linked immunosorbent assay (ELISA). Among them, the rapid fluorescence spot inhibition test (RFFIT) is adopted by WHO and "pharmacopoeia 2010 edition of the people's republic of china" as a gold standard method for detecting neutralizing antibodies against rabies viruses. The Rapid Fluorescence Focus Inhibition Test (RFFIT) is used as a gold standard method for detecting rabies virus neutralizing antibodies, and has the following characteristics: the rabies virus neutralizing antibody in human serum can be quantitatively detected, the level of the antibody for effectively resisting virus infection in vivo can be truly reflected, and the result is true and reliable. However, the test method is very complicated to operate, technicians needing strict professional training can perform the test work, the test period is long, in addition, virus suspension needs to be prepared in the test process, the biological safety problem exists, the requirement on equipment is high, a fluorescence microscope needs to be equipped, and the price is high. Fluorescent antibody virus neutralization assay (FAVN) and mouse neutralization assay (MNT) also have the disadvantages of high requirements on technicians, preparation of virus suspension, long detection time and inconvenience for rapid diagnosis and large-scale sample detection. The enzyme-linked immunosorbent assay (ELISA) has the remarkable advantages of simple and convenient operation, high sensitivity, strong specificity, short detection time, low requirement on equipment, suitability for detection of a large number of samples and the like, and can also be used for quantitatively detecting the content of rabies virus antibodies in human serum.

The antigen used by the existing kit (enzyme linked immunosorbent assay) for detecting the rabies virus antibody on the market is usually rabies virus whole virus particles, and the rabies virus whole virus particles are used as the envelope plate antigen of the kit, and the most defect is that the detected antibody is a whole virus antibody which comprises a neutralizing antibody and a non-neutralizing antibody. Because only neutralizing antibodies can resist rabies virus invasion, rabies is prevented, and non-neutralizing antibodies cannot resist rabies virus invasion. The World Health Organization (WHO) rabies expert Committee considers that sufficient protection is only obtained if the titer of neutralizing antibodies in serum is greater than or equal to 0.5IU/ml, and if the titer is less than 0.5IU/ml, the dosage must be boosted until the titer of neutralizing antibodies reaches the required level. Therefore, only neutralizing antibodies specifically detecting rabies virus can reflect the true antibody protection level in vivo. The G protein is glycoprotein of rabies virus, is related to infection and immunity of the rabies virus, and is the only protein of five structural proteins coded by the rabies virus, which can stimulate the body to produce neutralizing antibodies. The 3 major neutralizing antibody binding sites of GI, GII and GIII are present on the rabies G protein. Researchers try to successfully express G protein by using a genetic engineering method and a prokaryotic expression system, the G protein is used as an antigen protein raw material for detecting a rabies virus antibody, and a rabies virus antibody detection kit (an enzyme linked immunosorbent assay) is prepared for detecting the rabies virus neutralizing antibody. The defect is mainly because the target protein G protein is a glycoprotein, and during the expression and secretion process, sufficient glycosylation modification plays a crucial role in the stability, antigenicity and biological activity of the glycoprotein, and the prokaryotic expression system lacks a relatively complete post-translational modification function, and the difference between the expressed conformation of the G protein and the natural conformation is relatively large, so that the expressed G protein can not accurately reflect the true level of the rabies virus neutralizing antibody in the serum when being used as a diagnostic antigen raw material for detecting the rabies virus neutralizing antibody.

Therefore, a new rabies virus antibody detection kit is needed.

Disclosure of Invention

The invention aims to solve the technical problem of providing a preparation method and a kit for detecting the antigen protein of the rabies virus antibody, which can be produced in a large scale, detect other infectious disease positive serum, have no cross reaction, strong specificity, high sensitivity, good repeatability and wide linear range and avoid the biological safety risk.

The technical scheme for solving the technical problems is as follows: a preparation method of an antigen protein for detecting rabies virus antibodies comprises the following steps:

s1: infecting insect cells with the recombinant baculovirus;

s2: culturing and proliferating the infected insect cells;

s3: extracting the antigen protein of the rabies virus antibody from the cultured and proliferated insect cells.

The invention has the beneficial effects that: the envelope antigen G protein is glycoprotein of rabies virus, and is the only structural protein capable of stimulating in-vivo cellular immunity and inducing to generate a neutralizing antibody. The recombinant G protein is used as the envelope antigen of the kit envelope plate, and firstly, the problem that the real level of the effective protective antibody in vivo cannot be accurately reflected due to the fact that the rabies virus whole virus particles are used as the envelope antigen in the traditional method and the antibody detection result contains a neutralizing antibody and a non-neutralizing antibody is solved. And secondly, in the operation of preparing the recombinant G protein and detecting the antibody, the rabies virus live virus cannot be involved, so that the biological safety risk is avoided.

Further, in S3, the method includes the following steps:

s31 centrifuging 800g of the insect cells with pathological changes for 5min to precipitate and collect the insect cells;

s32, resuspending the insect cells by using 100mL of phosphate buffer containing 20mmol of imidazole, and crushing the insect cells by high-pressure homogenization;

s33 centrifuging 12000g insect cells in S32 for 20min, discarding the precipitate, and filtering the supernatant after the insect cells are broken and centrifuged and the supernatant of the insect cell culture by a 0.45 mu m filter membrane;

s34, passing the filtered cell lysate supernatant and the filtered culture supernatant through a column, and balancing the chromatographic column again;

s35 the supernatant of the insect cell after elution of the column with 200mmol of imidazole in phosphate buffer and the supernatant of the insect cell culture were disrupted and centrifuged, the desired antigen protein was extracted and collected at the peak of the UV value indicated by the instrument, 10% glycerol was added to the antigen protein to adjust the antigen protein concentration to 1mg/mL, and the antigen protein was stored at-80 ℃.

The further beneficial effects are as follows: the insect cell suspension culture process has high cell culture density, and can obviously improve the expression level of foreign protein compared with the traditional adherent standing culture method. The target protein G protein can be purified from the crushed cell supernatant and the culture solution supernatant, and the protein concentration is as high as more than 3.5 mg/L.

A kit for detecting rabies virus antibodies, which is characterized by using the antigen protein prepared by the preparation method.

The kit comprises the following reagents: the kit comprises a solution containing the antigen protein, an ELISA plate, an enzyme marker, a series of calibrators, a sample diluent, a concentrated washing solution and a chromogenic reagent.

The method comprises the following reagents: antigen coated plate coated by the antigen protein, enzyme marker, serial calibrator, sample diluent, concentrated washing liquid and color reagent.

The kit can be produced in a large scale, can detect other infectious disease positive serum, has no cross reaction, strong specificity, high sensitivity, good repeatability and wide linear range, and has a linear correlation coefficient R2 of more than 0.99 within the linear range of 0-10 IU/ml.

A use method of a kit for detecting rabies virus antibodies, which uses the kit, comprises the following steps:

1) taking out the antigen coated plate coated with the G protein from the kit, diluting a sample to be detected with a sample diluent according to a ratio of 1:100, adding 100 mu L of the sample to the antigen coated plate in each hole, and simultaneously setting 6 holes of a calibrator, and adding 100 mu L of the sample to each hole;

2) gently vibrating the sample in the hole, sticking a sealing film, incubating at 37 ℃ for 60 minutes, removing the sealing film, throwing off the solution in the hole, washing the plate for 5 times by using diluted washing liquid, wherein each hole is 300 mu L, and finally drying on absorbent paper;

3) adding 100 mu L of enzyme marker into each hole, pasting a sealing film, placing at 37 ℃ for incubation for 30 minutes, removing the sealing film, washing for 5 times, and adopting the method as the step 2);

4) adding 50 mu L of color development liquid A and 50 mu L of color development liquid B into each hole, mixing uniformly, sticking a sealing film, placing the mixture in a dark place at 37 ℃ for color development for 15 minutes, removing the sealing film, adding 50 mu L of stop solution into each hole, mixing uniformly, and measuring the OD value of each hole by using an enzyme-labeling instrument with the double wavelength of 450/630nm within 30 minutes;

5) and establishing a standard curve of the absorbance value and the antibody concentration, substituting the absorbance of the sample to be detected into a standard curve equation, and solving the antibody content of the G protein in the corresponding sample.

The invention has the beneficial effects that: the detection limit of the antibody content of the G protein is 0.01584IU/ml, the Coefficient of Variation (CV) among pores is less than or equal to 4.58 percent, and the relative deviation of accuracy is less than or equal to 6.47 percent. And (3) performing correlation analysis on the detection result of a rabies neutralizing antibody detection gold standard method 'Rapid Fluorescence Focus Inhibition Test (RFFIT)' recommended by WHO, wherein two groups of data are linearly correlated, and the correlation coefficient R2 is 0.912.

Drawings

FIG. 1 is a schematic block diagram of a method for preparing an antigen protein for detecting rabies virus antibodies;

FIG. 2 is a functional block diagram of the sub-step of S1 in FIG. 1;

FIG. 3 is a functional block diagram of the sub-step of S3 in FIG. 1;

FIG. 4 is a linear range analysis chart of the measurement system in example 4;

FIG. 5 is a graph showing comparative correlation analysis of the method in example 4.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

Example 1

The preparation method of the antigen protein for detecting the rabies virus antibody shown in figure 1 comprises the following steps:

s1: infecting the insect cells with the recombinant baculovirus to obtain infected insect cells;

s2: culturing the infected insect cells for culture and proliferation;

s3: the antigen protein of the rabies virus antibody was extracted from the insect cell in S2.

In S1, cloning the G protein gene segment containing the rabies virus neutralizing antibody binding site to a baculovirus expression vector and infecting insect cells to obtain infected insect cells, wherein the insect cells are Sf9 cells.

The DNA coding sequence of the G protein gene segment containing the binding site of the rabies virus neutralizing antibody is the sequence of SEQ ID NO 1.

As shown in fig. 2, S1 includes the steps of:

s11: standing and culturing Sf9 cells in a Kirschner flask for resuscitation;

s12: subculturing the recovered Sf9 cells, and gradually amplifying the inoculum size until the inoculum size is gradually increased to shake flask culture, wherein the initial density is not lower than 2.5 multiplied by 105/mL after subculturing;

s13: when the growth density of Sf9 cells reached more than 2.0X 106 cells/mL, the Sf9 cells were infected with a baculovirus expression vector carrying a G protein expression cassette.

After the infected insect cells are cultured for 96-108h, more than 80 percent of the cells have obvious lesions.

As shown in fig. 3, S3 includes the following steps:

Example 2

Designing primers according to a G protein gene sequence of a rabies virus CVS-11 strain recorded in Genbank, introducing EcoRI and HindIII enzyme cutting sites into upstream and downstream primers, and leading the upstream primer to be 5'-CCGGAATTCATGGTTCCTCAGGT-3'; the downstream primer was 5'-GGGTTCGAATTACAGTCTGATCT-3' (synthesized by Shanghai Bioengineering Co., Ltd.). A prokaryotic expression recombinant vector pET23a-G (constructed and stored by Wuhan life science and technology Co., Ltd.) containing a G protein DNA fragment (SEQ ID NO:1) of a rabies virus CVS-11 strain is used as a PCR template, the G protein DNA fragment is amplified by PCR, is cloned to a donor plasmid pFastBacHTB of a baculovirus expression system which is also subjected to the same double enzyme digestion after double enzyme digestion, a recombinant donor plasmid pFastBacHTB-G is obtained after the correct expression frame is identified by sequencing, the recombinant donor plasmid is transformed into a DH10Bac competent cell, the recombinant shuttle plasmid Bacmid-G is obtained after 2 times of resistance, blue and white screening and PCR analysis, and the recombinant shuttle plasmid Bacmid-G is transfected into an Sf9 cell for 48h and is shown to be successfully transfected by cytomorphological observation. And (3) harvesting the recombinant baculovirus particles, and infecting Sf9 cells by two rounds to obtain the recombinant baculovirus expression vector containing the G protein expression frame with high titer.

After Sf9 cells are recovered by still culture in a Kirschner flask, the initial density after passage is not lower than 2.5 multiplied by 10⁵The inoculation amount of each strain/mL is gradually enlarged to 1000mL of a shake flask, the liquid volume of the shake flask is 500mL, the culture medium is Sf900 II serum-free culture medium, the culture temperature is 27 ℃, and the rotation speed of a shaking table is (90-110) r/min. When the cell growth density reaches 2.0X 10⁶At each mL, the cells are infected by using the constructed G protein recombinant baculovirus expression vector with the multiplicity of infection (MOI) of 5-10.

Culturing for 96-108h, and centrifuging for 5min at 800g after 80% of cells have obvious lesions to collect the cells. Resuspending the cells in 100mL phosphate buffer (pH7.4) containing 20mmol of imidazole, homogenizing the disrupted cells under high pressure, centrifuging 12000g for 20min, discarding the precipitate, filtering the cell disruption centrifuged supernatant and the cell culture supernatant through a 0.45 μm filter, adding 4mL Ni2+ -NTAResin to a 10mL chromatographic column, equilibrating the chromatographic column with 20mmol of imidazole phosphate buffer (pH7.4), passing the filtered cell lysate supernatant and the culture supernatant through the column, again equilibrating the chromatographic column, eluting with 200mmol of imidazole phosphate buffer (pH7.4), collecting the protein at the peak of the UV value indicated by the instrument, and determining the protein concentration of the eluate by Bradford method. Adding 10% (volume fraction) glycerol to adjust protein concentration to 1mg/mL, and storing at-80 deg.C.

Example 3

The rabies virus antibody detection kit in the embodiment comprises the following components:

1) the above G protein antigen;

2) an ELISA plate;

3) an enzyme label;

4) a series of calibrators;

5) a sample diluent;

6) concentrating the washing solution;

6) a color developing solution A;

7) a color developing solution B;

8) and (4) stopping the solution.

Wherein,

the enzyme label is mouse anti-human IgG labeled with horseradish peroxidase, and the preparation method is as follows: 5mg of horseradish peroxidase (HRP) is weighed and dissolved in 1mL of double distilled water, 500. mu.L of newly configured 0.06mol/L NaIO4 is added, the mixture is placed at 4 ℃ for 30 minutes, the solution is in early green, and 0.5mL of glycol (0.16mol/L) is added for reaction at room temperature and in dark place for 30 minutes. Then, 1mL of 5mg/mL of mouse anti-human IgG was added thereto, and the mixture was dialyzed overnight at 4 ℃ in 0.05mol/L of carbonate buffer solution having a pH of 9.5. After aspiration, 5mg/mL of newly prepared NaBH40.2mL was added, the mixture was left at 4 ℃ for 2 hours, an equal volume of saturated ammonium sulfate solution was added, the mixture was left at 4 ℃ for 30 minutes, centrifuged at 7000rpm for 10 minutes, the supernatant was discarded, resuspended in physiological saline, and dialyzed overnight in 0.15mol/L PBS (pH7.4). The anti-human IgG marked in the dialysis bag is collected for working concentration calibration, and then the anti-human IgG is subpackaged in 20mL bottles and stored at 4 ℃ for later use.

The series of calibrators are prepared as follows: to control the difference in accuracy of different batches of kits, an enterprise working calibrator is established as a reference for preparing each batch of kit calibrator before preparing a kit series calibrator so as to reduce batch-to-batch difference. First, serum samples collected by Wuhan Life technologies, Inc. were assayed by the Rapid fluorescence Range inhibition assay (RFFIT) (inactivated at 60 ℃ for 1 hour). The samples are all human serum collected after completing basic immunization of rabies vaccines. 10 parts of serum with high positive value of neutralizing antibody measured by RFFIT is selected and mixed to be used as calibration substance of calibration kit series. According to a 10IU/ml kit calibrator calibrated by WHO rabies virus human antibody international standard (30IU/ml), diluting to 0.5IU/ml by using a sample diluent in a double ratio to prepare a kit series calibrator (Q1-Q6): 0. 0.5, 1.0, 2.5, 5.0 and 10IU/ml (0IU/ml calibrator is blank sample diluent), simultaneously measuring by using the enterprise calibrator and the kit calibrator, and calculating the titer ratio of the kit calibrator and the enterprise calibrator, wherein the titer ratio is required to be between 0.90 and 1.10. Subpackaging into 1 ml/tube, and storing at 4 deg.C for use.

The preparation method of the sample diluent comprises the following steps: weighing 5g of Bovine Serum Albumin (BSA), 0.5ml of Tween 20(Tween-20), 8.0g of sodium chloride (NaCl), 0.2g of potassium chloride (KCl), 0.24g of monopotassium phosphate (KH2PO4) and 1.8g of dipotassium phosphate (K2HPO4), adding water for injection to dissolve and fixing the volume to 1000 ml.

Preparation of concentrated washings (20 ×): 160g of sodium chloride (NaCl), 4g of potassium chloride (KCl), 4.8g of monopotassium phosphate (KH2PO4), 36g of dipotassium phosphate (K2HPO4) and 10ml of Tween 20(Tween-20) are weighed, and are added with water for injection to dissolve and fix the volume to 1000 ml.

The developing solution A was a citrate buffer containing 50mg/ml of urea hydrogen peroxide, and the developing solution B was a citric acid/sodium citrate buffer containing 0.2mg/ml of TMB and having a pH of 5.0.

The stop solution was 2M H2SO4 solution.

The kit comprises an ELISA plate coated with rabies G protein as a ready-to-use antigen coated plate. The preparation method of the antigen coated plate comprises the following steps: diluting the purified G protein by affinity medium chromatography to 0.5 mu G/mL with a sodium bicarbonate solution with pH of 9.6, adding 100 mu L/well into an ELISA plate, standing overnight at 4 ℃, removing the coating solution, adding 300 mu L of diluted washing buffer solution into each well, repeatedly washing for 3 times, finally patting dry on absorbent paper, adding 120 mu L of phosphate buffer solution (pH7.4) containing 5mg/mL BSA into each well for blocking, standing and blocking overnight at 4 ℃, removing the blocking solution, patting dry on the absorbent paper, standing and drying for 5 hours under the drying condition that the temperature is 25-28 ℃ and the humidity is lower than 30%. And after drying, putting the coated ELISA plate into an aluminum foil bag containing a drying agent, sealing, and storing at 2-8 ℃ for later use.

Example 4

Application method of rabies virus antibody detection kit (enzyme-linked immunosorbent assay)

1) Taking out the antigen coated plate coated with G protein from the kit, diluting the sample to be detected with sample diluent at a ratio of 1:100, adding 100 μ L of the sample to the antigen coated plate per well, and simultaneously setting 6 wells (0, 0.5, 1.0, 2.5, 5.0, 10IU/mL) of the calibrator and adding 100 μ L per well;

3) adding 100 mu L of enzyme marker into each hole, sticking a sealing film, placing the hole at 37 ℃ for incubation for 30 minutes, removing the sealing film, and washing for 5 times, wherein the method is the same as the step (2);

4) adding 50 μ L of color developing solution A and 50 μ L of color developing solution B into each well, mixing, sticking sealing film, developing in dark at 37 deg.C for 15 min, removing sealing film, adding 50 μ L of stop solution into each well, and mixing. Measuring the OD value of each hole by using an enzyme-labeling instrument with double wavelength of 450/630nm within 30 minutes;

5) and establishing a standard curve of the absorbance value and the antibody concentration, and substituting the absorbance of the sample to be detected into a standard curve equation to obtain the antibody content in the corresponding sample.

The content of rabies virus antibody (IgG) is less than 0.5IU/ml, the complete protection titer is not reached, and the rabies virus antibody does not have complete protection effect on human bodies.

The content of rabies virus antibody (IgG) is more than or equal to 0.5IU/ml, the complete protection titer is achieved, and the full protection effect on human body is achieved.

4. Verification of detection performance result of kit

1) Linearity of measuring system

A fixed value serum sample with the concentration of the positive antibody of the rabies virus antibody close to 10IU/ml is serially diluted into 6 concentrations within the range of 0-10 IU/ml, wherein the low value concentration is close to the lower limit of a linear range. Operating according to the kit instruction, repeatedly detecting each concentration for 2 times, calculating the average value of the concentration, and mixing the result with the dilution ratio according to the maximum valueFitting by a small two-multiplication to obtain a linear correlation coefficient R²0.9978. The results are shown in Table 1 and FIG. 4. The result proves that the rabies virus antibody detection kit (enzyme linked immunosorbent assay) has good linear relation in the detection range (0-10 IU/ml). Indicating the reliability of the established ELISA detection method.

TABLE 1 determination of IU values of serum samples with different concentrations and fixed values by the kit

2) Minimum limit of detection

Detecting with zero concentration calibrator as sample, repeating the measurement for 20 times to obtain absorbance value (OD value) of 20 measurement results, calculating average value (M) and Standard Deviation (SD) to obtain A value corresponding to M +2SD, and substituting the A value corresponding to M +2SD into the above equation according to calibration curve equation of calibrator used in kit to obtain corresponding concentration value, which is the lowest detection limit. The lowest detection limit of the kit is determined to be 0.01584 IU/ml.

3) Accuracy of

Using a kit sample diluent to dilute a WHO rabies virus human antibody international standard (30IU/ml) to enable the final antibody concentration to be about 10IU/ml and 5IU/ml, using the diluted WHO rabies virus human antibody international standard as a sample to detect according to the kit instruction steps, repeatedly measuring each sample for 3 times, and calculating the average value M of the measurement results according to the formula: the measured deviation is (M-theoretical)/theoretical × 100%. The relative deviation of the two samples was measured to be 5.27% and 6.47%, respectively. The high accuracy of the present measurement system was confirmed as shown in table 2 and fig. 5.

TABLE 2 deviation of accuracy measurement of this kit

4) Repeatability of

The high-concentration and low-concentration rabies virus antibody positive serum samples are selected, repeated detection is carried out for 10 times respectively, the average value and the standard deviation of the measurement result are calculated, the obtained variation Coefficients (CV) are respectively 4.58% and 3.96%, and the result shows that the repeatability of the detection system is good.

5) Accuracy assessment in comparison to Rapid fluorescence Range inhibition test (RFFIT)

The Rapid Fluorescence Focus Inhibition Test (RFFIT) is taken as a gold standard method for detecting the neutralizing antibody of the rabies virus approved by the World Health Organization (WHO), and can truly reflect the level of the neutralizing antibody which is generated by an organism after the rabies vaccine inoculation and can resist the rabies virus. 40 serum samples injected with rabies vaccine are randomly selected by the company, and the detection result is compared by using a self-made rabies virus antibody detection kit and a Rapid Fluorescence Focus Inhibition Test (RFFIT) to evaluate the detection accuracy of the kit.

TABLE 3 comparison of test results of two methods

The results of the two methods were subjected to regression analysis as shown in table 3, the linear regression equation was y 0.8698x +3.9545, the two data sets were linearly correlated, and the correlation coefficient R was obtained²0.9122, the correlation curve is as above. According to the results, the results obtained by the kit and the rapid fluorescence range inhibition test (RFFIT) have good correlation, so that the detection accuracy of the kit is further verified.

In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are therefore not to be considered limiting of the invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

In the present invention, unless otherwise expressly stated or limited, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through an intermediate. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Sequence listing

<120> preparation method and kit for antigen protein for detecting rabies virus antibody

<160>0

<170>SIPOSequenceListing 1.0

Claims

1. A preparation method of an antigen protein for detecting rabies virus antibodies is characterized by comprising the following steps:

s1: infecting insect cells with the recombinant baculovirus;

s2: culturing and proliferating the infected insect cells;

2. The method according to claim 1, wherein in S1, the G protein gene fragment containing the binding site of the rabies virus neutralizing antibody is cloned into a baculovirus expression vector and infects insect cells, such as Sf9 cells.

3. The method for preparing an antigen protein for detecting rabies virus antibody according to claim 2, wherein the DNA coding sequence of the G protein gene fragment containing the binding site of the rabies virus neutralizing antibody is the sequence of SEQ ID NO. 1.

4. The method for preparing an antigen protein for detecting rabies virus antibody according to claim 2, wherein S1 comprises the following steps:

s11: standing and culturing Sf9 cells in a Kirschner flask for resuscitation;

s12: subculturing the recovered Sf9 cells, wherein the initial density after subculturing is not less than 2.5 multiplied by 10⁵The inoculation amount of each mL is gradually amplified until the mixture is cultured in a shake flask;

s13: when the growth density of Sf9 cells reaches 2.0X 10⁶At a dose of more than mL, the Sf9 cells were infected with a baculovirus expression vector carrying a G protein expression cassette.

5. The method according to claim 1, wherein the infected insect cells are cultured for 96-108h at S2, and more than 80% of the insect cells show obvious lesions.

6. The method for preparing an antigen protein for detecting rabies virus antibody according to claim 1, wherein the step of S3 comprises the following steps:

7. A kit for detecting rabies virus antibodies, which uses the antigen protein prepared by the preparation method according to any one of claims 1 to 6.

8. The kit for detecting rabies virus antibodies according to claim 7, comprising the following reagents: the kit comprises a solution containing the antigen protein, an ELISA plate, an enzyme marker, a series of calibrators, a sample diluent, a concentrated washing solution and a chromogenic reagent.

9. The kit for detecting rabies virus antibodies according to claim 7, comprising the following reagents: antigen protein coated antigen coated plate, enzyme label, serial calibration product, sample diluent, concentrated washing liquid and color reagent.

10. A method for using a kit for detecting rabies virus antibodies, which is characterized in that the method for using the kit of claim 9 comprises the following steps:

1) taking out the antigen coated plate coated with the antigen protein from the kit, diluting a sample to be detected with a sample diluent according to a ratio of 1:100, adding 100 mu L of the sample to the antigen coated plate in each hole, and simultaneously setting 6 holes of a calibrator, and adding 100 mu L of the sample to each hole;