CN112213484A

CN112213484A - Method for detecting in vitro efficacy of mycoplasma hyopneumoniae inactivated vaccine based on ELISA method

Info

Publication number: CN112213484A
Application number: CN202011433678.0A
Authority: CN
Inventors: 徐慧敏; 卢晓楠; 于萍萍; 王美君; 汤波; 韩昱鹏; 刘飞; 刘奇; 张渊魁; 王敏
Original assignee: Beijing Biomedical Technology Center Of Zhaofenghua Biotechnology Nanjing Co Ltd; Zhaofenghua Biotechnology Fuzhou Co ltd; Zhaofenghua Biotechnology Nanjing Co Ltd; Beijing Kemufeng Biological Pharmaceutical Co ltd
Current assignee: Beijing Biomedical Technology Center Of Zhaofenghua Biotechnology Nanjing Co Ltd; Zhaofenghua Biotechnology Fuzhou Co ltd; Zhaofenghua Biotechnology Nanjing Co Ltd; Beijing Kemufeng Biological Pharmaceutical Co ltd
Priority date: 2020-12-10
Filing date: 2020-12-10
Publication date: 2021-01-12

Abstract

The invention belongs to the technical field of animal biological product inspection, and particularly relates to a method for inspecting in vitro relative efficacy of mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain) based on an indirect competitive inhibition ELISA detection method. The invention relates to a method for detecting in vitro relative efficacy of a mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain) based on an indirect competitive inhibition ELISA method, which comprises the steps of inoculating the mycoplasma hyopneumoniae DJ-166 strain into a suitable culture medium for culture, concentrating and purifying the obtained culture, inactivating the obtained culture with thimerosal to prepare an antigen and an inactivated reference vaccine, and performing indirect competitive inhibition ELISA antigen detection on the vaccine to be detected and the reference vaccine, namely in vitro relative efficacy ELISA detection. The detection method disclosed by the invention is simple to operate, short in time consumption, good in sensitivity and specificity, good in stability, specificity and repeatability of a detection result, and convenient, rapid, specific and sensitive to meet the development trend.

Description

Method for detecting in vitro efficacy of mycoplasma hyopneumoniae inactivated vaccine based on ELISA method

Technical Field

The invention belongs to the technical field of animal biological product inspection, and particularly relates to a method for inspecting in vitro relative efficacy of mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain) based on an indirect competitive inhibition ELISA detection method.

Background

Mycoplasma hyopneumoniae (MPS) is commonly referred to as mycoplasma hyopneumoniae, also known as swine enzootic pneumonia, and is a chronic contact respiratory infectious disease caused by mycoplasma hyopneumoniae. The clinical symptoms of the mycoplasma pneumonia of the swine are mainly tremble and asthma, the death rate of the swine is not high generally, but the feed conversion rate is seriously reduced, and the growth and the development are poor for a long time, so that huge economic loss is caused. At present, the immunization of the mycoplasma hyopneumoniae vaccine is one of the important measures for preventing and controlling MPS, and the vaccination can effectively reduce the lung loss, improve the feed conversion rate and shorten the marketing time.

At present, a plurality of vaccine companies at home and abroad are in the market to produce and sell the mycoplasma hyopneumoniae inactivated vaccine. At present, the traditional method for testing the in vitro relative efficacy of the mycoplasma hyopneumoniae inactivated vaccine is a virus attack protection test, namely, the protection efficacy of the vaccine is determined by strong virus attack after the vaccine is immunized by pigs and comparing with a control group. Although the method can intuitively evaluate the in-vitro efficacy of the vaccine, the in-vitro efficacy of the vaccine is long in duration of animal experiments, and the animal experiments need to be attacked during the experiments, so that the biological safety risk exists, the accuracy and reliability of detection results are directly influenced due to various factors such as species, nest, age, weight, sex, health condition, feeding environment, feed nutrition and the like of animals, and the repeatability of detection is inevitably influenced; and the defects of higher test cost and larger difference between challenge protection test batches due to the use of animals, operators, preparation of challenge bacteria liquid, feeding environment and other conditions in the efficacy test. More importantly, the detection and result period of the method is as long as 2 months, so that the research and development period of new vaccines is prolonged, the effective period of the vaccines is indirectly shortened, the stock pressure of the vaccines is increased, and the production and sale of the vaccines are seriously influenced. Therefore, the method for detecting the efficacy of the mycoplasma hyopneumoniae inactivated vaccine is developed to meet the requirements of rapidness, sensitivity and good repeatability, is favorable for improving the reliability of a detection result, further ensures the product quality, and has positive significance.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide a method for detecting the in vitro relative efficacy of the mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain) based on an indirect competitive inhibition ELISA detection method, the detection method has the advantages of simple operation, good specificity, high sensitivity and short time consumption, can replace the traditional in vivo efficacy animal test of pigs, solves the adverse factors of multiple influencing factors, poor repeatability and long detection period of 2 months brought by tested animals, can reduce the amount of vaccine stock in a production line, prolongs the effective period of the vaccine, ensures the quality of the vaccine, reduces the detection cost and labor intensity, improves the detection efficiency, completes all in vitro relative efficacy tests of the vaccine and data arrangement and judgment results in two days, and can realize high-throughput vaccine efficacy detection;

the second problem to be solved by the present invention is to provide a reference vaccine preparation method and conditions for long-term stable storage for establishing the above detection method;

the third problem to be solved by the invention is to provide a preparation method of competitive mycoplasma enveloped antigen, positive serum and negative serum for establishing the detection method;

the fourth problem to be solved by the present invention is to provide a dilution method of a vaccine to be tested and a reference vaccine for establishing the above detection method;

the fifth problem to be solved by the present invention is to provide a method for determining the whole incubation time for establishing the above-mentioned detection method;

the sixth problem to be solved by the present invention is to provide a method for determining a whole course diluent for establishing the above-mentioned detection method;

a seventh problem to be solved by the present invention is to provide a calculation and analysis method for establishing the detection result of the above-described detection method;

an eighth problem to be solved by the present invention is to provide a method for establishing the use of the above detection method in the preparation of a mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain).

In order to solve the technical problems, the method for detecting the in vitro efficacy of the mycoplasma hyopneumoniae inactivated vaccine based on the ELISA method comprises the following steps:

(1) taking a mycoplasma hyopneumoniae inactivated vaccine to be detected and a reference vaccine to be detected for later use;

(2) diluting the purified mycoplasma hyopneumoniae coating antigen with a coating solution, adding the diluted mycoplasma hyopneumoniae coating antigen into an ELISA plate for coating treatment, washing the plate after the coating treatment is finished, adding a sealing solution for incubation in a constant-temperature incubator, and washing the plate for later use;

(3) respectively adding vaccines to be detected and reference vaccines from different sources into different holes of the processed ELISA plate, and respectively adding detection primary antibody, standard negative serum, standard positive serum and blank control into other blank holes;

(4) placing the enzyme label plate after sample addition in a constant temperature incubator for incubation, respectively adding an enzyme label detection secondary antibody marked by HRP into each hole after plate washing, carrying out constant temperature incubation, adding a developing solution after plate washing, and adding a stop solution after incubation in a dark place at room temperature to stop reaction;

(5) measuring the OD value of each reaction solution in each hole under the condition of 450nm, and calculating the calculated OD_450nmThe values are input into Rel Pot4.0 software to calculate RP values to obtain numerical relationship values and numerical relationship graphs between antigen contents to judge whether the in vitro relative potency of the vaccine is qualified.

Specifically, in the step (1), the reference vaccine is a vaccine with minimum protection amount of antigen in a qualified challenge test.

Specifically, in the step (2):

controlling the dilution concentration of the mycoplasma hyopneumoniae to be 20 +/-5 mu g/ml, controlling the liquid adding amount to be 100 mu l/hole, and controlling the temperature of the coating treatment step to be 4 ℃ for coating for 16-18 hours;

controlling the adding amount of the sealing liquid to be 100 mu L/hole, and culturing at the constant temperature of 37 +/-2 ℃ for 1-2 hours.

Specifically, in the step (3), the adding amount of the vaccine to be detected and the reference vaccine is 200 μ l/hole.

Specifically, in the step (3):

the primary detection antibody is rabbit anti-mycoplasma hyopneumoniae positive serum diluted by PBS;

the standard negative serum is rabbit anti-mycoplasma hyopneumoniae negative serum diluted by PBS;

the standard positive serum is rabbit anti-mycoplasma hyopneumoniae positive serum diluted by PBS;

the blank was PBS.

Specifically, the step (3) further comprises the step of performing gradient dilution on the reference vaccine and/or the vaccine to be detected, and then adding the reference vaccine and/or the vaccine to be detected into the elisa plate for different concentration detection.

Specifically, in the step (3), the dilution gradient is an original-fold gradient, and the dilution gradient is a gradient of 1: 2. 1: 4. 1: 8. 1: 16. 1: 32. 1:64 and 1: serial dilution of 128 by 2 fold.

Specifically, in the step (4):

the enzyme-labeled detection secondary antibody is a horseradish peroxidase-labeled goat anti-rabbit secondary antibody diluted by PBS, and the liquid adding amount is 100 mu l/hole;

the liquid adding amount of the color development liquid is 100 ul/hole;

the amount of the stop solution added is 50ul per hole.

Specifically, in the step (5), the test validity determination includes:

a: positive control OD_450nmThe average value should be within the range of 0.7-2.0;

b: negative control OD_450nmThe average value should be less than 0.4;

c: test invalidation criteria: if one of the above criteria is not met, the test is invalid.

Specifically, in the step (5), the qualified judgment criteria of the vaccine are as follows:

a: the test effectiveness standard is met;

b: setting the RP value of a reference vaccine to be 1.0, setting the RP value of the vaccine to be detected to be more than or equal to 1.0, and ensuring that the relative efficacy of the vaccine to be detected is qualified;

c: if the RP value of the vaccine to be detected is less than 1.0, the vaccine should be re-detected for 1 time; and judging that the relative efficacy of the vaccine to be detected is qualified if the RP value of the result to be rechecked is more than or equal to 1.0, or judging that the relative efficacy of the vaccine to be detected is unqualified if the RP value of the result to be rechecked is not less than or equal to 1.0.

The invention relates to a method for detecting in vitro relative efficacy of a mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain) based on an indirect competitive inhibition ELISA method, which comprises the steps of inoculating the mycoplasma hyopneumoniae DJ-166 strain into a suitable culture medium for culture, concentrating and purifying the obtained culture, inactivating the obtained culture with thimerosal to prepare an antigen and an inactivated reference vaccine, and performing indirect competitive inhibition ELISA antigen detection on the vaccine to be detected and the reference vaccine, namely in vitro relative efficacy ELISA detection. The detection method disclosed by the invention is simple to operate, short in time consumption, good in sensitivity and specificity, good in stability, specificity and repeatability of a detection result, and convenient, rapid, specific and sensitive to meet the development trend.

The method for detecting the in vitro relative efficacy of the mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain) has good sensitivity and specificity, can detect the antigen content interval of 2ug/ml-5000ug/ml, realizes high-throughput detection, can completely meet the technical requirement of in vitro efficacy detection of vaccine production, and has no cross reaction with common porcine viruses PCV2, PPV, PRV, TGEV, PEDV, CSFV and PRRSV. The method for detecting the in vitro relative efficacy of the mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain) can complete the detection of the relative efficacy of the vaccine in about 6 hours, effectively reduces the stock of the vaccine, prolongs the effective period of the vaccine, reduces the production cost of the vaccine and the labor intensity of animal tests, particularly can increase the production vaccine amount when the vaccine is stressed, and can create more output values. The method can monitor the relative efficacy of the vaccine in the whole process, overcomes the adverse factors of more influencing factors, poor repeatability and long verification period of 2 months caused by using test animals, overcomes the influence of various influencing factors such as unexpected death of the animals in the middle of the test and the like, can thoroughly replace the traditional in-vivo detection method of the vaccine of the pigs, and has important significance for further ensuring the quality of the vaccine.

The method for detecting the in vitro relative efficacy of the mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain) adopts the principle of double parallel line biological assay to make indirect competitive inhibition ELISA to detect the in vitro relative efficacy of the mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain), and the dilution (original times, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and 1: 128) of the vaccine to be detected and the corresponding OD_450nmShows good linear regression R between logarithms of values²Over 0.99, the RP values are all over 1.0 after software calculationThe slope (slope score) is above 0.97, the detection result shows better specificity and repeatability, the detection method is convenient to operate, quick to complete and reliable in result, can become a substitute method for detecting the relative efficacy of the vaccine in an animal test in the production of the mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain), and has positive popularization value.

Drawings

In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,

FIG. 1 is a graph showing the results of a higher P/N value in a matrix titration of the envelope antigen and a detection primary antibody (rabbit anti-Mycoplasma hyopneumoniae positive serum);

FIG. 2 is a graph showing the results of the matrix titration of the primary antibody (rabbit anti-Mycoplasma hyopneumoniae positive serum) and the secondary antibody with higher P/N values;

FIG. 3 is a graph of the results of optimizing the seal time with higher P/N values;

FIG. 4 is a graph showing the results of the optimization of incubation times for the optimal detection of vaccine and detection of primary antibody with higher P/N values;

FIG. 5 is a graph showing the results of the optimal detection of the optimal secondary antibody with the optimal incubation time and the higher P/N value;

FIG. 6 is a graph showing the results of optimizing the color development time and obtaining a higher P/N value;

FIG. 7 is a graph of in vitro potency RP values of the vaccine to be tested ZYT-191;

FIG. 8 is a graph of in vitro potency slope (slope) of the vaccine to be tested ZYT-191;

FIG. 9 is a graph of in vitro potency RP values of the vaccine to be tested ZYT-192;

FIG. 10 is a graph of in vitro potency slope (slope) for the vaccine to be tested ZYT-192.

Detailed Description

In the following examples of the present invention, the coating solution (carbonate buffer solution CBS concentration 0.05mol/L, pH 9.6), PBS (phosphate buffer solution), PBST (phosphate buffer solution containing penta tween 80), blocking solution (PBS containing 5% skim milk powder), primary antibody detection and secondary antibody detection diluent PBS (phosphate buffer solution), color developing solution, and stop solution are all conventional solutions in the prior art.

Example 1

This example was used to prepare a Mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain) and its reference vaccine (batch number ZYTCK-1801).

The mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain) reference vaccine (batch number: ZYTCK-1801) is produced by an applicant according to the existing standard of Chinese veterinary pharmacopoeia, and the mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain) to be detected (batch numbers: ZYT-191 and ZYT-192) is also produced by the applicant according to the existing standard of Chinese veterinary pharmacopoeia.

The reference vaccine ZYTCK-1801 and the mycoplasma hyopneumoniae inactivated vaccine (DJ 166) to be detected (batch numbers: ZYT-191 and ZYT-192) inoculate the mycoplasma hyopneumoniae DJ-166 with a proper culture medium for culture, the culture is harvested, the culture is concentrated and purified, and then the antigen is prepared by inactivating the thiomersal, a plurality of antigen concentration gradients are prepared, and proper adjuvants are respectively added to emulsify the antigen into the vaccine.

Reference vaccine quality control

The characteristics are as follows: yellowish or light white liquid, wherein the lower layer has precipitate after long-term storage, and the precipitate is shaken to form uniform suspension;

and (4) checking the loading quantity: the inspection is carried out according to the current Chinese veterinary pharmacopoeia, and the requirements are met;

and (4) sterile inspection: checking according to the existing Chinese animal pharmacopoeia, and allowing sterile growth;

according to the existing Chinese animal pharmacopoeia, the characters (light yellow), the loading inspection (meeting the regulations) and the sterile test (sterile growth) are all in accordance with the regulations.

Safety and efficacy testing of reference vaccines

And (4) safety inspection: 5 healthy susceptible pigs of 3-4 weeks old were injected intramuscularly at the neck with 4.0ml of vaccine for 14 days. Systemic and local adverse reactions caused by vaccine injection should not occur (if there is a body temperature reaction, the temperature of the test pig should not exceed 1.5 ℃ of the basal body temperature within 2 days after injection);

and (3) testing the efficacy: 10 healthy susceptible pigs of 3-4 weeks old were divided into 2 groups of 5 pigs each. The group 1 is an immunization group, each head and neck part is injected with 2.0ml of vaccine through muscle, the group 2 is a challenge control group, and pigs of each group are separately fed. 28 days after immunization, the immunized pigs and the virus-counteracting control pigs are respectively subjected to virus counteracting by using virulent HB strains for mycoplasma hyopneumoniae test, 10ml (100 MID containing 0.4g of diseased lung tissues) is injected into each trachea, each group of pigs are separately fed, and the feed does not contain antibiotics. And (5) killing the pig at 25-28 days after virus attack, recording the pneumonia lesion score of the pig according to a 28-score method, and calculating the pneumonia lesion reduction rate. The reduction rate of the pneumonia lesions of the pigs in the immune group reaches 60 percent and is qualified by judging the vaccine efficacy test. Judging according to disease judgment standards: the vaccine with the lowest antigen content of the pneumonia lesions of the immunized pigs reaching 60 percent or more and the challenge control pigs having at least 4 pneumonia lesions can be used as a reference vaccine.

Pneumonia lesion reduction rate = (control swine pneumonia arithmetic mean score-immune swine pneumonia arithmetic mean score/control swine pneumonia arithmetic mean value) × 100%.

Storage and effective period: storing at below-70 deg.C for four years.

Example 2

The preparation method of the mycoplasma competitive envelope antigen in the embodiment comprises the following steps: the mycoplasma hyopneumoniae DJ-166 strain is concentrated and purified to obtain a bacterial liquid, and the bacterial liquid is subjected to inactivation and centrifugation and is combined by TritonX-100 and KI solution to obtain the mycoplasma hyopneumoniae strain. The kit is used for detecting the in vitro relative efficacy of the mycoplasma hyopneumoniae inactivated vaccine (DJ-166 strain) and indirectly competing and inhibiting an ELISA test envelope antigen.

Quality control of coating antigen

The characteristics are as follows: dissolving and shaking uniformly to obtain milky white liquid;

and (4) sterile inspection: sterile growth is required;

and (3) measuring the titer: and carrying out agar diffusion test with mycoplasma hyopneumoniae positive serum, wherein the agar diffusion titer is not less than 1: 2;

protein concentration determination: detecting by using a commercial BCA kit, wherein the protein concentration is not lower than 1.0 mg/ml;

specificity: clear white precipitate lines should appear in the serum positive to the mycoplasma hyopneumoniae, and no precipitate lines should appear in the serum positive to the mycoplasma hyopneumoniae and the mycoplasma hyorhinis.

Storage and effective period: storing at below 70 deg.C for three years.

Example 3

The preparation method of the positive serum in the embodiment comprises the following steps: the mycoplasma hyopneumoniae inactivated vaccine is used for immunizing healthy rabbits, the immunization is performed for 1 time in the same dosage and way after 2 weeks, and the titer of the agar-agar antibody is not lower than 1:16 hours, collecting blood aseptically, separating serum, filtering, sterilizing, inactivating, purifying, and freeze-drying under vacuum. The positive serum for the indirect competitive inhibition ELISA test is used for detecting the in vitro relative efficacy of the mycoplasma hyopneumoniae inactivated vaccine (DJ-166 strain), and can be lyophilized in batches.

Positive serum freeze-drying quality control standard

The characteristics are as follows: white spongy loose lumps;

and (4) sterile inspection: sterile growth is required;

and (3) measuring the titer: and performing agar diffusion test with mycoplasma hyopneumoniae antigen, wherein the agar diffusion potency is not lower than 1: 2;

specificity: clear white precipitation lines should appear with the mycoplasma hyopneumoniae antigen, the positive reaction is shown, and no precipitation lines should appear with the mycoplasma hyopneumoniae antigen and the mycoplasma hyorhinis antigen, the negative reaction is shown;

and (3) residual moisture determination: the residual water content is less than or equal to 4.0 percent;

and (3) measuring the vacuum degree: 5/5 should glow purple or white.

Storage and effective period: storing at below-70 deg.C for five years.

The method for preparing negative serum in this example comprises the following steps: the rabbit heart blood is prepared by aseptically collecting heart blood of healthy rabbits, centrifugally separating serum, filtering, sterilizing, inactivating, and freeze-drying under vacuum. The negative serum for indirect competitive inhibition ELISA test is used for in vitro relative efficacy detection of mycoplasma hyopneumoniae inactivated vaccine (DJ-166 strain), and can be lyophilized in batches.

Quality control standard of negative serum freeze-dried product

The characteristics are as follows: white spongy loose lumps;

and (4) sterile inspection: sterile growth is required;

and (3) measuring the titer: diluting according to the marked amount, and performing an agar diffusion test with the mycoplasma hyopneumoniae antigen, wherein no precipitation line is required to appear;

specificity: after being diluted according to the marked amount, the antigen and the mycoplasma hyopneumoniae, the mycoplasma hyofloccosum and the mycoplasma hyorhinis do not have any precipitation line;

and (3) measuring the vacuum degree: 5/5 should glow purple or white.

Storage and effective period: storing at below-70 deg.C for five years.

Example 4

The establishment and optimization of the method for detecting the in vitro relative efficacy of the mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain) based on the indirect competitive inhibition ELISA detection method specifically comprises the following steps:

(1) diluting mycoplasma coated antigen with coating solution (sodium carbonate 1.59g, sodium bicarbonate 2.93g, and distilled water 1L) to 20ug/ml, adding into 96-well enzyme-labeled plate, coating at 4 deg.C for 16-18 hr at 100 ul/well; after returning to room temperature, wash the plate 3 times with PBST (phosphate buffer containing penta tween 80) with 3 min intervals, and finally patt dry on absorbent paper; then, adding PBS (phosphate buffer solution) containing 5% skimmed milk powder in a confining liquid, incubating at a constant temperature of 37 ℃ in a incubator with 100 mu L/hole, washing the plate for 3 times, wherein each time is 3 minutes apart, and finally, patting the plate dry on absorbent paper;

(2) diluting the prepared reference vaccine and the vaccine to be detected, namely the antigen to be detected, in a dilution plate, respectively adding 200 mu l of the reference vaccine into A3, A4 and A5 holes on a 96-well plate, respectively adding the vaccine to be detected 1 (ZYT-191) into A6, A7 and A8 holes, respectively adding the vaccine to be detected 2 (ZYT-192) into A9, A10 and A11 holes, wherein the liquid adding amount is 200 mu l per hole;

mu.l of PBS was added to each of the remaining wells (i.e., B3-B11 to H3-H11), the reference vaccine and the test vaccine were diluted in successive 2-fold ratios (longitudinal direction), and 50. mu.l of each liquid was transferred to the corresponding well of a 96-well Mycoplasma hyopneumoniae-coated reaction plate. 50. mu.l of a primary assay antibody (rabbit anti-Mycoplasma hyopneumoniae positive serum diluted with PBS) was added to each well of A3-A11 to H3-H11; standard negative control (1: 4 rabbit anti-Mycoplasma hyopneumoniae negative serum diluted with PBS), standard positive control (1: 50 rabbit anti-Mycoplasma hyopneumoniae positive serum diluted with PBS) and blank control (PBS) were added to columns A1-H1, A2-H2 and A12-H12, respectively;

(3) placing the reaction plate on a shaking table, incubating the reaction plate in a constant-temperature incubator at 37 ℃ at a rotating speed of 100r/min, then washing the plate for 3 times, separating 3 minutes every time, and finally drying the plate on absorbent paper; then, HRP-labeled secondary enzyme-labeled detection antibody (horseradish peroxidase-labeled secondary goat-anti-rabbit antibody) diluted with PBS was added thereto, 100 ul/well was incubated at 37 ℃ in a constant temperature incubator for 1 hour, the plate was washed 3 times with 3-minute intervals, and finally, the plate was blotted dry on absorbent paper. Taking out the TMB color development liquid to return to room temperature, controlling the liquid adding amount to be 100 ul/hole, adding the enzyme label plate, and incubating and developing at room temperature in a dark place; after the reaction is finished, the reaction is stopped by adding stop solution into the reaction vessel with the addition amount of 100. mu.L/well.

The reaction solution in each well was measured for OD450nm at 450nm and calculated.

In order to determine the optimal reaction conditions, in this example, under different reaction conditions, the coated antigen and the detection primary antibody and the detection secondary antibody were diluted in series and then subjected to matrix titration, with higher P/N value and R²The optimum use concentration of the above-mentioned substances is determined by using the above-mentioned evaluation index of 0.99, and the measurement results are shown in FIGS. 1 and 2, respectively.

After the using concentrations of the optimal detection primary antibody and the optimal detection secondary antibody are determined, respectively detecting the influence of different incubation times (30 min, 60min, 90min and 120 min) of the confining liquid, the detection vaccine, the detection primary antibody and the detection secondary antibody on the detection effect based on the parameters under the concentrations, and selecting the higher P/N value as the optimal incubation time; and finally, measuring the influence of different color development time (10 min, 15min and 20 min) on the detection effect of indirect competitive inhibition ELISA, and selecting the one with higher P/N value as the optimal color development time. The results of the optimum reaction time for each step are shown in FIGS. 3-6, respectively.

Tests prove that the optimal coating concentration of the mycoplasma coating antigen is 20 ug/ml; the optimal dilution factor of the primary antibody is detected to be 1: 25; the optimal dilution factor of the detected secondary antibody is 1: 15000; the optimal action time of sealing, detecting the primary antibody, detecting the secondary antibody and developing solution is 1.5h, 1h and 15min respectively. According to the optimal action conditions, the optimal dilution mode of the reference vaccine and the vaccine to be detected is determined as follows, concentration gradient dilution is carried out for original times, and the ratio of 1: 2. 1: 4. 1: 8. 1: 16. 1: 32. 1:64 and 1:128, etc. in a 2-fold serial multiple dilution.

Example 5

This example describes the specific implementation of a method for testing the in vitro relative efficacy of a mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain) based on an indirect competitive inhibition ELISA test.

Diluting mycoplasma coated antigen to 20ug/ml with coating solution, adding into 96-well enzyme-linked immunosorbent assay plate, coating at 4 deg.C for 16-18 hr at 100 ul/well; after returning to room temperature, wash the plate 3 times with PBST (phosphate buffer containing penta tween 80) with 3 min intervals, and finally patt dry on absorbent paper; subsequently, a blocking solution containing 5% nonfat dry milk in PBS (phosphate buffered saline), 100. mu.L/well, incubation in a 37 ℃ incubator for 1.5 hours, washing the plate 3 times with 3 minute intervals, and finally patting dry on absorbent paper.

Diluting the prepared reference vaccine and the vaccine to be detected, namely the antigen to be detected, in a dilution plate, adding 200 mu l of the reference vaccine into each of A3, A4 and A5 holes on a 96-well plate, adding the vaccine to be detected 1 (ZYT-191) into each of A6, A7 and A8 holes, adding the vaccine to be detected 2 (ZYT-192) into each of A9, A10 and A11 holes, and adding the liquid amount to be 200 mu l per hole.

Mu.l of PBS was added to each of the remaining wells (i.e., B3-B11 to H3-H11), the reference vaccine and the test vaccine were diluted in successive 2-fold ratios (longitudinal direction), and 50. mu.l of each liquid was transferred to the corresponding well of a 96-well Mycoplasma hyopneumoniae-coated reaction plate. Add 50 μ l of assay primary antibody (1: 25 rabbit anti-Mycoplasma hyopneumoniae positive serum diluted with PBS) to each well of A3-A11-H3-H11; standard negative controls (1: 4 rabbit anti-Mycoplasma hyopneumoniae negative serum diluted with PBS), standard positive controls (1: 50 rabbit anti-Mycoplasma hyopneumoniae positive serum diluted with PBS) and blank controls (PBS) were added to columns A1-H1, A2-H2 and A12-H12, respectively. The loading schematic for each well is shown in table 1 below.

TABLE 1 OD determination of the vaccine ZYT-191 to be tested and the vaccine ZYT-192 to be tested_450nmSchematic diagram of value loading

The reaction plate was incubated on a shaker at a constant temperature incubator at 37 ℃ for 1.5 hours at a rotational speed of 100r/min, the plate was washed 3 times with 3 minute intervals each time, and finally patted dry on absorbent paper. Then, a 15000-fold dilution of HRP-labeled secondary enzyme-labeled detection antibody (horseradish peroxidase-labeled secondary goat-anti-rabbit antibody) in PBS was added thereto, the mixture was incubated at 100 ul/well in a 37 ℃ incubator for 1 hour, the plate was washed 3 times at 3-minute intervals, and finally the mixture was blotted dry on absorbent paper. Taking out the TMB color development liquid, returning to the room temperature, controlling the liquid adding amount to be 100 ul/hole, adding the enzyme label plate, and incubating for 15 minutes in a dark place at the room temperature; after the reaction is finished, the reaction is stopped by adding stop solution into the reaction vessel with the addition amount of 100. mu.L/well.

The reaction solution in each well was measured for OD450nm at 450nm, and the following calculation was performed based on the measured OD450 nm:

(1) A1-H1 well negative control OD_450nmValue is added and the average value is the negative control OD_450nmAverage value;

(2) A2-H2 well positive control OD_450nmThe values are added and the average value is the positive control OD_450nmAverage value;

(3) blank OD of A9-H9 well_450nmValue addition and averaging, i.e. blank OD_450nmAverage value.

The average value of blank control OD450nm is subtracted from OD450nm values of 8 different dilutions of each triple duplicate of the reference vaccine and the vaccine to be detected, and the calculated values are input into Relpot 4.0 software to calculate the relative efficacy RP value and slope (slope score) of the vaccine to be detected.

Result judgment

The test effectiveness judgment comprises the following steps:

b: negative control OD_450nmThe average value should be less than 0.4;

The vaccine qualification criteria include:

a: the test effectiveness standard is met;

c: if the RP value of the vaccine to be detected is less than 1.0, the vaccine should be re-detected for 1 time; the RP value of the rechecking result is more than or equal to 1.0, and the relative efficacy of the vaccine to be detected is judged to be qualified; otherwise, judging that the relative efficacy of the vaccine to be detected is unqualified.

The data in Table 2 below were obtained from the OD450nm values measured for each well reaction solution.

TABLE 2 OD of vaccine ZYT-191 and vaccine ZYT-192 to be tested_450nmValue of

Control OD calculated from the data in Table 2 above_450nmAverage 1.128, negative control OD_450nmAverage of 0.083, blank control OD_450nmThe average value of (2) is 0.065, and the comparison is true, so that the test is effective.

The reference vaccine and the OD of the vaccine to be detected are_450nmValue minus blank OD_450nmMean values, input to Relpot 4.0 software for in vitro potency RP values and slope (slope score) calculations, are shown in Table 3 below and in FIGS. 7, 8, 9 and 10, where RP in FIG. 7: 1.27, RP in FIG. 9: 1.13.

TABLE 3 in vitro potency RP values and slopes (slope score) of the vaccines to be tested

And judging according to the result: RP 1.27 and slope 0.97 of the vaccine ZYT-191 to be detected; the RP value of the vaccine to be detected ZYT-192 is 1.13, and the slope is 0.99. And (3) detecting that the in vitro relative efficacy tests of the vaccines to be detected ZYT-191 and ZYT-192 are qualified by taking ZYTCK-1801 as a reference vaccine.

Example 6

This example deals with the antigen content and the response OD of the vaccine to be examined_450nmThe linear relationship of the values was evaluated.

The method is used for carrying out three-time parallel detection on the mycoplasma hyopneumoniae inactivated vaccine (DJ 166 strain) to be detected, and the dilutions (original times, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and 1: 128) of the vaccine to be detected are used as vertical coordinates and corresponding OD (OD) is obtained_450nmValue and OD_450nmLinear regression analysis with the logarithm of the value as abscissa, R²The values, RP values and slope results are shown in table 4 below.

TABLE 4 antigen content and corresponding OD of the vaccine to be tested_450nmComparison of values by linear regression analysis

Note: the 8 dilution points are fold, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1: 128.

Therefore, two batches of vaccine samples to be detected which are detected in three times of parallel detection show good linear regression R²All over 0.99, all RP values are more than 1.0, all slopes are over 0.97, and the method can be used for establishing a biological assay method.

Example 7

This example verifies the reliability of the double parallel line detection method.

According to the linear regression analysis result in the above example 6, the 6 points with the best linearity are taken for analysis, and a double parallel line detection method is established. According to the basic requirements of statistical analysis, the method for detecting the double parallel lines (6, 6) is characterized in that the linear regression is very significant (p is less than 0.01), and the three parameter tests deviating from the parallel curve, the quadratic curve and the reverse quadratic curve are not significant (p is more than 0.05). The 6.6-double-hole detection method F (1, 8) established in the application is 0.01-12.31, and F (1, 8) is 0.05-5.78, namely when F is more than 12.31, p is less than 0.01, and the method is very remarkable; when F is less than 5.78, p is more than 0.05 and is not significant. And when the above conditions are met, the verification is passed.

The inactivated vaccines of the vaccine samples to be tested in the efficacy test established by the invention (batch numbers ZYT-191 and ZYT-192) are subjected to two-batch three-person (A, B, C) test, the statistics of the detection results of the RP value and the slope value of the in-vitro relative efficacy test method of the vaccine disclosed by the invention are shown in table 5, the verification of the in-vitro relative efficacy test method of the vaccine disclosed by the invention is shown in table 6, and the analysis of the RP value and the slope value of the efficacy test in the same batch is shown in table 7.

TABLE 5 vaccine in vitro relative potency test method RP value and slope value detection results

Table 6 verification of the vaccine in vitro relative potency test method

TABLE 7 analysis of RP and slope values for in vitro relative potency assay of vaccines in the same batch

2 batches of 3-person detection of the vaccine in vitro relative efficacy test of the invention are verified, and the RP value and the slope value of the vaccine in the same batch are 1.38 percent and 1.02 percent of variation coefficient (CV%) of the ZYT-191 RP value and the slope in 6 detection results respectively; the RP value and the coefficient of variation (CV%) of the slope of the vaccine to be detected ZYT-192 are respectively 3.67% and 1.01%. The coefficient of variation (CV%) is less than 10%, which shows that the test result is stable and has good repeatability. The RP values and slope values for all batches were 1.27 and 1.00 maximum, 110 and 0.97 minimum, respectively, with an average of 1.20 and 0.99, respectively, and a standard deviation of 6.3% and 1.1%, respectively.

Example 8 evaluation of effects of application

5 batches of Mycoplasma hyopneumoniae inactivated vaccine (DJ 166) products to be detected, which are provided with batch numbers of ZYT-192-1, ZYT-192-2, ZYT-192-3, ZYT-192-4 and ZYT-192-5, are detected by the existing method, and are simultaneously compared with the results of the in vivo relative efficacy test of the animals (see Table 8).

TABLE 8 statistical analysis of the correlation of in vitro relative potency assay with in vivo vaccine in animals

As can be seen from Table 8, the standard deviation SD and the coefficient of variation CV of the RP value and the slope value of the in vitro vaccine relative efficacy test of 5 batches of vaccine products to be tested are both obviously lower than those of the in vivo vaccine relative efficacy test of animals, and the coefficient of variation (CV%) of the in vivo vaccine relative efficacy test of the animals is 4.55% and is far higher than that of the in vitro vaccine relative efficacy test, which indicates that the in vitro relative efficacy test of the invention is more stable and has better repeatability.

Example 9

5 batches of vaccine products (ZYT-192-1, ZYT-192-2, ZYT-192-3, ZYT-192-4 and ZYT-192-5) to be tested prepared in the laboratory are stored at 2-8 ℃ for 3, 6, 9, 12, 18 and 21 months, respectively sampled to carry out an in vitro relative efficacy test and an in vivo animal vaccine relative efficacy test, and are subjected to statistical analysis and comparison, and the results are shown in Table 9.

TABLE 9 comparative analysis of the results of the in vitro relative potency assay of the sandwich ELISA vaccines with the relative potency assay of the vaccines in animals

As shown by the results in the table 9, 6 sampling indirect competitive inhibition ELISA relative effectiveness tests and animal in-vivo vaccine relative effectiveness tests of the 5 batches of vaccines to be detected are synchronously detected by taking an RP value not less than 1.0 and a slope not less than 0.97 as determination standards, the results are shown in the table 9 below, standard deviation SD and coefficient of variation CV values of the RP value and the slope value of the in-vitro vaccine relative effectiveness tests are obviously lower than those of the animal in-vivo vaccine relative effectiveness tests, the two test results are consistent, and all the 7 sampling 2 test methods of the 5 batches of vaccines to be detected are qualified. Compared with the traditional animal in-vivo vaccine relative efficacy test, 2 months are needed from the beginning of each vaccine sampling to the end of the test, the sandwich ELISA in-vitro vaccine relative efficacy test method can be completed in about 6 hours, and the method has the advantages of high efficiency, trouble saving and labor saving.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. A method for testing the in vitro efficacy of a mycoplasma hyopneumoniae inactivated vaccine based on an ELISA method is characterized by comprising the following steps:

2. The method for testing the in vitro efficacy of the inactivated mycoplasma hyopneumoniae vaccine according to claim 1, wherein in the step (1), the reference vaccine is a vaccine with minimum protective amount of antigen in a qualified challenge test.

3. The method for testing the in vitro efficacy of the inactivated mycoplasma hyopneumoniae vaccine according to claim 2 based on the ELISA method, wherein in the step (2):

controlling the dilution concentration of the mycoplasma hyopneumoniae to be 20 +/-5 mu g/ml, controlling the liquid adding amount to be 100 mu l/hole, and controlling the temperature of the step of treating the coating to be 4 ℃ for coating for 16-18 hours;

4. The method for testing the in vitro efficacy of the inactivated mycoplasma hyopneumoniae vaccine based on the ELISA method of claim 3, wherein in the step (3), the adding amount of the vaccine to be tested and the reference vaccine is 200 μ l/well.

5. The method for testing the in vitro efficacy of the inactivated mycoplasma hyopneumoniae vaccine according to claim 4 based on the ELISA method, wherein in the step (3):

the blank was PBS.

6. The method for testing the in vitro efficacy of the inactivated mycoplasma hyopneumoniae vaccine based on the ELISA method according to claim 5, wherein the step (3) further comprises the step of performing gradient dilution on the reference vaccine and/or the vaccine to be tested, and then adding the reference vaccine and/or the vaccine to be tested into the ELISA plate for testing at different concentrations.

7. The method for testing the in vitro efficacy of the inactivated mycoplasma hyopneumoniae vaccine based on the ELISA method according to claim 6, wherein in the step (3), the dilution gradient is an original-fold gradient and the dilution gradient is 1: 2. 1: 4. 1: 8. 1: 16. 1: 32. 1:64 and 1: serial dilution of 128 by 2 fold.

8. The method for testing the in vitro efficacy of the inactivated mycoplasma hyopneumoniae vaccine according to claim 7 based on the ELISA method, wherein in the step (4):

the liquid adding amount of the color development liquid is 100 ul/hole;

the amount of the stop solution added is 50ul per hole.

9. The method for testing the in vitro efficacy of the inactivated mycoplasma hyopneumoniae vaccine according to any one of claims 1-8 based on the ELISA method, wherein in the step (5), the test validity determination comprises:

b: negative control OD_450nmThe average value should be less than 0.4;

10. The method for testing the in vitro efficacy of the inactivated mycoplasma hyopneumoniae vaccine based on the ELISA method according to claim 9, wherein in the step (5), the qualified judgment criteria of the vaccine are as follows:

a: the test effectiveness standard is met;