CN112505333A

CN112505333A - Trichina competition ELISA antibody detection kit and detection method thereof

Info

Publication number: CN112505333A
Application number: CN202011376831.0A
Authority: CN
Inventors: 刘晓雷; 刘明远; 白雪; 杨勇; 王学林; 丁静; 刘琰
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2021-03-16
Anticipated expiration: 2040-11-30
Also published as: CN112505333B

Abstract

A trichina competition ELISA antibody detection kit and a detection method thereof belong to the technical field of serological immunoassay. In order to more stably and accurately detect the trichina infection of multiple hosts, the invention provides a trichina competition ELISA antibody detection kit, which comprises: the bottom plate is coated with a 96-hole reaction plate of trichina Ts-WN10 recombinant antigen, a biotin-labeled monoclonal antibody Ts-WN10-1H9 solution, a horseradish catalase HRP-labeled avidin solution, a washing solution, a TMB chromogenic substrate, a stop solution and negative quality control; wherein the negative quality control is a biotin-labeled monoclonal antibody Ts-WN10-1H9 solution with the concentration of 1 mug/mL. The kit prepared by the invention has the characteristics of strong specificity, high sensitivity and the like, and can be used for detecting the antibody of the multi-host trichinosis such as pig/mouse/human and the like.

Description

Trichina competition ELISA antibody detection kit and detection method thereof

Technical Field

The invention belongs to the technical field of serological immunodetection, and particularly relates to a trichina competition ELISA antibody detection kit and a detection method thereof.

Background

Trichinosis is a common disease of people and animals with serious harm, which not only can cause huge economic loss to the animal husbandry production, but also can pose a huge threat to the human health, and the trichinosis is mainly developed by eating or semi-eating meat (mainly pork) containing trichinosis by humans or animals.

For the examination of trichinosis of slaughtered animals, the examination method of the international animal health Organization (OIE) rules is a microscopic examination method and a sample collection digestion method, and at present, the two methods are also used in China. However, both methods have certain disadvantages, and the microscopic examination method is time-consuming, labor-consuming and has poor sensitivity, and the sensitivity is that the density of the worm bodies in the meat can be detected when 3 worm bodies per gram. Although the sample collection digestion method can greatly improve the detection rate to 1 worm per gram of meat, the method is still very complicated, and the digestion method is still required to be adopted for carrying out head-by-head detection on a positive group when a positive sample is found. From the perspective of sensitivity, meat missed by microscopy and sample collection digestion both present safety hazards and can cause human infection (human infection can be caused by intake of 75 worms). Scholars at home and abroad make a great deal of research on the trichinella serological detection method. At present, the trichina myolarva excretory secretion ES antigen is a standard antigen recommended by OIE and the international trichina committee for serological antibody detection. The sensitivity of the indirect ELISA detection method based on the ES antigen reaches 0.01 strip worm body per gram of meat, and the indirect ELISA detection method can be used for diagnosing trichina infection. However, ES antigen components are complex, preparation is cumbersome and strictly dependent on living animals, production cycle is long, quality control in and between batches is very difficult, and cross reaction and the like are problematic, thereby hindering practical application thereof.

Human infection with trichina is mainly through the live or semi-live feeding of meat from captive animals (mainly pigs) containing infective larvae, and therefore monitoring the level of trichina infection in herds is important for controlling human infection. The circulating infections of trichinella in captive animals are mainly caused by feeding activities of meat residues and by occasional predatory activities of rodents. Therefore, it is important to prevent and control infection of each host in rodent-swinery-human food chain. However, the indirect ELISA kit based on the ES antigen requires different kit components to be prepared according to the host, and thus, it is impossible to detect a plurality of hosts in a universal manner by one method. At present, a multi-host universal trichina antibody detection kit does not exist.

In addition to ES antigen, research has shown that immunological screening is carried out on trichina cDNA expression library 6h after infection, so as to obtain an antigen protein with high abundance and strong reactogenicity, and the antigen protein is named WN10 and codes cysteine protease inhibitor. WN10 gene accession number: EU263325, WN10 protein accession no: ABY 60755. In addition, researchers found that further immunoblotting and indirect ELSIA indicated that prokaryotic expression of recombinant Ts-WN10 antigen could be recognized by pig trichina infection in 17, 25 and 60 days of serum, indicating that Ts-WN10 was an ideal candidate antigen for trichina serological detection, and could be used to improve the serological detection method.

The solid phase competitive ELISA detection technology (cELISA) is an immunological detection technology developed on the basis of monoclonal antibody technology. In the conventional solid phase competition ELISA, an antigen is coated on a 96-well microplate, serum to be detected and a monoclonal antibody compete to be combined with the antigen at the bottom of the microplate, and a secondary antibody of an anti-monoclonal antibody marked by HRP is used for signal detection. In recent years, antibody labeling technology has been greatly developed, and single antibodies can be labeled with biotin, and signal detection can be performed by using a biotin-avidin system, so that the limitation of monoclonal antibody species can be eliminated. The ELISA methods for detecting trichinosis antibodies reported at present comprise indirect ELISA and a competitive ELISA, and the ES antigen of the secretion of the muscle larvae is adopted.

Disclosure of Invention

In order to more stably and accurately detect the trichina infection of multiple hosts, the invention provides a trichina competition ELISA antibody detection kit, which comprises:

the bottom plate is coated with a 96-hole reaction plate with trichina Ts-WN10 recombinant antigen,

diluting to 2 mug/mL of biotin-labeled monoclonal antibody Ts-WN10-1H9 solution by using 0.9% NaCL solution,

horse radish catalase HRP marked avidin solution,

the washing liquid is used for washing the surface of the workpiece,

a TMB color-developing substrate is used,

the stop solution is added to the reaction kettle,

and negative quality control;

wherein the negative quality control is a biotin-labeled monoclonal antibody Ts-WN10-1H9 solution with the concentration of 1 mug/mL.

Further limited, the monoclonal antibody Ts-WN10-1H9 is prepared from hybridoma cell strain WN10-1H9, and the microbial collection number of the hybridoma cell strain WN10-1H9 is CGMCC No. 18316.

Further defined, the horseradish catalase HRP-labeled avidin solution is ebioscienctmavidin-HRP.

Further defined, the TMB chromogenic substrate is

TMB single-component color developing solution.

Further defined, the stop solution is H with the concentration of 2M₂SO₄And (3) solution.

In one embodiment of the invention, the preparation steps of the trichina Ts-WN10 recombinant antigen are as follows:

adding recombinant bacteria BL21(DE3) -pET28a-Ts-WN10 into an LB culture medium, performing shake culture at 37 ℃ until OD600nm is 0.5-1, adding IPTG (isopropyl-beta-thiogalactoside) until the final concentration is 1mmol/L, and inducing at 37 ℃ for 6-8 hours;

centrifuging the induced bacterial liquid, then resuspending the bacterial liquid by using 30mL of 20mM Tris-HCL heavy suspension buffer solution, ultrasonically crushing the bacterial liquid on ice, and taking a precipitate after centrifugation;

resuspending with 30mL of precooled inclusion body washing solution, ultrasonically crushing on ice, and centrifuging to obtain precipitate;

fourthly, repeating the operation for at least 2 times;

fifthly, resuspending the sediment obtained by centrifugation with 30mL of precooled PBS washing solution containing urea, ultrasonically crushing the sediment on ice, and centrifugally obtaining the sediment;

sixthly, repeating the step five for at least 1 time;

seventhly, suspending the centrifugally harvested sediment by using a 5mLBinding buffer, dissolving the sediment at 4 ℃ overnight, centrifugally collecting supernatant, and filtering the supernatant to prepare for loading;

the content of the column renaturation process of the AKTA protein purifier is referred to GE healthcare handbook Purifying charling Proteins 77-79, a Purifying column adopts HisTrapHP 1mL, the column renaturation time after sample injection and loading is 2h, and 100 percent of the column renaturation time is replaced to solution of reforming buffer and then replaced to Elutionbuffer to elute target protein.

In one embodiment of the invention, the biotin-labeled monoclonal antibody Ts-WN10-1H9 is prepared by the following steps:

step 1, taking the abdominal cavity of a healthy BALB/c mouse aged 12 weeks, and injecting paraffin oil into the abdominal cavity, wherein each mouse is 0.5 mL;

step 2, intraperitoneal injection 1X 10 after 1 week ⁶2, extracting ascites when the abdominal cavity of a mouse is extremely swollen 7-10 days later than hybridoma cells WN10-1H9, wherein the microbial preservation number of the hybridoma cells WN10-1H9 is CGMCC No. 18316;

and 3, centrifuging the extracted ascites at 10000rpm for 10min to remove upper-layer grease and sediment, purifying the supernatant to obtain a monoclonal Antibody Ts-WN10-1H9, wherein the content of an Antibody Purification process of the AKTA Protein purifier is referred to GE healthcare manual Antibody Purification 125, and a Purification column adopts HiTrap Protein G HP 1 mL.

And 4, labeling the monoclonal antibody Ts-WN10-1H9 obtained in the step 3 with biotin by adopting a method of covalently combining protein epsilon-amino with acylated biotin.

In one embodiment of the invention, the 96-well reaction plate with the trichina Ts-WN10 recombinant antigen coated on the bottom of the plate is prepared by the following steps:

step one, diluting a trichina Ts-WN10 recombinant antigen to 3 mu g/mL by using a CBS buffer solution, coating 100 mu L of the diluted recombinant antigen in each hole of a 96-hole reaction plate, and coating for 16h at 4 ℃;

step two, discarding redundant coating liquid from the coated reaction plate, adding 300 mu L PBST cleaning solution into each hole, and washing for 3 times, 1min each time;

step three, adding 300 mu L of PBST blocking solution containing 1% BSA into each hole, and blocking for 1h at 37 ℃;

step four, discarding the confining liquid, adding 300 μ L PBST washing liquid into each hole, and washing for 3 times, each time for 1 min.

The invention also provides a method for detecting the trichina competition ELISA antibody detection kit, which comprises the following steps:

taking 50uL of serum to be detected and 50uL of monoclonal antibody Ts-WN10-1H9 solution with the concentration of 2 mug/mL, uniformly mixing, adding the mixture into a 96-well reaction plate coated with trichina Ts-WN10 recombinant antigen at the bottom of the plate, reacting for 1H at 37 ℃, and parallelly establishing negative quality control contrast in the plate;

after washing 3 times with PBST wash solution, 100uL of HRP-labeled avidin diluted with 1% BSA in PBST was added to each well and reacted at 37 ℃ for 30 min;

washing with PBST washing solution for 3 times;

adding 100uLTMB chromogenic substrate into each well, developing at 37 ℃ for 8min, stopping, reading the absorbance value OD at 450nm of the negative quality control_NCAnd the absorbance value OD of the serum to be detected at 450nm_sample；

Then, the inhibition ratio PI% (1-OD) was calculated_sample/OD_NC)％。

Further limited, the method for detecting the trichina competition ELISA antibody detection kit comprises the following steps:

for the pig serum sample, when PI% of the sample to be detected is more than or equal to 52%, the sample is judged to be positive;

when the PI% of the sample to be detected is less than 52%, judging the sample to be negative; simultaneous OD_NCJudging that the experiment is established if the value is more than or equal to 1.00;

for a mouse serum sample, judging the sample to be tested to be positive when PI% is more than or equal to 39%;

when the PI% of the sample to be detected is less than 39%, judging the sample to be detected to be negative; simultaneous OD_NCJudging that the experiment is established if the value is more than or equal to 1.00;

for a human serum sample, judging the human serum sample to be positive when PI% of the sample to be detected is not less than 41.12%;

when the PI% of the sample to be detected is less than 41.12%, determining that the sample is negative; simultaneous OD_NCAnd if the value is more than or equal to 1.00, judging that the experiment is established.

Advantageous effects

The prokaryotic expression recombinant Ts-WN10 antigen and the corresponding monoclonal antibody Ts-WN10-1H9 have the advantages of strong combination specificity, good purity, strong repeatability, convenient quality control and mass production. Therefore, the invention establishes the trichinosis antibody diagnosis ELISA based on the recombinant Ts-WN10 antigen and the monoclonal antibody, and simultaneously adopts a biotin-avidin system and a competition ELISA method to define different critical values aiming at the serum of different hosts so as to assemble a multi-host universal detection kit.

The ELISA kit prepared by the invention has the advantages of controllable quality, simple operation, easy result interpretation, multi-host universality and the like, does not have cross reaction with other parasite positive serum during detection, and has high specificity; and has the characteristics of long-term stability, strong practicability, easy storage and market development value. Has wide popularization space and has good market prospect.

Drawings

FIG. 1 is a schematic diagram of the detection principle of a trichina competition ELISA antibody detection kit;

FIG. 2 shows the results of the measurement of Cut-off values: a is the result of PI% statistical analysis of 270 healthy pig sera; b is the result of PI% statistical analysis of 30 sera of healthy mice; c is the result of PI% statistical analysis of 100 parts of healthy human serum and 47 parts of trichina patient serum; d is the result of ROC statistical analysis of PI% of 100 parts of healthy human serum and 47 parts of trichina patient serum;

FIG. 3 detection of antibody levels in pigs at different infection doses and times;

FIG. 4. competitive ELISA kit specific detection (human serum);

FIG. 5 detection of sera from mice infected with 200 ML/60 dpi dose of different species/genotype of Trichinella spiralis.

Detailed Description

In the aspect of trichina detection, a solid phase competition ELISA kit for detecting an anti-trichina antibody by using a monoclonal antibody and a single recombinant antigen as diagnostic antigens does not exist, and the invention researches the detection. The invention adopts a biotin-labeled monoclonal antibody Ts-WN10-1H9, and establishes a solid phase competition ELISA kit based on recombinant antigens of the monoclonal antibodies Ts-WN10-1H9 and Ts-WN10 by selecting various solid phase materials and optimizing reaction systems and reaction conditions. Coating the recombinant antigen Ts-WN10 at the bottom of a 96-well reaction plate, labeling biotin with a monoclonal antibody Ts-WN10-1H9, and assembling the trichinosis competition ELISA detection kit together with horseradish catalase HRP-labeled avidin, PBST washing liquid, TMB substrate, stop solution and negative quality control. The detection principle of the kit is shown in figure 1, a biotin-labeled monoclonal antibody Ts-WN10-1H9 competes with an antibody in serum to be detected to combine with a Ts-WN10 recombinant antigen in a 96-well reaction plate, and is trapped by a coating antigen at the bottom of the reaction plate; biotin marked on the monoclonal antibody can be specifically combined with avidin marked by HRP; HRP labeled on avidin catalyzes the color development of TMB substrate. After the color development was terminated, the absorbance value at 450nm was read. Reading the absorbance value OD of 450nm of negative quality control_NCAbsorbance value OD of 450nm of serum to be detected_sampleThe inhibition ratio PI% (1-OD) was calculated_sample/OD_NC) % of the total weight of the composition. For the pig serum sample, when PI% of the sample to be detected is more than or equal to 52%, the sample is judged to be positive; when the PI% of the sample to be detected is less than 52%, judging the sample to be negative; simultaneous OD_NCAnd if the value is more than or equal to 1.00, judging that the experiment is established. For a mouse serum sample, judging the sample to be tested to be positive when PI% is more than or equal to 39%; when the PI% of the sample to be detected is less than 39%, judging the sample to be detected to be negative; simultaneous OD_NCAnd if the value is more than or equal to 1.00, judging that the experiment is established. For a human serum sample, judging the human serum sample to be positive when PI% of the sample to be detected is not less than 41.12%; when the PI% of the sample to be tested is less than 41.12%,judging the test result to be negative; simultaneous OD_NCAnd if the value is more than or equal to 1.00, judging that the experiment is established.

The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings, and the present invention is not limited to the following embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

Main experimental materials and sources:

ni purification column histrap ph was purchased from GE corporation, usa; a solvent TMB substrate Solution was obtained from TIANGEN; horse Radish Peroxidase (HRP) -labeled avidin was purchased from seimer feishel scientific.

Experimental animals:

12 week old BALB/c mice were provided by Vinca hundred million laboratory animal technology, Inc. New Zealand rabbits are offered by Vinca hundred million laboratory animal technology, Inc. Other reagents or instrumentation are commercially available without specific reference.

Example 1. trichina competition ELISA antibody detection kit.

The trichina competition ELISA antibody detection kit comprises: the bottom plate is coated with a 96-hole reaction plate of trichina Ts-WN10 recombinant antigen, and is diluted to 2 mu g/mL biotin-labeled monoclonal antibody Ts-WN10-1H9 solution, horseradish catalase HRP-labeled avidin solution, washing liquid, TMB chromogenic substrate, stop solution and negative quality control by 0.9% NaCL solution; wherein the negative quality control is a biotin-labeled monoclonal antibody Ts-WN10-1H9 solution with the concentration of 1 mug/mL; the monoclonal antibody Ts-WN10-1H9 is prepared from a hybridoma cell strain WN10-1H9, the microbial preservation number of the hybridoma cell strain WN10-1H9 is CGMCC No.18316, the cell strain is preserved in the China general microbiological culture Collection center in 2019, 8 and 15 months, and the preservation address isThe institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing; the horseradish catalase HRP marked avidin solution is eBioscenCETMavidin-HRP; the TMB chromogenic substrate is

TMB single-component color developing liquid; the stop solution is H with the concentration of 2M₂SO₄And (3) solution.

1) The preparation method of the trichina Ts-WN10 recombinant antigen comprises the following steps:

adding 1mL of recombinant bacterium BL21(DE3) -pET28a-Ts-WN10 into 100mL of LB culture medium, performing shake culture at 37 ℃ until OD600nm is 0.5-1, adding IPTG (isopropyl-beta-thiogalactoside) until the final concentration is 1mmol/L, and inducing at 37 ℃ for 6-8 h;

secondly, centrifuging the induced bacterial liquid, then resuspending the bacterial liquid by using 30mL of resuspension buffer solution (20mM Tris-HCL PH8.0), ultrasonically crushing the bacterial liquid on ice, and taking a precipitate after centrifugation;

③ resuspending the mixture with 30mL of precooled inclusion body washing solution (2M urea, 20mM Tris-HCL, 0.3M NaCL PH8.0), ultrasonically crushing the mixture on ice, and centrifuging the crushed mixture to obtain a precipitate;

fourthly, repeating the operation for at least 2 times;

fifthly, resuspending the sediment obtained by centrifugation with 30mL of precooled PBS washing solution containing urea (0.01M PBS containing 4M urea), ultrasonically crushing on ice, and centrifugally obtaining the sediment;

sixthly, repeating the step five for at least 1 time;

seventhly, centrifugally harvesting and depositing, re-suspending the sediment by using 5mLBinding buffer (8M urea, 20mMTris-HCL, 0.3M NaCL and 5mM imidazole PH8.0), dissolving the sediment at 4 ℃ for one night, centrifugally collecting supernatant, and filtering the supernatant to prepare the supernatant;

the content of the column renaturation process of the AKTA protein purification instrument is referred to GE healthcare handbook Purifying and charling Proteins 77-79, a purification column adopts 1mL of HisTrapHP, the column renaturation time after injection and loading is 2h, 100 percent of the column renaturation process is replaced to solution of Refoldingbuffer (20mM Tris-HCL, 0.3M NaCL, 5mM imidazole and 1mM 2-mercaptoethanol PH8.0), and then the column renaturation process is replaced to solution of elusion buffer (20mM Tris-HCL, 0.3M NaCL and 500mM imidazole PH8.0) to elute target Proteins.

2) The preparation method of the biotin-labeled monoclonal antibody Ts-WN10-1H9 comprises the following steps:

And 4, adopting a method of covalent bonding of protein epsilon-amino and acylated biotin to label the monoclonal antibody Ts-WN10-1H9 obtained in the step 3 with biotin, wherein the biotin is a conventional commercial reagent.

3) The preparation method of the 96-well reaction plate with the trichina Ts-WN10 recombinant antigen coated at the bottom of the plate comprises the following steps:

Example 2, detection method of trichina competition ELISA antibody detection kit.

1) The detection method of the serum sample comprises the following steps:

uniformly mixing 50 mu L of biotin-labeled monoclonal antibody Ts-WN10-1H9 solution and 50 mu L of serum to be detected in an EP tube, adding the mixture into a 96-well reaction plate pre-coated with Ts-WN10 recombinant antigen, carrying out competitive reaction for 1H at 37 ℃, and establishing negative quality control (1 mu g/mL, 100 mu L) in the plate;

discarding the reaction solution, adding 300 μ of LPBST washing solution into each hole, and washing for 1min for 3 times;

HRP-labeled avidin was diluted 1:400 with 1% BSA in PBST, 100. mu.L of the diluted avidin was added to each reaction well, and the mixture was incubated at 37 ℃ for 30 min;

add 100u LTMB substrate into each hole, develop color for 8min at 37 deg.C, then add 50u L stop solution into each hole to stop the color reaction, read the absorbance value at 450 nm.

The result judgment should be carried out within 30min, and the absorbance value OD at 450nm of the negative quality control should be read_NCAbsorbance value OD of 450nm of serum to be detected_sampleThe inhibition ratio PI% (1-OD) was calculated_sample/OD_NC) Percent, for a pig serum sample, when PI% of a sample to be detected is more than or equal to 52%, the sample is judged to be positive; when the PI% of the sample to be detected is less than 52%, judging the sample to be negative; simultaneous OD_NCJudging that the experiment is established if the value is more than or equal to 1.00; for a mouse serum sample, judging the sample to be tested to be positive when PI% is more than or equal to 39%; when the PI% of the sample to be detected is less than 39%, judging the sample to be detected to be negative; simultaneous OD_NCJudging that the experiment is established if the value is more than or equal to 1.00; for a human serum sample, judging the human serum sample to be positive when PI% of the sample to be detected is not less than 41.12%; when the PI% of the sample to be detected is less than 41.12%, determining that the sample is negative; simultaneous OD_NCAnd if the value is more than or equal to 1.00, judging that the experiment is established.

2) Determination of cut-off value.

The kit is used for respectively measuring 270 parts of serum of healthy pigs, 30 parts of serum of healthy mice, 100 parts of serum of healthy people and 47 parts of serum of trichinosis patients, and reading the negative quality control OD (absorbance) value at 450nm_NCAbsorbance value OD of 450nm of serum to be detected_sampleThe inhibition ratio PI% (1-OD) was calculated_sample/OD_NC) % of the total weight of the composition. For the pig serum samples, PI% of 270 healthy pig sera is subjected to statistical analysis, the average value and the standard deviation are calculated, the sum of the average value and 2 times of the standard deviation is taken as the cut-off value, and the cut-of value is obtained through calculationThe f value is 52%, as shown in A in FIG. 2, when the PI% of the sample to be tested is more than or equal to 52%, the sample is judged to be positive; when the PI% of the sample to be detected is less than 52%, judging the sample to be negative; simultaneous OD_NCAnd if the value is more than or equal to 1.00, judging that the experiment is established. For a mouse serum sample, carrying out statistical analysis on PI% of 30 parts of healthy mouse serum, calculating an average value and a standard deviation, taking the average value plus 2 times of the standard deviation as a cut-off value, and calculating to obtain a cut-off value of 39%, wherein as shown in B in figure 2, when the PI% of a sample to be detected is more than or equal to 39%, the sample is judged to be positive; when the PI% of the sample to be detected is less than 39%, judging the sample to be detected to be negative; simultaneous OD_NCAnd if the value is more than or equal to 1.00, judging that the experiment is established. For human serum samples, ROC statistical analysis was performed on PI% of 100 parts of healthy human serum and 47 parts of serum from trichina patients, and the cut-off value of the test strip was calculated to be 41.12% as shown in C and D in fig. 2. When the PI% of the sample to be detected is not less than 41.12%, judging the sample to be detected to be positive; when the PI% of the sample to be detected is less than 41.12%, determining that the sample is negative; simultaneous OD_NCAnd if the value is more than or equal to 1.00, judging that the experiment is established.

3) Sensitivity, specificity and stability of trichina competition ELISA antibody detection kit

Testing the sensitivity:

for porcine serum samples: the trichina competitive ELISA kit is used for detecting the trichina antibody in the pig serum under different infection conditions, and the detection result is compared with a commercial enzyme-linked immunosorbent antibody detection method ES ELISA (Qiagen, swing). Ensure that the kits used in the assay are from the same batch. The commercial ES ELISA kit is a product of the same batch, and the detection steps and the result judgment standard are carried out according to the kit instruction.

The competitive ELISA kit and the ES ELISA respectively detect 50 parts of pig infected trichina serum, and the result shows that 48 parts of serum is positive in ES ELISA detection and 47 parts of serum is positive in competitive ELISA detection, wherein 2 parts of serum in ES ELISA is negative in the competitive ELISA detection, trichina is negative in the competitive ELISA detection, 1 part of serum is positive in ES ELISA and negative in the competitive ELISA. 60 negative sera, ES ELISA all negative, competition ELISA60 negative. The two methods have strong consistency and goodness of fit in the aspect of detecting the trichinosis of animals; the differences in sensitivity and specificity of both the competition ELISA kit and the ES ELISA were not statistically significant. The sensitivity and specificity of the invention are equivalent to those of the swine trichinosis antibody detection method (ES ELISA) recommended by OIE, and the invention has the advantages of multi-host general type, controllable quality and the like which are not possessed by ES ELISA, has wide popularization space and has great market prospect.

For human serum samples: 47 parts of trichina patient serum from Yunnan and 100 parts of healthy Human serum are detected by using a trichina competition ELISA kit, and the detection result is compared with a commercial enzyme-linked immunosorbent antibody detection method ES ELISA (ABCam, Human). Ensure that the kits used in the assay are from the same batch. The commercial ES ELISA kit is a product of the same batch, and the detection steps and the result judgment standard are carried out according to the kit instruction.

The competitive ELISA kit and the ES ELISA respectively detect 47 serums of the human infected trichinella, and the result shows that 47 serums of the ES ELISA kit are positive, and 47 serums of the ES ELISA kit are positive. 100 negative sera, ES ELISA all negative, and competition ELISA 100 negative. By ROC analysis, when the threshold value of the established competition ELISA method is 41.12%, the specificity of the established competition ELISA method for the samples is 100%, the sensitivity is 100%, and the AUC is 1.000, which indicates that the competition ELISA can well distinguish the negative/positive serum of the human trichina. The ES ELISA and the competitive ELISA have strong coincidence rate and goodness of fit in the aspect of detecting human trichinosis, and have no statistical significance in the differences of sensitivity and specificity. The sensitivity and specificity of the invention are equivalent to those of the human trichinosis antibody detection method (ES ELISA) recommended by OIE, and the invention has the advantages of multi-host general type, controllable quality and the like which are not possessed by ES ELISA, has wide popularization space and has great market prospect.

② the specificity of the trichina competition ELISA antibody detection kit:

for the swine serum samples, swine positive sera of other parasitic infections and swine sera immunized with conventional vaccines were tested. The swine Asian tapeworm, toxoplasma gondii, taenia solium and clonorchis sinensis positive swine serum and the swine serum (the trichina aggregate digestion detection result of 6 pigs is negative) which is subjected to conventional vaccine immunization (comprising pseudorabies vaccine, Langerhans vaccine, circovirus vaccine, classical swine fever vaccine and foot and mouth disease vaccine) in 6-head large-scale farms are subjected to specificity analysis. The trichina competition ELISA kit disclosed by the invention is negative in detection of the pig serum, but the detection result of the commercialized ES ELISA kit shows that the trichina competition ELISA kit has cross reaction with the clonorchis sinensis pig positive serum, namely the trichina competition ELISA antibody detection kit disclosed by the invention does not have cross reaction with other parasites and has higher specificity than the ES ELISA recommended by OIE.

For human serum samples, human positive sera for other parasitic infections and human serum for enterovirus infections were tested. The experimental results are shown in FIG. 4, which includes 2 parts of human encystia and 8 parts of human serum positive for clonorchis sinensis, 3 parts of human serum for hand-foot-and-mouth disease, 3 parts of serum from a patient infected with Coxsackie virus and 20 parts of serum from a patient infected with other enteroviruses. The trichina competition ELISA kit disclosed by the invention is negative in detection of the human serum, but the detection result of the commercialized ES ELISA kit (ABCam) shows that the trichina competition ELISA kit has cross reaction with 2 parts of clonorchis sinensis human positive serum, and the 2 parts of human serum which has cross reaction with ES antigen has cross reaction bands through western-bolt identification. Namely, the trichina competition ELISA antibody detection kit does not have cross reaction with other parasite and enterovirus infection samples, and has higher specificity than the ES ELISA recommended by OIE.

③ the detection of the antibody level of the trichina infected by pigs at different dosages and time points:

the experimental results of the detection of the anti-trichina antibodies of the pig sera at different time points after the trichina infection are shown in fig. 3, the number of the diaphragma larvae is 8.7-11.6lpg in each gram of diaphragma when the pig sera are subjected to moderate infection (600/pig) by a digestion method, the trichina infection can be detected by a commercial ES ELISA kit (Qiagen, swing) in 30 days, and the trichina infection can be detected by a competitive ELISA kit in 35 days. At the time of mild infection (400 pieces/piece), the number of larvae per gram of diaphragm muscle is detected to be 0.96-0.45lpg by a digestion method, the trichinella infection can be detected by a commercial ES ELISA kit (Qiagen, swing) for 45 days, and the trichinella infection can be detected by a competitive ELISA kit for 45-60 days. At the time of low infection (200 pieces/piece), the digestion method detects that the number of larvae per gram of diaphragm muscle is 0.07-0.44lpg, the infection of trichinella spiralis can be detected in 60-90 days by the ES ELISA, and the infection of trichinella spiralis can be detected in 60-90 days by the competitive ELISA kit. Compared with ES ELISA, the detection sensitivity of the competition ELISA based on the Ts-WN10 recombinant antigen and the Ts-WN10-1H9 monoclonal antibody is equivalent to that of a commercial ES ELISA kit.

Fourthly, detecting the antibody of the trichina infected by the mice with different species/genotypes:

mice were infected with 17 isolates of trichina, 200 muscle larvae per mouse, and serum antibodies were detected on day 60 of infection. The 17 isolates included: t1(ISS534), T1(ISS4), T1(ISS533), T2(ISS70), T3(ISS235), T4(ISS141), T4(ISS13), T4(ISS470), T5(ISS35), T5(ISS415), T6(ISS34), T7(ISS37), T8(ISS124), T9(ISS408), T10(ISS572), T11(ISS1029) and T12(ISS 1826). The results of the experiment are shown in a in fig. 5, indicating that T1(ISS534), T1(ISS4), T1(ISS33), T7(ISS37) and T8(ISS124) were strongly positive, and T6(ISS34) and T12(ISS1826) were also positive, but close to the critical value of 39%, as detected by the competition ELISA kit. Meanwhile, through the western-blot identification of the crude worm proteins of 17 isolates, the result is shown as B in figure 5, and the monoclonal antibody Ts-WN10-1H9 can perform specific reaction with the crude worm proteins of the isolates corresponding to the positive serum. Therefore, the competitive ELISA based on the Ts-WN10 recombinant antigen and the Ts-WN10-1H9 monoclonal antibody can be applied to the antibody detection of multiple species/genotype trichina infection, has wide popularization space and has great market prospect.

Claims

1. A trichina competition ELISA antibody detection kit is characterized in that the trichina competition ELISA antibody detection kit contains:

horse radish catalase HRP marked avidin solution,

the washing liquid is used for washing the surface of the workpiece,

a TMB color-developing substrate is used,

the stop solution is added to the reaction kettle,

and negative quality control;

2. The trichina competition ELISA antibody detection kit of claim 1, wherein the monoclonal antibody Ts-WN10-1H9 is prepared from hybridoma cell strain WN10-1H9, and the microbial deposit number of hybridoma cell strain WN10-1H9 is CGMCC No. 18316.

3. The trichina competition ELISA antibody detection kit of claim 1, wherein the horseradish catalase HRP labeled Avidin solution is ebioscience (tm) Avidin-HRP.

4. The trichina competition ELISA antibody detection kit of claim 1, wherein the TMB chromogenic substrate is TMB

TMB single-component color developing solution.

5. The trichina competition ELISA antibody detection kit of claim 1, wherein the stop solution is H with a concentration of 2M₂SO₄And (3) solution.

6. The trichina competition ELISA antibody detection kit of any one of claims 1 to 5, wherein the trichina Ts-WN10 recombinant antigen is prepared by the steps of:

fourthly, repeating the operation for at least 2 times;

sixthly, repeating the step five for at least 1 time;

seventhly, suspending the centrifugally harvested sediment by using 5mL Binding buffer, dissolving the sediment at 4 ℃ overnight, centrifugally collecting supernatant, and filtering the supernatant to prepare for loading;

the content of the column renaturation process of the AKTA protein purifier is referred to GE healthcare handbook Purifying charling Proteins 77-79, a Purifying column adopts HisTrapHP 1mL, the column renaturation time after sample injection and loading is 2h, and 100 percent of the column renaturation time is replaced to solution of reforming buffer and then replaced to solution of elusion buffer to elute target protein.

7. The trichina competition ELISA antibody assay kit of any one of claims 1 to 5, wherein the biotin-labeled monoclonal antibody Ts-WN10-1H9 is prepared by the steps of:

step 2, intraperitoneal injection 1X 10 after 1 week⁶2, extracting ascites when the abdominal cavity of a mouse is extremely swollen 7-10 days later than hybridoma cells WN10-1H9, wherein the microbial preservation number of the hybridoma cells WN10-1H9 is CGMCC No. 18316;

8. The trichina competition ELISA antibody assay kit of any one of claims 1 to 5, wherein the 96-well reaction plate coated with the trichina Ts-WN10 recombinant antigen at the bottom of the plate is prepared by the steps of:

9. The method for detecting the trichina competition ELISA antibody detection kit of claim 1, which is characterized in that 50uL of serum to be detected and 50uL of monoclonal antibody Ts-WN10-1H9 solution with the concentration of 2 μ g/mL are uniformly mixed, added into a 96-well reaction plate coated with trichina Ts-WN10 recombinant antigen at the bottom of the plate, reacted for 1H at 37 ℃, and negative quality control controls are parallelly established in the plate;

washing with PBST washing solution for 3 times;

Then, the inhibition ratio PI% (1-OD) was calculated_sample/OD_NC)％。

10. The method for detecting trichina competition ELISA antibody detection kit of claim 9, wherein the method is characterized in that