CN103837688A - Indirect enzyme-linked immuno sorbent assay (ELISA) kit for detecting canine and feline Toxoplasma gondii antibodies - Google Patents
Indirect enzyme-linked immuno sorbent assay (ELISA) kit for detecting canine and feline Toxoplasma gondii antibodies Download PDFInfo
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- CN103837688A CN103837688A CN201410118911.4A CN201410118911A CN103837688A CN 103837688 A CN103837688 A CN 103837688A CN 201410118911 A CN201410118911 A CN 201410118911A CN 103837688 A CN103837688 A CN 103837688A
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Abstract
The invention discloses an indirect enzyme-linked immuno sorbent assay (ELISA) kit for detecting canine and feline Toxoplasma gondii antibodies, relates to an immunological detection technology for human and animal infectious diseases and is applicable to qualitative detection of canine and feline Toxoplasma gondii antibodies. The kit comprises a Toxoplasma gondii recombinant antigen GRA7 pre-coated ELISA plate, wherein the sample diluent is 0.01 mol/L of PBS (with the pH of 7.2) containing 0.5 percent of BSA and 0.05 percent of NaN3; the enzyme bonder is a horse radish peroxidase (HRP)-rabbit anti-dog/cat IgG bonder; the substrate developing solution is a TMB developing solution; the stopping solution is a 2mol/L of sulfuric acid solution, positive control and negative control. The kit is high in sensitivity, high in specificity and high in repeatability and can serve as a method for detecting canine and feline Toxoplasma gondii antibodies.
Description
Technical field
The invention discloses the indirect ELISA reagent kit that a kind of dog cat toxoplasma antibody detects, belong to technique for gene engineering and diagnostic reagent field.
Background technology
Toxoplasmosis is a kind of important zoonotic parasitic diseases that Infection of Toxoplasma Gondii causes, causes people and animal miscarriage, monster, stillborn foetus, and serious harm public health security and animal husbandry are produced.Cat is Infection of Toxoplasma Gondii final host, and dog is Infection of Toxoplasma Gondii intermediate host, the propagation of Infection of Toxoplasma Gondii with infect in play an important role.Infection of Toxoplasma Gondii there is no effective vaccine at present, and the monitoring of dog cat arch insect infection is significant for the prevention and control of disease.
The diagnosis of arch insect infection mainly comprises the methods such as aetology, molecular biology and immunology.Aetology method is time-consuming, effort, susceptibility are low.Molecular biology method comprises that the technology such as PCR, DNA probe and genetic chip have higher susceptibility and specificity, but needs expensive instrument and equipment, and operating process is more complicated.Immunological detection method comprises stain test (DT), IFA (IFAT), indirect hemagglutination test (IHA), direct agglutination test (DAT), improvement aggegation experiment (MAT), enzyme linked immunosorbent assay (ELISA), need to, with the polypide living, there is bio-safety risk in DT wherein; IFAT has higher susceptibility and specificity, but needs special instrument and equipment, is not suitable for clinical use; IHA and LAT are without species specificity, but its susceptibility is lower; MAT testing result is considered to goldstandard, but it is lower that dog Infection of Toxoplasma Gondii is detected to rate.ELISA susceptibility, specificity are higher, are suitable for large sample amount and detect.
At present Infection of Toxoplasma Gondii ELISA detects antigen used and is mostly polypide soluble antigen, and its composition is uncertain, and between antigen preparation batch, difference is larger, is difficult for standardization.Along with Protocols in Molecular Biology development, recombinant antigen has unrivaled advantage as the diagnostic antigen of Infection of Toxoplasma Gondii, as antigenic component is determined, and easily standardization, high specificity etc.Toxoplasma tachyzoite has 3 kinds of secretory organelles, microneme, clava and dense granule.Dense granule albumen (dense granule protein, GRA) be mainly to play an important role in the time that worm bubble is received in polypide modification and in thin Intracellular survival, to polypide at thin Intracellular survival with copied relevant, can cause the strong immune response of host T cell B cell, wherein GRA7 secretion process continues all stage of tachyzoite infection host, it is the principal ingredient of excrete (ES) antigen, can stimulate a large amount of secretions of interferon and TNF, having good immunogenicity, is a kind of potential vaccine, diagnosis candidate antigens molecule.The indirect ELISA reagent kit that the GRA7 that the invention discloses to recombinate detects as dog cat toxoplasma antibody, compared with other method, has stronger susceptibility and specificity.
Summary of the invention
The invention provides a kind of indirect ELISA reagent kit that detects dog cat toxoplasma antibody taking recombinant protein GRA7 as antigen, its object is to overcome the shortcoming and defect such as the specificity that prior art exists is strong, false positive rate is high, for the qualitative detection of toxoplasma antibody.
The indirect ELISA reagent kit of dog cat toxoplasma antibody provided by the invention comprises recombinant antigen GRA7 pre-coated elisa plate, sample diluting liquid, positive control, negative control, enzyme conjugates, cleansing solution, substrate nitrite ion and stop buffer.
Kit preparation method of the present invention comprises the following steps:
1, the expression and purification of Infection of Toxoplasma Gondii restructuring GRA7 albumen: use round pcr amplification GRA 7 gene of Toxoplasma gondii, be cloned into prokaryotic expression carrier pET-28a (+), construction recombination plasmid pET-GRA7, cuts identification and analysis and sequencing through enzyme.Recombinant plasmid pET-GRA7 is transformed to e. coli bl21, and after IPTG induction, expression product carries out SDS-PAGE analysis, occurs specific expressed band at about 29kDa place, consistent with expection.After recombinant bacterium cracking, centrifugal 6 min of 9000 rpm, get respectively cleer and peaceful precipitation and carry out SDS-PAGE, and result shows that destination protein is mainly with insolubility form expression (Fig. 1).Western blotting analyzes, and expression product can react with Infection of Toxoplasma Gondii positive serum (Fig. 2).Recombinant protein is after Ni-NTA purifying, and purity reaches more than 90% (Fig. 3).
2, antigen coated: the recombinant protein GRA7 of purifying is diluted to 5 μ g/mL with the carbonate buffer solution of pH 9.6, coated 96 hole ELISA Plate, every hole 50 μ L, 4 DEG C are spent the night, and wash 5 times with cleansing solution, every all over 5min; The confining liquid that adds 5% skimmed milk power to be mixed with, every hole 100 μ L, 37 DEG C of incubators, 1h; With cleansing solution washing 5 times, every all over 5min, 4 DEG C save backup subsequently.
3, the preparation of the anti-dog of rabbit or cat IgG and horseradish peroxidase (HRP) mark: extract according to a conventional method purifying dog or cat IgG, immunizing rabbit, reaches 1:32 and get serum when above when rabbit anti-serum ELISA tires; Ammonium sulfate precipitation purifying; With improvement periodate oxidation method carry out mark; Last enzyme labeling thing adds the neutral glycerine of 5% bovine serum albumin(BSA), 1% casein and 50%, measures after working concentration, and-20 DEG C save backup.
4, the preparation of above-mentioned various solution: 1. sample diluting liquid: 5% BSA, 0.01 mol/L of 0.05% NaN3, the phosphate buffer (PBS) of pH 7.2; 2. cleansing solution: at 0.01 M PBS solution (the 3.58g Na of 1000 ml
2hPO
4, 0.27 g KH
2pO
4, 8 g NaCl, 0.2 g KCl) in add 0.5 ml Tween-20 (Tween-20);
confining liquid: 100 ml liquid are washed and added 5g skimmed milk power, i.e. 5% skimmed milk power confining liquid in liquid;
stop buffer: getting 54.3ml concentration is that 95-98% concentrated sulphuric acid adding distil water is to 1000ml.
The present invention compared with prior art its good effect to be to have specificity good, highly sensitive, reproducible, can be used as dog cat toxoplasma antibody conventional sense method.
attached caption
Fig. 1: the SDS-PAGE analysis result that Infection of Toxoplasma Gondii recombinant protein GRA7 expresses. 1. albumen Marker; 2-4. recombinant protein GRA7; 5. blank.
Fig. 2: the Western blotting that Infection of Toxoplasma Gondii recombinant protein GRA7 expresses analyzes. 1. albumen Marker; 2. recombinant protein GRA7; 3. negative control.
Fig. 3: Infection of Toxoplasma Gondii recombinant protein GRA7 purification result .1. albumen Marker; 2, the 3. recombinant protein GRA7. of purifying
The indirect ELSIA kit of table 1. dog toxoplasma antibody testing result
The indirect ELSIA kit of table 2. cat toxoplasma antibody testing result
Embodiment
embodiment 1:the indirect ELSIA kit of dog toxoplasma antibody operation steps
(1) sample diluting liquid is pressed dog serum after 1:50 dilution, added in the hole of GRA7 pre-coated elisa plate every hole 50 μ L, 37 DEG C of incubators, 1h;
(2) cleansing solution washing 5 times, every all over 5min;
(3) add the anti-dog IgG of HRP mark rabbit (working concentration), every hole 50 μ L, 37 DEG C of incubators, 1h;
(4) cleansing solution washing 5 times, every all over 5min;
(5) add tmb substrate nitrite ion, every hole 50 μ L, 37 DEG C of lucifuge colour developing 20min of incubator;
(6) add stop buffer, every hole 50 μ L;
(7) utilize microplate reader to measure light absorption (OD) value at 450nm;
(8) result is judged: to survey each hole OD value after blank hole zeroing, if be greater than 2.1 times of negative control OD value of regulation, positive.
embodiment 2: the indirect ELSIA kit of cat toxoplasma antibody operation steps
(1) sample diluting liquid is pressed cat serum after 1:64 dilution, added in the hole of GRA7 pre-coated elisa plate every hole 50 μ L, 37 DEG C of incubators, 1h;
(2) cleansing solution washing 5 times, every all over 5min;
(3) add the anti-cat IgG of HRP mark rabbit (working concentration), every hole 50 μ L, 37 DEG C of incubators, 1h;
(4) cleansing solution washing 5 times, every all over 5min;
(5) add tmb substrate nitrite ion, every hole 50 μ L, 37 DEG C of lucifuge colour developing 20min of incubator;
(6) add stop buffer, every hole 50 μ L;
(7) utilize microplate reader to measure light absorption (OD) value at 450nm;
(8) result is judged: to survey each hole OD value after blank hole zeroing, if be greater than 2.1 times of negative control OD value of regulation, positive.
embodiment 3:the indirect ELSIA kit of dog toxoplasma antibody evaluation
To be defined as 259 parts of the dog toxoplasma antibody positive, negative serums by MAT method and IFAT method, with this kit detection.Testing result is as table 1.Statistical analysis shows, ELISA testing result and actual result are without significant difference (P > 0.05; Kappa=0.8631,95% CI:0.7803,0.9459), result concordance rate is 96.1%.The indirect ELSIA kit of dog toxoplasma antibody testing result susceptibility is 88.6%, and specificity is 97.7%.
embodiment 4:the indirect ELSIA kit of cat toxoplasma antibody evaluation
To be defined as 185 parts of the cat toxoplasma antibody positive, negative serums by MAT method and IFAT method, with this kit detection.Testing result is as table 2.Statistical analysis shows, ELISA testing result and actual result are without significant difference (P > 0.05; Kappa=0.9195,95% CI:0.8500,0.9890), result concordance rate is 97.3%.The indirect ELSIA kit of cat toxoplasma antibody testing result susceptibility is 92.5%, and specificity is 98.6%.
The indirect ELSIA kit of table 1. dog toxoplasma antibody testing result
The indirect ELSIA kit of table 2. cat toxoplasma antibody testing result
Claims (2)
1. detect an indirect ELISA reagent kit for dog cat toxoplasma antibody, described detection kit comprises: Infection of Toxoplasma Gondii recombinant antigen GRA7 pre-coated elisa plate, and package amount is 0.25 μ g; Sample diluting liquid is the 0.01mol/L(pH 7.2 containing 0.5%BSA and 0.05% NaN3) PBS; Enzyme conjugates is the anti-dog of horseradish peroxidase-rabbit or cat IgG bond; Substrate nitrite ion is TMB nitrite ion; Stop buffer is 2 mol/L sulfuric acid solutions, positive control, negative control.
2. dog cat toxoplasma antibody indirect ELISA reagent kit according to claim 1, the preparation that it is characterized in that envelope antigen is by the round pcr Infection of Toxoplasma Gondii dense granule Protein G RA7 gene that increases, be cloned in prokaryotic expression carrier pET-28a, build pET-GRA7, transform Escherichia coli, through IPTG abduction delivering, after Ni-NTA purifying as Infection of Toxoplasma Gondii detectable antigens.
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Cited By (5)
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| CN104292329A (en) * | 2014-09-15 | 2015-01-21 | 沈鹤霄 | Preparation method of antibody of toxoplasma gondii compact granular protein 6 |
| CN104965086A (en) * | 2015-05-21 | 2015-10-07 | 华南农业大学 | Dog toxoplasma gondii antibody indirect ELISA detection kit |
| CN104965087A (en) * | 2015-05-25 | 2015-10-07 | 中国农业科学院上海兽医研究所 | Method for efficiently detecting toxoplasma acute infection, and target protein thereof |
| CN106970210A (en) * | 2017-02-22 | 2017-07-21 | 中国农业科学院上海兽医研究所 | A kind of toxoplasmosis indirect ELISA diagnostic reagent kit |
| CN114720696A (en) * | 2022-04-02 | 2022-07-08 | 应节(南京)智能科技有限公司 | Kit for detecting canine/feline toxoplasma antibody and blocking ELISA (enzyme-linked immunosorbent assay) detection method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104292329A (en) * | 2014-09-15 | 2015-01-21 | 沈鹤霄 | Preparation method of antibody of toxoplasma gondii compact granular protein 6 |
| CN104965086A (en) * | 2015-05-21 | 2015-10-07 | 华南农业大学 | Dog toxoplasma gondii antibody indirect ELISA detection kit |
| CN104965087A (en) * | 2015-05-25 | 2015-10-07 | 中国农业科学院上海兽医研究所 | Method for efficiently detecting toxoplasma acute infection, and target protein thereof |
| CN104965087B (en) * | 2015-05-25 | 2017-06-27 | 中国农业科学院上海兽医研究所 | The method and its target proteinses of a kind of efficient detection Infection of Toxoplasma Gondii acute infection |
| CN106970210A (en) * | 2017-02-22 | 2017-07-21 | 中国农业科学院上海兽医研究所 | A kind of toxoplasmosis indirect ELISA diagnostic reagent kit |
| CN106970210B (en) * | 2017-02-22 | 2018-08-03 | 中国农业科学院上海兽医研究所 | A kind of toxoplasmosis indirect ELISA diagnostic reagent kit |
| CN114720696A (en) * | 2022-04-02 | 2022-07-08 | 应节(南京)智能科技有限公司 | Kit for detecting canine/feline toxoplasma antibody and blocking ELISA (enzyme-linked immunosorbent assay) detection method |
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Application publication date: 20140604 |