CN102288768A - Toxoplasma gondii IgG (immunoglobulin G) antibody immunoblotting kit and preparation method thereof - Google Patents
Toxoplasma gondii IgG (immunoglobulin G) antibody immunoblotting kit and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a toxoplasma gondii IgG (immunoglobulin G) antibody immunoblotting kit and a preparation method thereof and relates to a kit. The kit is provided with a vector board, a nitrocellulose membrane, a toxoplasma gondii IgG antibody detection line and a control line. The toxoplasma gondii IgG antibody detection line and the control line are arranged on the nitrocellulose membrane sequentially. A toxoplasma gondii recombinant antigen is enveloped at the position of the toxoplasma gondii IgG antibody detection line. A human IgG antibody is enveloped at the position of the control line. An anti-human gamma-chain antibody is marked by horse radish peroxidase. The preparation method of the toxoplasma gondii IgG antibody immunoblotting kit comprises the following steps of: preparing the toxoplasma gondii recombinant antigen; carrying out sample application on the nitrocellulose membrane; preparing an anti-human IgG specific fragment gamma-chain monoclonal antibody; marking the anti-human IgG specific fragment gamma-chain monoclonal antibody with the horse radish peroxidase; and preparing the immunoblotting kit. In the detecting process, a required specimen amount is small, a specific instrument is not required and a result is directly interpreted by naked eyes. Moreover, the toxoplasma gondii IgG antibody immunoblotting kit is simple, convenient and rapid to detect, has strong specificity and high sensitivity, is accurate and reliable and has low cost and wide application.
Description
Technical field
The present invention relates to a kind of kit, especially relate to a kind of Toxoplasma Gondi IgG antibody and detect HP immunoblotting kit and preparation method thereof.
Background technology
Toxoplasmosis (Toxoplasmosis) is that the people beast of a kind of serious harm human health of causing of toxoplasma gondii (Toxoplasmagondii) suffers from parasitic disease altogether, the arc worm of its pathogen can colonize in the karyocyte of people and multiple animal, the general susceptible of crowd and animal.This disease is distributed widely in all over the world, according to statistics the whole world have approximately 1,000,000,000 people by arch insect infection ([1] Xi Linlin, Li Wei. arc worm pathogenesis, virulence and genotype progress [J]. Chinese Pathogen Biology magazine, 2009,4 (11): 859-861.).Toxoplasmosis distributes very extensive in China, each provinces of the whole nation all have the report of arch insect infection, the average infection rate of China's normal population is ([2] Liu Min about 4%～9%, Chen Xiaoguang. the analysis on epidemic of Chinese population toxoplasmosis [J]. parasite and medical insect journal, 2010,17 (3): 131-134.), special population such as tumor patient, mental patient, birth defect infant, immunosupress, immunodeficiency patient infection lead higher.Normal adult of thumping majority immunologic function or children are normal asymptomatic or light symptoms is only arranged after by arch insect infection fortunately, and multipotency self-healing and obtain permanent immunity.But after the fetus of congenital infection, children and immunodeficiency person were infected, then prognosis was very serious.It is an important intercurrent disease that causes AIDS patient's death.Human immunodeficiency virus (HIV) the infected has 20%～80% to merge arch insect infection approximately, the more important thing is that it also is one of important pathogen that causes human congenital malformation, defective, feeblemindedness, stillborn foetus, premature labor.
Toxoplasmosis diagnostic method comprise directly and carry out etiological diagnosis from arc worms of separate tissue such as internal organs, blood, cerebrospinal fluid, need several days even a few time-of-week, waste time and energy, be worth little in actual applications.Conventional sense mainly is to use various serological methods clinically, detect its special IgG and IgM ([3] Zhou Bin, Zhang Jue, Wang Ke, Deng. the foundation and the Preliminary Clinical [J] thereof of Toxoplasma Gondi IgG and IgM antibody double-tagging time resolved fluoro-immunoassay. Chinese laboratory medicine magazine, 2010,33 (010): 957-959.), serological method can be diagnosed congenital, acute and chronic toxoplasmosis, but because the IgG titre of its resisting toxoplasmosis of human or animal of most of immunodeficiencies can not rise or high IgM titre can not occur, so can not diagnose toxoplasmosis with serological method to the human or animal who suffers from immunodeficiency effectively.In addition, serum antibody titer just can progressively descend after antigen disappears the long duration, so can not be applied to treatment effectiveness evaluation.Detect IgG antibody at present and have higher false positive, and problems such as the general poor sensitivity of IgM antibody test ([4] Han Jingyun, Liu Qian, Guo Jian. the technical progress of arch insect infection laboratory diagnosis [J]. laboratory medicine, 2009,24 (5): 393-395).Therefore, the early diagnosis of disease is helped little, is badly in need of setting up a kind of high susceptibility that has, and inexpensive, quick, simple to operate, do not need specific installation, be more suitable in clinical method of early diagnosis.
The diagnosis of toxoplasmosis and epidemiology survey mainly depend on serological test, normal human's toxoplasma gondii infection mostly is subclinical infection, when immunity of organisms descends, polypide breeds in a large number, invade and remove exo-erythrocytic each histocyte endoparasitism, cause the extensive inflammation of various tissues, thereby clinical symptoms occurs.Can inducing producing specificity antibody behind the arc worm of human infection.Infecting early stage IgM antibody increases, IgM fades away after infecting 4 months, high concentration IgG antibody ([5] KASPER D C appears after infecting 1 month, PRUSA A R, HAYDE M, et al.Evaluation of the vitros eciq immunodiagnostic system for detection of anti-toxoplasma immunoglobulin g and immunoglobulin m antibodies for confirmatory testing for acute toxoplasma gondii infection in pregnant women[J] .Journal of clinical microbiology, 2009,47 (1): 164-168).Therefore, Toxoplasma Gondi IgG antibody is an important indicator of toxoplasmosis diagnosis and epidemiology survey.
Early stage serological method uses arch insect circulating antigen.The arch insect circulating antigen of research and diagnosis usefulness is to obtain with the arch insect infection mouse peritoneal, antigen amount few and impure (often being mixed with host protein) big, that obtain that this method spends, so false positive also happens occasionally.Along with the clone in succession who reaches toxoplasma antigen that popularizes of Protocols in Molecular Biology, it is more and more that recombinant antigen is applied to arc worm experiment.Surface antigen (SAG1 (P30), SAG2 (P22), SAG3 (P43), SAG4 (P18)), polypide clava antigen (ROP1, ROP2), dense granule albumen ([6] Xiong Meihua such as (GRA1, GRA7) of the arc worm that research at present is many, Wang Xiuzhen, Liu Luxia, Deng. the extraction of toxoplasma tachyzoite antigen and analysis of protein [J]. Chinese preventing and treating verminosis magazine, 2001,14 (3): 237-238.).The recombinant antigen that adopts recombinant DNA technology to prepare can overcome the shortcoming of complete toxoplasma antigen, can prepare endless special recombinant toxoplasma antigen fast, economically.
Existing toxoplasma antibody detection method comprises ELISA, Western-blot etc., and its specificity is all higher.The kit that adopts the Western-blot method to make, general Routine Test Lab all can be finished, and does not need specific installation, and interpretation is also simple and clear.Existing commercially available Western-blot reagent is import reagent, costs an arm and a leg.Anti-system form in the face of severe not only needs special detection means accurately, also needs a kind of reagent of more economical practicality to come examination, so that provide countermeasure for clinical with the disease prevention and control.
Summary of the invention
The purpose of this invention is to provide a kind of Toxoplasma Gondi IgG antibody HP immunoblotting kit and preparation method thereof.
Described Toxoplasma Gondi IgG antibody HP immunoblotting kit is provided with carrier board, nitrocellulose membrane, Toxoplasma Gondi IgG antibody detection line and control line; Described Toxoplasma Gondi IgG antibody detection line and control line are located on the nitrocellulose membrane successively; , wrapped by the human IgG antibody by arc worm recombinant antigen at Toxoplasma Gondi IgG antibody detection line place bag at the control line place; The anti-people γ of horseradish peroxidase-labeled chain antibody.
Described arc worm recombinant antigen is SAG1 (P30), SAG2 (P22), ROP2, GRA7 recombinant antigen.
Described carrier board can adopt the PVC plate.
The preparation method of described Toxoplasma Gondi IgG antibody HP immunoblotting kit may further comprise the steps:
1) the arc worm recombinant antigen of preparation
Adopt gene clone technology, the DNA of pcr amplification coding toxoplasma antigen, and make its expression in the insertion Escherichia coli, get arc worm recombinant antigen;
2) point sample of nitrocellulose filter
Bag by the human IgG antibody, is dried at control line place bag by arc worm recombinant antigen on nitrocellulose filter IgG detection line;
3) the anti-human IgG specific fragment γ chain monoclonal antibody of preparation
With human IgG specific fragment γ chain is antigen immune Balb/c mouse, and by hybridoma technology, screening obtains the hybridoma cell strain of the anti-human IgG monoclonal antibody of stably excreting;
4) horseradish peroxidase (HRP) mark of anti-human IgG specific fragment γ chain monoclonal antibody
Adopt the sodium periodate method to carry out horseradish peroxidase (HRP) mark;
5) preparation HP immunoblotting kit
Nitrocellulose membrane is sticked on the carrier board upper surface, Toxoplasma Gondi IgG antibody detection line and control line are located on the nitrocellulose membrane successively, wrap by arc worm recombinant antigen at Toxoplasma Gondi IgG antibody detection line place, wrap coated human IgG antibody at the control line place, be cut into strip with cutting cutter, dress up the Toxoplasma Gondi IgG antibody HP immunoblotting kit with the anti-people γ chain monoclonal antibody and the substrate mutual group thereof of enzyme labeling.
In step 1), 2), 5) in, described arc worm recombinant antigen is SAG1 (P30), SAG2 (P22), ROP2, GRA7 etc.
In step 2) in, described arc worm recombinant antigen and human IgG antibody's concentration can be 1～4mg/mL; The point sample amount can be 1 μ L/cm.
In step 3), adopt the method identification of M cAb of ELISA to tire at 1: 10
7More than.
In step 4), the substrate of described horseradish peroxidase is made up of the diaminobenzidine of the hydrogen peroxide and 0.07% (W/V) of 0.03% (W/V).
The invention provides a kind of employing immunoblot assay and set up Toxoplasma Gondi IgG antibody detectable bar, can be used for the detection of Toxoplasma Gondi IgG antibody in the samples such as whole blood, serum, blood plasma and cerebrospinal fluid.The required specimen amount of this detection method is minimum, does not need specific apparatus, the direct sentence read result of naked eyes, and detect easy fast, high specificity, highly sensitive, accurately and reliably, cost is low, is widely used.
Description of drawings
Fig. 1 is that the structure of film bar embodiment in the Toxoplasma Gondi IgM antibody HP immunoblotting kit of the present invention is formed synoptic diagram.
Fig. 2 is the experimental result pattern diagram.In Fig. 2, H is the synoptic diagram before using, and G is invalid test (product quality problem), the negative result of F, and A～E is the Toxoplasma Gondi IgG positive findings; 2 is arc worm SAG1 (P30)-IgG antibody detection line, and 3 is arc worm SAG2 (P22)-IgG antibody detection line, and 4 is arc worm ROP2-IgG antibody detection line, and 5 is arc worm GRA7-IgG antibody detection line, and 6 is control line.
Embodiment
Following examples will the present invention is further illustrated in conjunction with the accompanying drawings.
Referring to Fig. 1, Toxoplasma Gondi IgG antibody HP immunoblotting kit embodiment of the present invention is provided with carrier board 1, Toxoplasma Gondi IgG antibody detection line 2～5, control line 6 and nitrocellulose membrane (NC film) 7.
Nitrocellulose membrane 7 sticks on carrier board 1 upper surface, and Toxoplasma Gondi IgG antibody detection line 2～5 and control line 6 are located on the nitrocellulose membrane 7 successively; Wrap respectively by arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at Toxoplasma Gondi IgG antibody detection line place, wrap by the human IgG antibody at control line 6 places.
Described carrier board adopts the PVC plate.
The preparation method of described Toxoplasma Gondi IgG antibody HP immunoblotting kit may further comprise the steps:
1) preparation SAG1 (P30), SAG2 (P22), ROP2 and the arc worm recombinant antigen of GRA7
Adopt gene clone technology, the pcr amplification DNA of arc worm leptospira antigen that encodes, and insert in the Escherichia coli and make its expression, arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7.
2) point sample of nitrocellulose filter
Bag by the human IgG antibody, is dried at control line place bag by SAG1 (P30), SAG2 (P22), ROP2 and the arc worm recombinant antigen of GRA7 on nitrocellulose filter IgG detection line.
3) the anti-human IgG specific fragment γ chain monoclonal antibody of preparation
With human IgG specific fragment γ chain is antigen immune Balb/c mouse, and by hybridoma technology, screening obtains the hybridoma cell strain of the anti-human IgG monoclonal antibody of stably excreting.Adopt the method identification of M cAb of ELISA to tire at 1: 10
7More than.
4) horseradish peroxidase (HRP) mark of anti-human IgG specific fragment γ chain monoclonal antibody
Adopt the sodium periodate method to carry out horseradish peroxidase (HRP) mark.
5) preparation HP immunoblotting kit
Nitrocellulose membrane is sticked on the carrier board upper surface, and Toxoplasma Gondi IgG antibody detection line and control line are located on the nitrocellulose membrane successively; Wrap by arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at Toxoplasma Gondi IgG antibody detection line place, wrap by the human IgG antibody at the control line place, the anti-people γ chain monoclonal antibody and the substrate mutual group thereof that are cut into strip and enzyme labeling with cutting cutter are dressed up the Toxoplasma Gondi IgG antibody HP immunoblotting kit.
Below provide the clinical samples that adopts the Toxoplasma Gondi IgG antibody HP immunoblotting kit to detect the patient:
1) sample adopts the 0.01mol/LPBS damping fluid to carry out dilution in 1: 50.
2) sealing: adopt 5% skimmed milk power (preparation of 0.01mol/L PBST damping fluid), waving incubation 30min on the shaking table in room temperature (18～25 ℃) as confining liquid.
3) serum incubation: take out required film bar, put it in the incubation groove, and numbering.In the incubation groove, add 1.5ml diluted sample respectively.Waving incubation 30min on the shaking table in room temperature (18～25 ℃).
4) clean: inhale and remove liquid in the groove, waving on the shaking table with 1.5ml 0.01mol/L PBS buffer solution for cleaning film bar 3 times each 5min.
5) add enzyme conjugates: in the incubation groove, add the enzyme conjugates (the anti-human IgG specific fragment γ chain monoclonal antibody that adopts horseradish peroxidase-labeled is for detecting antibody) that 1.5ml has diluted, waving incubation 30min on the shaking table in room temperature (18～25 ℃).
6) clean: inhale and remove liquid in the groove, waving on the shaking table with 1.5ml 0.01mol/L PBS buffer solution for cleaning film bar 3 times each 5min.
7) substrate incubation: in the incubation groove, add 1.5ml substrate solution (DAB) respectively, waving incubation 10min on the shaking table in room temperature (18～25 ℃).
8) stop: inhale and remove liquid in the groove, clean the film bar 3 times, each 1min with distilled water.
The result judges: will detect the film bar and be placed on the result and judge in the template air-dry judged result afterwards.Any protein targets marks existing specific band, all can be judged as the positive, but auxiliary diagnosis toxoplasmosis (see figure 2).
Below provide the performance calibrating of Toxoplasma Gondi IgG antibody HP immunoblotting kit:
1) visual examination: kit does not have breakage, and bag is by smooth, the clean pollution-free spot of reaction film, free from flaw, and adhesive tape does not have and comes unglued, and does not have and cuts oblique phenomenon.
2) positive sample coincidence rate: use the positive control serum of the different titers of the Toxoplasma Gondi IgG positive to adopt the calibrating of Toxoplasma Gondi IgG antibody HP immunoblotting kits for each 50 parts, calculate positive coincidence rate.The clinical samples that definite employing ELISA (import reagent) method of positive control serum is determined.
3) negative sample coincidence rate:, calculate the feminine gender coincidence rate with 50 parts of negative control serum calibratings.The clinical samples that definite employing ELISA (import reagent) method of negative control serum is determined.
4) criticize interior difference: same batch of Toxoplasma Gondi IgG antibody HP immunoblotting kit, detect with characteristic serum, require the shade unanimity of positive serum testing result demonstration colour band, the feminine gender as a result that negative serum detects.
5) differences between batches: different batches Toxoplasma Gondi IgG antibody HP immunoblotting kit, detect with characteristic serum, require the shade unanimity of positive serum testing result demonstration colour band, the feminine gender as a result that negative serum detects.
6) interference test: testing result is not subjected to the interference of sample hemolysis (n=50), piarhemia (n=50) and jaundice (n=50).
7) cross reaction: adopt this Toxoplasma Gondi IgG antibody HP immunoblotting kit, adopt the serum of systemic loupus erythematosus (n=30), rheumatoid disease (n=30), autoallergic autoimmunity systemic diseases such as (n=30) to detect, do not find cross reaction.
8) Detection of Stability: use the Arrhenius rule, the Toxoplasma Gondi IgG antibody HP immunoblotting kit placed 37 ℃ detect after 20 days, more than every index do not have marked change, guarantee that finished product preserves under the drying at room temperature condition, the term of validity is 18 months.
Below provide specific embodiment.
Embodiment 1
Nitrocellulose membrane is sticked on the carrier board upper surface, and Toxoplasma Gondi IgG antibody detection line and control line are located on the nitrocellulose membrane successively; Wrap by arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at Toxoplasma Gondi IgG antibody detection line place, wrap by the human IgG antibody at the control line place, the anti-people γ chain monoclonal antibody and the substrate mutual group thereof that are cut into strip and enzyme labeling with cutting cutter are dressed up the Toxoplasma Gondi IgG antibody HP immunoblotting kit.
1) sample adopts the 0.01mol/LPBS damping fluid to carry out dilution in 1: 50.
2) sealing: adopt 5% skimmed milk power (preparation of 0.01mol/L PBST damping fluid), waving incubation 30min on the shaking table in room temperature (18～25 ℃) as confining liquid.
3) serum incubation: take out required film bar, put it in the incubation groove, and numbering.In the incubation groove, add 1.5ml diluted sample respectively.Waving incubation 30min on the shaking table in room temperature (18～25 ℃).
4) clean: inhale and remove liquid in the groove, waving on the shaking table with 1.5ml 0.01mol/L PBS buffer solution for cleaning film bar 3 times each 5min.
5) add enzyme conjugates: in the incubation groove, add the enzyme conjugates (the anti-human IgG specific fragment γ chain monoclonal antibody that adopts horseradish peroxidase-labeled is for detecting antibody) that 1.5ml has diluted, waving incubation 30min on the shaking table in room temperature (18～25 ℃).
6) clean: inhale and remove liquid in the groove, waving on the shaking table with 1.5ml 0.01mol/L PBS buffer solution for cleaning film bar 3 times each 5min.
7) substrate incubation: in the incubation groove, add 1.5ml substrate solution (DAB) respectively, waving incubation 10min on the shaking table in room temperature (18～25 ℃).
8) stop: inhale and remove liquid in the groove, clean the film bar 3 times, each 1min with distilled water.
The result judges: will detect the film bar and be placed on the result and judge in the template air-dry judged result afterwards.Any protein targets marks existing specific band, all can be judged as the positive, but auxiliary diagnosis toxoplasmosis (see figure 2).
Similar to embodiment 1, its difference is that the nitrocellulose membrane detection line only is made up of SAG2 (P22), ROP2 and the arc worm recombinant antigen of GRA7, does not contain the arc worm recombinant antigen of SAG1 (P30).The result judges identical with embodiment 1.
Similar to embodiment 1, its difference is that the nitrocellulose membrane detection line only is made up of SAG1 (P30), ROP2 and the arc worm recombinant antigen of GRA7, does not contain the arc worm recombinant antigen of SAG2 (P22).The result judges identical with embodiment 1.
Similar to embodiment 1, its difference is that the nitrocellulose membrane detection line only is made up of SAG1 (P30), SAG2 (P22) and GRA7 recombinant antigen, does not contain the ROP2 recombinant antigen.The result judges identical with embodiment 1.
Similar to embodiment 1, its difference is that the nitrocellulose membrane detection line only is made up of SAG1 (P30), SAG2 (P22) and the arc worm recombinant antigen of ROP2, does not contain the arc worm recombinant antigen of GRA7.The result judges identical with embodiment 1.
Similar to embodiment 1, its difference is that sample to be checked is a samples of CSF, and the result judges identical with embodiment 1.
Embodiment 7
Performance verification test: prepare the Toxoplasma Gondi IgG antibody HP immunoblotting kit by embodiment 1 scheme, carry out performance verification again.
1) visual examination: kit does not have breakage, and bag is by smooth, the clean pollution-free spot of reaction film, free from flaw, and adhesive tape does not have and comes unglued, and does not have and cuts oblique phenomenon.
2) positive sample coincidence rate: use the positive reference sample of the different titers of the Toxoplasma Gondi IgG positive to adopt the calibrating of Toxoplasma Gondi IgG antibody HP immunoblotting kits for each 50 parts, calculate positive coincidence rate.The clinical samples that definite employing ELISA (advancing product reagent) method of positive reference sample is determined.
3) negative sample coincidence rate:, calculate negative match-rate with 50 parts of negative reference sample calibratings.The clinical samples that definite employing ELISA (advancing product reagent) method of negative reference sample is determined.
4) criticize interior difference: same batch of Toxoplasma Gondi IgG antibody HP immunoblotting kit, detect with the characteristic sample, require the shade unanimity of positive sample testing result demonstration colour band, the feminine gender as a result that negative sample detects.
5) differences between batches: different batches Toxoplasma Gondi IgG antibody HP immunoblotting kit, detect with the characteristic sample, require the shade unanimity of positive sample testing result demonstration colour band, the feminine gender as a result that negative sample detects.
6) cross reaction: adopt this Toxoplasma Gondi IgG antibody HP immunoblotting kit, carry out the detection of systemic loupus erythematosus (n=30), rheumatoid disease (n=30), autoallergic autoimmunity systemic diseases such as (n=30), do not find cross reaction.
7) Detection of Stability: use the Arrhenius rule, the Toxoplasma Gondi IgG antibody HP immunoblotting kit placed 37 ℃ detect after 20 days, more than every index do not have marked change, guarantee that finished product preserves under the drying at room temperature condition, the term of validity is 18 months.
Claims (8)
1. the Toxoplasma Gondi IgG antibody HP immunoblotting kit is characterized in that being provided with carrier board, nitrocellulose membrane, Toxoplasma Gondi IgG antibody detection line, control line, and Toxoplasma Gondi IgG antibody detection line and control line are located on the nitrocellulose membrane successively; , wrapped by the human IgG antibody by arc worm recombinant antigen at Toxoplasma Gondi IgG antibody detection line place bag at the control line place; The anti-people γ of horseradish peroxidase-labeled chain antibody.
2. Toxoplasma Gondi IgG antibody HP immunoblotting kit as claimed in claim 1 is characterized in that described arc worm recombinant antigen is SAG1 (P30), SAG2 (P22), ROP2, GRA7.
3. Toxoplasma Gondi IgG antibody HP immunoblotting kit as claimed in claim 1 is characterized in that described carrier board adopts the PVC plate.
4. the preparation method of Toxoplasma Gondi IgG antibody HP immunoblotting kit as claimed in claim 1 is characterized in that may further comprise the steps:
1) the arc worm recombinant antigen of preparation
Adopt gene clone technology, the pcr amplification DNA of arc worm leptospira antigen that encodes, and insert in the Escherichia coli and make its expression, arc worm recombinant antigen;
2) point sample of nitrocellulose filter
Bag by the human IgG antibody, is dried at control line place bag by arc worm recombinant antigen on nitrocellulose filter IgG detection line;
3) the anti-human IgG specific fragment γ chain monoclonal antibody of preparation
With human IgG specific fragment γ chain is antigen immune Balb/c mouse, and by hybridoma technology, screening obtains the hybridoma cell strain of the anti-human IgG monoclonal antibody of stably excreting;
4) horseradish peroxidase-labeled of anti-human IgG specific fragment γ chain monoclonal antibody
Adopt the sodium periodate method to carry out horseradish peroxidase-labeled;
5) preparation HP immunoblotting kit
Nitrocellulose membrane is sticked on the carrier board upper surface, Toxoplasma Gondi IgG antibody detection line and control line are located on the nitrocellulose membrane successively, wrap by arc worm recombinant antigen at Toxoplasma Gondi IgG antibody detection line place, wrap by the human IgG antibody at the control line place, be cut into strip with cutting cutter, dress up the Toxoplasma Gondi IgG antibody HP immunoblotting kit with the anti-people γ chain monoclonal antibody and the substrate mutual group thereof of enzyme labeling.
5. the preparation method of Toxoplasma Gondi IgG antibody HP immunoblotting kit as claimed in claim 4 is characterized in that in step 1), and described arc worm recombinant antigen is SAG1 (P30), SAG2 (P22), ROP2, GRA7.
6. the preparation method of Toxoplasma Gondi IgG antibody HP immunoblotting kit as claimed in claim 4 is characterized in that in step 2) in, described arc worm recombinant antigen and human IgG antibody's concentration is 1～4mg/mL; The point sample amount is 1 μ L/cm.
7. the preparation method of Toxoplasma Gondi IgG antibody HP immunoblotting kit as claimed in claim 4 is characterized in that in step 3), adopts the method identification of M cAb of ELISA to tire at 1: 10
7More than.
8. the preparation method of Toxoplasma Gondi IgG antibody HP immunoblotting kit as claimed in claim 4, it is characterized in that in step 4) the substrate of described horseradish peroxidase is made up of 0.03% hydrogen peroxide and 0.07% diaminobenzidine by mass/volume number percent.
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| CN103837688A (en) * | 2014-03-27 | 2014-06-04 | 吉林农业大学 | Indirect enzyme-linked immuno sorbent assay (ELISA) kit for detecting canine and feline Toxoplasma gondii antibodies |
| CN104745536A (en) * | 2015-03-02 | 2015-07-01 | 南方医科大学 | Monoclonal antibody hybridoma 3A6 and monoclonal antibody for toxoplasma protein TgVPI |
| CN104745536B (en) * | 2015-03-02 | 2018-03-09 | 南方医科大学 | A kind of toxoplasma protein TgVP1 monoclonal antibody hybridomas 3A6 and monoclonal antibody |
| CN106053817A (en) * | 2016-05-03 | 2016-10-26 | 吉林大学 | A double-antibody sandwich kit for detecting a toxoplasma gondii circulating antigen and a preparing method thereof |
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