CN202256346U - Toxoplasma IgM antibody immunoblotting kit - Google Patents
Toxoplasma IgM antibody immunoblotting kit Download PDFInfo
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- CN202256346U CN202256346U CN2011203511871U CN201120351187U CN202256346U CN 202256346 U CN202256346 U CN 202256346U CN 2011203511871 U CN2011203511871 U CN 2011203511871U CN 201120351187 U CN201120351187 U CN 201120351187U CN 202256346 U CN202256346 U CN 202256346U
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Abstract
Toxoplasma gondii IgM antibody immunoblotting kit relates to a kit. The kit is provided with a carrier plate, a nitrocellulose membrane, a toxoplasma IgM antibody detection line and a contrast line; the toxoplasma IgM antibody detection line and the control line are sequentially arranged on the nitrocellulose membrane; coating a toxoplasma recombinant antigen at a toxoplasma IgM antibody detection line, and coating a human IgM antibody at a control line; horse radish peroxidase is labeled with anti-human mu chain antibody. Preparing a toxoplasma gondii recombinant antigen; spotting of the nitrocellulose membrane; preparing an anti-human IgM specific fragment mu-chain monoclonal antibody; labeling the anti-human IgM specific fragment mu chain monoclonal antibody with horseradish peroxidase; an immunoblotting kit was prepared. During detection, the required sample amount is extremely small, special instruments are not needed, results can be directly interpreted by naked eyes, and the kit is simple, convenient and quick to detect, high in specificity, high in sensitivity, accurate and reliable, low in cost and wide in application.
Description
Technical field
The utility model relates to a kind of kit, especially relates to a kind of Toxoplasma Gondi IgM antibody and detects HP immunoblotting kit.
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; All there are the report of arch insect infection in each province, the whole nation, city, autonomous region; The average infection rate of China's normal population about 4%～9% ([2] Liu Min, 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 the AIDS patient dead.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.
The diagnostic method of existing toxoplasmosis comprises directly and carries out etiological diagnosis from arc worms of separate tissue such as internal organs, blood, cerebrospinal fluid, needs several days even a few time-of-week, wastes time and energy, and in practical application, is worth little.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 Diagnosis of 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; Mostly normal human's toxoplasma gondii infection is subclinical infection; When immunity of organisms descended, polypide bred in a large number, invaded and removed 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 process, general Routine Test Lab all can be accomplished, 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.In the face of severe anti-system form, not only need special detection means accurately, also need a kind of reagent of more economical practicality to come examination, so that with the disease prevention and control countermeasure is provided for clinical.
Summary of the invention
The purpose of the utility model provides a kind of Toxoplasma Gondi IgM antibody HP immunoblotting kit.
The utility model is provided with carrier board, nitrocellulose membrane, Toxoplasma Gondi IgM antibody detection line and control line; Said Toxoplasma Gondi IgM antibody detection line and control line are located on the nitrocellulose membrane successively; Encapsulate arc worm recombinant antigen at Toxoplasma Gondi IgM antibody detection line place, the place encapsulates human IgM antibody at control line; The anti-people μ of horseradish peroxidase-labeled chain antibody.
Said arc worm recombinant antigen is SAG1 (P30), SAG2 (P22), ROP2, GRA7 recombinant antigen.
Said carrier board can adopt the PVC plate.
Below provide the preparation method of the utility model:
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; Said arc worm recombinant antigen is SAG1 (P30), SAG2 (P22), ROP2, GRA7 etc.
2) point sample of nitrocellulose filter
On nitrocellulose filter IgM detection line, encapsulate arc worm recombinant antigen, the place encapsulates human IgM antibody at control line, dries; Said arc worm recombinant antigen is SAG1 (P30), SAG2 (P22), ROP2, GRA7 etc.; The concentration of said arc worm recombinant antigen and human IgM antibody can be 1～4mg/mL; The point sample amount can be 1 μ L/cm.
3) the anti-people IgM specific fragment μ chain monoclonal antibody of preparation
With people IgM specific fragment μ chain is antigen immune Balb/c mouse, and through hybridoma technology, screening obtains the hybridoma cell strain of the anti-people IgM of stably excreting monoclonal antibody; Adopt the method identification of M cAb of ELISA to tire more than 1: 107.
4) horseradish peroxidase (HRP) mark of anti-people IgM specific fragment μ chain monoclonal antibody
Adopt the sodium periodate method to carry out horseradish peroxidase (HRP) mark; The substrate of said horseradish peroxidase is made up of the diaminobenzidine of the hydrogen peroxide and 0.07% (W/V) of 0.03% (W/V).
5) preparation HP immunoblotting kit
Nitrocellulose membrane is sticked on the carrier board upper surface; Toxoplasma Gondi IgM antibody detection line and control line are located on the nitrocellulose membrane successively; Encapsulate arc worm recombinant antigen at Toxoplasma Gondi IgM antibody detection line place; The place encapsulates human IgM antibody at control line, is cut into strip with cutting cutter, dresses up the Toxoplasma Gondi IgM antibody HP immunoblotting kit with the anti-people μ chain monoclonal antibody and the substrate mutual group thereof of enzyme labeling; Said arc worm recombinant antigen is SAG1 (P30), SAG2 (P22), ROP2, GRA7 etc.
The utility model provides a kind of employing immunoblot assay to set up the toxoplasmosis IgM antigen testing reagent bar, can be used for the detection of Toxoplasma Gondi IgM 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 the utility model embodiment 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 IgM positive findings; 2 is arc worm SAG1 (P30)-IgM antibody detection line, and 3 is arc worm SAG2 (P22)-IgM antibody detection line, and 4 is arc worm ROP2-IgM antibody detection line, and 5 is arc worm GRA7-IgM antibody detection line, and 6 is control line.
Embodiment
Following examples will combine accompanying drawing that the utility model is further described.
Referring to Fig. 1, the utility model embodiment is provided with carrier board 1, Toxoplasma Gondi IgM 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 IgM antibody detection line 2～5 is located on the nitrocellulose membrane 7 with control line 6 successively; Encapsulate arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 respectively at Toxoplasma Gondi IgM antibody detection line place, 6 places encapsulate human IgM antibody at control line.
Said carrier board adopts the PVC plate.
Below provide the preparation method of the utility model:
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
On nitrocellulose filter IgM detection line, encapsulate SAG1 (P30), SAG2 (P22), ROP2 and the arc worm recombinant antigen of GRA7, the place encapsulates human IgM antibody at control line, dries.
3) the anti-people IgM specific fragment μ chain monoclonal antibody of preparation
With people IgM specific fragment μ chain is antigen immune Balb/c mouse, and through hybridoma technology, screening obtains the hybridoma cell strain of the anti-people IgM of stably excreting monoclonal antibody.Adopt the method identification of M cAb of ELISA to tire more than 1: 107.
4) horseradish peroxidase (HRP) mark of anti-people IgM specific fragment μ 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 IgM antibody detection line and control line are located on the nitrocellulose membrane successively; Encapsulate arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at Toxoplasma Gondi IgM antibody detection line place; The place encapsulates human IgM antibody at control line; The anti-people μ 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 IgM antibody HP immunoblotting kit.
Below provide the clinical samples that adopts the utility model 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, at every turn 5min.
5) add enzyme conjugates: in the incubation groove, add the enzyme conjugates (the anti-people IgM specific fragment μ 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, at every turn 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 the utility model:
1) visual examination: kit does not have breakage, encapsulates 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: the positive control serum with the positive different titers of Toxoplasma Gondi IgM adopts the calibrating of Toxoplasma Gondi IgM antibody HP immunoblotting kit for each 50 parts, calculates positive coincidence rate.The clinical samples that definite employing ELISA (import reagent) method of positive control serum is confirmed.
3) negative sample coincidence rate:, calculate negative match-rate with 50 parts of negative control serum calibratings.The clinical samples that definite employing ELISA (import reagent) method of negative control serum is confirmed.
4) criticize interior difference: same batch of Toxoplasma Gondi IgM antibody HP immunoblotting kit, detect with characteristic serum, require the positive serum testing result to show that the shade of colour band is consistent, result's feminine gender that negative serum detects.
5) differences between batches: different batches Toxoplasma Gondi IgM antibody HP immunoblotting kit, detect with characteristic serum, require the positive serum testing result to show that the shade of colour band is consistent, the result that negative serum detects is negative.
6) interference test: testing result does not receive the interference of sample hemolysis (n=50), piarhemia (n=50) and jaundice (n=50).
7) cross reaction: adopt this Toxoplasma Gondi IgM 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.
8) Detection of Stability: use the Arrhenius rule, the Toxoplasma Gondi IgM antibody HP immunoblotting kit is placed 37 ℃ detect after 20 days, above each item index does not have marked change, guarantees that finished product preserves under the drying at room temperature condition, and 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 IgM antibody detection line and control line are located on the nitrocellulose membrane successively; Encapsulate arc worm recombinant antigen SAG1 (P30), SAG2 (P22), ROP2 and GRA7 at Toxoplasma Gondi IgM antibody detection line place; The place encapsulates human IgM antibody at control line; The anti-people μ 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 IgM 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, at every turn 5min.
5) add enzyme conjugates: in the incubation groove, add the enzyme conjugates (the anti-people IgM specific fragment μ 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, at every turn 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 with embodiment 1, its difference is that the nitrocellulose membrane detection line only is made up of the arc worm recombinant antigen of SAG2 (P22), ROP2 and GRA7, does not contain the arc worm recombinant antigen of SAG1 (P30).The result judges identical with embodiment 1.
Similar with embodiment 1, its difference is that the nitrocellulose membrane detection line only is made up of the arc worm recombinant antigen of SAG1 (P30), ROP2 and GRA7, does not contain the arc worm recombinant antigen of SAG2 (P22).The result judges identical with embodiment 1.
Embodiment 4
Similar with 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 with 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.
Embodiment 6
Similar with 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 IgM antibody HP immunoblotting kit by embodiment 1 scheme, carry out performance verification again.
1) visual examination: kit does not have breakage, encapsulates 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: the positive reference sample with the positive different titers of Toxoplasma Gondi IgM adopts the calibrating of Toxoplasma Gondi IgM antibody HP immunoblotting kit for each 50 parts, calculates positive coincidence rate.The clinical samples that definite employing ELISA (advancing article reagent) method of positive reference sample is confirmed.
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 article reagent) method of negative reference sample is confirmed.
4) criticize interior difference: same batch of Toxoplasma Gondi IgM antibody HP immunoblotting kit, detect with the characteristic sample, require the positive sample testing result to show that the shade of colour band is consistent, result's feminine gender that negative sample detects.
5) differences between batches: different batches Toxoplasma Gondi IgM antibody HP immunoblotting kit, detect with the characteristic sample, require the positive sample testing result to show that the shade of colour band is consistent, the result that negative sample detects is negative.
6) cross reaction: adopt this Toxoplasma Gondi IgM 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 IgM antibody HP immunoblotting kit is placed 37 ℃ detect after 20 days, above each item index does not have marked change, guarantees that finished product preserves under the drying at room temperature condition, and the term of validity is 18 months.
Claims (2)
1. the Toxoplasma Gondi IgM antibody HP immunoblotting kit is characterized in that being provided with carrier board, nitrocellulose membrane, Toxoplasma Gondi IgM antibody detection line, control line, and Toxoplasma Gondi IgM antibody detection line and control line are located on the nitrocellulose membrane successively.
2. Toxoplasma Gondi IgM antibody HP immunoblotting kit as claimed in claim 1 is characterized in that said carrier board adopts the PVC plate.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011203511871U CN202256346U (en) | 2011-09-19 | 2011-09-19 | Toxoplasma IgM antibody immunoblotting kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011203511871U CN202256346U (en) | 2011-09-19 | 2011-09-19 | Toxoplasma IgM antibody immunoblotting kit |
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| CN202256346U true CN202256346U (en) | 2012-05-30 |
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| CN2011203511871U Expired - Fee Related CN202256346U (en) | 2011-09-19 | 2011-09-19 | Toxoplasma IgM antibody immunoblotting kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106153894A (en) * | 2016-08-12 | 2016-11-23 | 江苏泽成生物技术有限公司 | A kind of detection kit measuring Toxoplasma Gondi IgM antibody and detection method thereof |
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2011
- 2011-09-19 CN CN2011203511871U patent/CN202256346U/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106153894A (en) * | 2016-08-12 | 2016-11-23 | 江苏泽成生物技术有限公司 | A kind of detection kit measuring Toxoplasma Gondi IgM antibody and detection method thereof |
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| GR01 | Patent grant | ||
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| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120530 Termination date: 20130919 |