CN102095861A - Western blot kit for syphilis specific total antibodies and anticardiolipin antibodies and preparation method of kit - Google Patents
Western blot kit for syphilis specific total antibodies and anticardiolipin antibodies and preparation method of kit Download PDFInfo
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Abstract
The invention relates to a kit, in particular to a western blot kit for syphilis specific total antibodies and anticardiolipin antibodies and a preparation method of the kit. The kit is provided with a carrier plate, a nitrocellulose membrane, detection lines for syphilis specific total antibodies and anticardiolipin antibodies and a comparison line, wherein the detection lines and the comparison line are arranged on the nitrocellulose membrane; syphilis specific recombinant antigens and anticardiolipin antigens are coated at the detection lines for the total antibodies and anticardiolipin antibodies; anti-human Ig (immunoglobulin G) antibodies are coated at the comparison line; and horse radish peroxidase is labeled with the anti-human Ig antibodies. The method comprises the following steps: preparing the syphilis specific recombinant antigens; preparing the anticardiolipin antigens; applying samples of the nitrocellulose membrane; preparing anti-human total specific segment Ig chain monoclonal antibodies; labeling the horse radish peroxidase with the anti-human Ig monoclonal antibodies; and preparing the western blot kit. The western blot kit can be used for detecting the syphilis specific total antibodies and the anticardiolipin antibodies in the specimens of whole blood, serum, blood plasma, cerebrospinal fluid and the like. The invention has the advantages of small required specimen quantity, strong specificity, high sensitivity and is simple and fast in detection, and accurate and reliable in detection, and special instruments are not needed.
Description
Technical field
The present invention relates to a kind of kit, especially relate to a kind of syphilis specific total antibodies and cuorin antibody mediated immunity trace kit and preparation method thereof.
Background technology
Syphilis (Syphilis) is that a kind of (its pathogen is a microspironema pallidum for Treponema pallidum, the sexually transmitted disease that TP) causes, belongs to Spirochaetaceae by microspironema pallidum.Approach such as the main trafficability characteristic contact of microspironema pallidum, blood transfusion, wound or placenta are propagated.Microspironema pallidum enters blood near the lymph node the infected area, sends out whole body, and nearly all tissue of body and organ are got involved, and clinical manifestation is a general, can be divided into different clinical stages, comprises first phase, second phase, three phases and latent period.The World Health Organization (WHO) is prophesy optimistically once: " because high sensitivity detection method and therapeutic scheme are efficiently arranged, syphilis is a kind of sexually transmitted disease that can succeed and control by the public health measure ".Regrettably, so far syphilis still is worldwide public health problem, lack effective administrative control measure, annual nearly 1,200 ten thousand the patient in the whole world, 600,000 pregnant woman patients ([1] Lin Lirong, Yang Bo, Pan Xitao wherein, Deng. the selection of patient's syphilis serology detection method is propagated in potential blood source. Chinese hospital infection magazine .2010,20 (10): 1491-1494).In fact, the infection present situation of syphilis is possibly than more allowing people's pessimism in the imagination.
Investigation finds that syphilis the person be present among the general population more widely.
Microspironema pallidum still can not carry out in vitro culture, and controller used in syphilis diagnosis and epidemiology survey mainly depend on serological test, comprises that specific antibody and reagin detect two major types.Syphilis specific antibody IgM (TP-IgM) and IgG (TP-IgG) antibody are respectively at 2 weeks and the back generation of 4 weeks, even the patient is through enough treatments, it still can long-term existence, even ([2] Lin Lirong that do not disappear all the life, but ice, Fu Zuogen, etc. patient syphilis serology TRUST/TPPA and the antibody combined detection of IgM are propagated in potential blood source. Chinese skin cypridology magazine .2010,152 (05): 446-448); And that another kind of antibody materials reagin produces is later, generally produce in 5～7 weeks of infected back, and late syphilis, syphilis treatment later stage and latent syphilis may be negative, so the positive rate of syphilis specific antibody, susceptibility is significantly higher than reagin.After TP-IgM is syphilis, the specific antibody that body occurs at first.As long as there is microspironema pallidum alive to exist, its TP-IgM will maintain certain level.([3] Martina H such as Martina H, Daan W N, Mart M, et al.Comparison of a Treponema pallidum IgM immunoblot with a 19Sfluorescent treponemal antibody absorption test for the diagnosis of congenital syphilis[J] .Diagnostic Microbiology and Infectious Disease, 2007,59:61-66.) think that TP-IgM is a syphilis early infection and a movable serologic marker, ([4] Li Burong such as Li Burong, He Juntao, Zhang Yi, Deng. the clinical meaning [J] of microspironema pallidum IgM antibody test. The Fourth Military Medical University's journal, 2007,28 (16): 1495-1497) think that TP-IgM is the same with TP-DNA, representing syphilis infectiousness index.Under the prerequisite of the recent anti-syphilis treatment of eliminating, TP-IgM is not if turn out cloudy, and remaining microspironema pallidum of possibility or treatment are not thorough in the prompting body.The cloudy commentaries on classics person of TP-IgM changes positive when following up a case by regular visits to again, show once more syphilization ([5] Rawstron SA, Mehta S, Bromberg K, et al.Evaluation of a Treponema pallidum2specificIgM enzyme immunoassay and Treponema pallidum western blot antibody detection in thediagnosis ofmaternal and congenital syphilis[J] .Sex Transm Dis, 2004,31 (2): 123-126).Although the TP-IgM feminine gender can not be got rid of infectiousness fully, the TP-IgM positive must point out this patient to have infectiousness.The appearance of TP-IgG will be later than IgM, the energy long-term existence, even do not disappear all the life, therefore, TP-IgG is an important indicator ([6] Li-Rong Lin of controller used in syphilis diagnosis and epidemiology survey, Zuo-Gen Fu, Bing Dan, et al.Development of a colloidalgold-immunochromatography assay to detect Immunoglobulin G Antibodies to Treponemapallidum with TPN17 and TPN47.Diagnostic Microbiology and Infectious Disease, 2010,68:193-200.).
Early stage serological method uses complete microspironema pallidum as antigen, the TP of research and diagnosis usefulness obtains with TP infected rabbits testis, this method exists that cost is big, the TP amount that obtains less and impure (being mixed with host protein), have shortcomings such as cross reaction with other pathogen, so false positive also happens occasionally.Along with the clone in succession who reaches treponemal antigen that popularizes of Protocols in Molecular Biology, it is more and more that recombinant antigen is applied to the syphilis experiment.The many TP antigen of research has TPN17, TPN47, TPN15, TPN44.5, TPN36, TP0453, TP0684 and TPr family at present.The recombinant antigen that adopts recombinant DNA technology to prepare can overcome the shortcoming of complete TP antigen, can prepare endless special reorganization TP antigen fast, economically.
The syphilis specific antibody detection method comprises microspironema pallidum specific antibody GAT (TPPA), enzyme linked immunosorbent assay (ELISA), FTA-ABS test (FTA-ABS) and immunoblot assay (Western-Blot) etc., and its specificity is all higher.Usually, TPPA, ELISA be as clinical primary dcreening operation, and when the serology positive, or clinical diagnosis needs to adopt FTA-ABS and Western-blot as validation test when having any problem.Wherein, FTA-ABS needs fluorescent microscope, and its result judges and need do some training very often and just can finish than the professional of rich experiences that therefore, its application is subjected to certain restriction.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.
Summary of the invention
The purpose of this invention is to provide a kind of syphilis specific total antibodies and cuorin antibody mediated immunity trace kit and preparation method thereof.
Syphilis specific total antibodies of the present invention and cuorin antibody mediated immunity trace kit are provided with carrier board, nitrocellulose membrane, syphilis specific total antibodies and cuorin antibody detection line, control line, and syphilis specific total antibodies and cuorin antibody detection line and control line are located on the nitrocellulose membrane successively; Wrap by syphilis specific recombinant antigen and cuorin antigen at syphilis specific total antibodies and cuorin antibody detection line place, wrap at the control line place by people Ig antibody; The anti-people Ig of horseradish peroxidase-labeled antibody.
Described syphilis specific recombinant antigen can be TPN15, TPN17, TPN37, TPN44.5, TPN47 etc.
Described carrier board can adopt the PVC plate.
The preparation method of described syphilis specific total antibodies and cuorin antibody mediated immunity trace kit may further comprise the steps:
1) preparation syphilis specific recombinant antigen
Adopt gene clone technology, the DNA of pcr amplification coding treponemal antigen, and make its expression in the insertion Escherichia coli, get the syphilis specific recombinant antigen;
2) preparation cuorin antigen
The pig myocardium tissue is air-dry behind low-temperature quick-freezing, grinds to form behind the powdery with methyl alcohol and chloroform extracting, and extract is by silica gel column chromatography wash-out, the cuorin antigen of dna purity more than 95%;
3) point sample of nitrocellulose filter
Bag by people Ig antibody, is dried at control line place bag by syphilis specific recombinant antigen and cuorin antigen on the total antibody detection line of nitrocellulose filter;
4) the total specific fragment Ig of the anti-people of preparation chain monoclonal antibody
With the total specific fragment Ig of people is antigen immune Balb/c mouse, and by hybridoma technology, screening obtains the hybridoma cell strain of the anti-people Ig of stably excreting monoclonal antibody;
5) horseradish peroxidase (HRP) mark of anti-people Ig monoclonal antibody
Adopt the sodium periodate method to carry out horseradish peroxidase (HRP) mark;
6) preparation HP immunoblotting kit
Nitrocellulose membrane is sticked on the carrier board upper surface, and syphilis specific total antibodies and cuorin antibody detection line and control line are located on the nitrocellulose membrane successively; Wrap by syphilis specific recombinant antigen and cuorin antigen at syphilis specific total antibodies and cuorin antibody detection line place, wrap by people Ig antibody at the control line place, the anti-people Ig antibody and the substrate mutual group thereof that are cut into strip and enzyme labeling with cutting cutter are dressed up syphilis specific total antibodies and cuorin antibody mediated immunity trace kit.
In step 1), 3), 6) in, described syphilis specific recombinant antigen is TPN15, TPN17, TPN37, TPN44.5, TPN47 etc.
In step 2) in, described methyl alcohol and chloroform, methyl alcohol by volume: chloroform can be 2: 1.
In step 4), adopt the method identification of M cAb of ELISA to tire at 1: 10
7More than.
In step 5), the concentration of described syphilis specific recombinant antigen and the total antibody of people can be 1～4mg/mL, and the point sample amount can be 1 μ L/cm; The concentration of described cuorin antigen can be 0.003%-0.03%, and the point sample amount can be 1 μ L/cm.
In step 6), the substrate of described horseradish peroxidase can be 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 syphilis specific total antibodies and cardiolipin antibody detection kit, can be used for syphilis specific total antibodies and cuorin detection of antibodies 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 syphilis specific total antibodies of the present invention and the cuorin antibody mediated immunity trace kit is formed synoptic diagram.
Fig. 2 is the experimental result pattern diagram.In Fig. 2, J is the synoptic diagram before using, and I is invalid test (product quality problem), the negative result of H, and A～F is a TP-specific total antibodies positive findings; G is the anticardiolipin antibody positive findings.2 is the total antibody detection line of syphilis specific TPN-15-, 3 is the total antibody detection line of syphilis specific TPN-17-, 4 is the total antibody detection line of syphilis specific TPN-37-, 5 is the total antibody detection line of syphilis specific TPN-44.5-, 6 is the total antibody detection line of syphilis specific TPN-47-, 7 is the cuorin antibody detection line, and 8 is control line.
Embodiment
Following examples will the present invention is further illustrated in conjunction with the accompanying drawings.
Referring to Fig. 1, syphilis specific total antibodies of the present invention and cuorin antibody mediated immunity trace kit embodiment are provided with carrier board 1, syphilis specific total antibodies and cuorin antibody detection line 2～7, control line 8 and nitrocellulose membrane (NC film) 9.
Nitrocellulose membrane 9 sticks on carrier board 1 upper surface, and syphilis specific total antibodies and cuorin antibody detection line 2～7 and control line 8 are located on the nitrocellulose membrane 9 successively; Wrap respectively by syphilis specific recombinant antigen TPN15, TPN17, TPN37, TPN44.5, TPN47 and cuorin antigen at syphilis specific total antibodies and cuorin antibody detection line place, wrap by people Ig antibody at control line 8 places.
Described carrier board adopts the PVC plate.
The preparation method of described syphilis specific total antibodies and cuorin antibody mediated immunity trace kit may further comprise the steps:
1) preparation TPN15, TPN17, TPN37, TPN44.5, TPN47 syphilis specific recombinant antigen
Adopt gene clone technology, the DNA of pcr amplification coding treponemal antigen, and make its expression in the insertion Escherichia coli, get TPN15, TPN17, TPN37, TPN44.5, TPN47 syphilis specific recombinant antigen.
2) preparation cuorin antigen
The pig myocardium tissue is air-dry behind low-temperature quick-freezing, grinds to form behind the powdery with methyl alcohol and chloroform extracting, and extract is by silica gel column chromatography wash-out, the cuorin antigen of dna purity more than 95%.
3) point sample of nitrocellulose filter
Bag by people Ig antibody, is dried at control line place bag by TPN15, TPN17, TPN37, TPN44.5, TPN47 syphilis specific recombinant antigen and cuorin antigen on the total antibody detection line of nitrocellulose filter.
4) the total specific fragment Ig of the anti-people of preparation chain monoclonal antibody
With the total specific fragment Ig of people is antigen immune Balb/c mouse, and by hybridoma technology, screening obtains the hybridoma cell strain of the anti-people Ig of stably excreting monoclonal antibody.Adopt the method identification of M cAb of ELISA to tire at 1: 10
7More than.
5) horseradish peroxidase (HRP) mark of anti-people Ig monoclonal antibody
Adopt the sodium periodate method to carry out horseradish peroxidase (HRP) mark.
6) preparation HP immunoblotting kit
Nitrocellulose membrane is sticked on the carrier board upper surface, and syphilis specific total antibodies and cuorin antibody detection line and control line are located on the nitrocellulose membrane successively; Wrap by TPN15, TPN17, TPN37, TPN44.5, TPN47 syphilis specific recombinant antigen and cuorin antigen at syphilis specific total antibodies and cuorin antibody detection line place, wrap by people Ig antibody at the control line place, the anti-people Ig antibody and the substrate mutual group thereof that are cut into strip and enzyme labeling with cutting cutter are dressed up syphilis specific total antibodies and cuorin antibody mediated immunity trace kit.
Below provide the clinical samples that adopts syphilis specific total antibodies and cuorin antibody mediated immunity trace kit to detect the patient:
1) sample adopts 0.01mol/L PBS damping fluid to carry out dilution in 1: 50.
2) sealing: adopt 5% skimmed milk power (preparation of 0.01mol/LPBST damping fluid) as confining liquid, waving incubation 30min on the shaking table in room temperature (18～25 ℃).
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-people Ig 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, is diagnosed as the syphilis (see figure 2).
Below provide the performance calibrating of syphilis specific total antibodies and cuorin antibody mediated immunity trace 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: the positive control serum with the different titers of the total antibody of TP-, the TRUST positive adopts syphilis specific total antibodies and the calibrating of cuorin antibody mediated immunity trace kit for each 50 parts, calculates positive coincidence rate.Definite employing FTA-ABS of positive control serum (German Ou Meng company) method and the definite clinical samples of TRUST (Xiamen Ying Kexin wound).
3) negative sample coincidence rate:, calculate positive coincidence rate with 50 parts of negative control serum calibratings.Definite employing TPPA (Japanese fuji Co., Ltd.) of negative control serum and the clinical samples that TRUST (Xiamen Ying Kexin wound) method is determined.
4) criticize in difference: same batch of syphilis specific total antibodies and cuorin antibody mediated immunity trace kit, detect with characteristic serum, require the positive serum testing result to show the shade unanimity of colour band, the feminine gender as a result that negative serum detects.
5) differences between batches: different batches syphilis specific total antibodies and cuorin antibody mediated immunity trace 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 syphilis specific total antibodies and cuorin antibody mediated immunity trace 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, syphilis specific total antibodies and cuorin antibody mediated immunity trace kit are placed 37 ℃ to be detected 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 syphilis specific total antibodies and cuorin antibody detection line and control line are located on the nitrocellulose membrane successively; Wrap by TPN15, TPN17, TPN37, TPN44.5, TPN47 syphilis specific total antibodies and cuorin antigen at syphilis specific total antibodies and cuorin antibody detection line place, wrap by people Ig antibody at the control line place, the anti-people Ig and the substrate mutual group thereof that are cut into strip and enzyme labeling with cutting cutter are dressed up syphilis specific total antibodies and cuorin antibody mediated immunity trace kit.
1) sample adopts 0.01mol/L PBS damping fluid to carry out dilution in 1: 50.
2) sealing: adopt 5% skimmed milk power (preparation of 0.01mol/LPBST damping fluid) as confining liquid, waving incubation 30min on the shaking table in room temperature (18～25 ℃).
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-people Ig 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, is diagnosed as the syphilis (see figure 2).
Similar to embodiment 1, its difference is that nitrocellulose membrane only is made up of TPN15, TPN17, TPN37, TPN44.5 syphilis specific total antibodies and cuorin antigen, does not contain the TPN47 syphilis specific total antibodies.The result judges identical with embodiment 1.
Similar to embodiment 1, its difference is that nitrocellulose membrane only is made up of TPN15, TPN17, TPN37, TPN47 syphilis specific total antibodies and cuorin antigen, does not contain the TPN44.5 syphilis specific total antibodies.The result judges identical with embodiment 1.
Similar to embodiment 1, its difference is that nitrocellulose membrane only is made up of TPN15, TPN17, TPN44.5, TPN47 syphilis specific total antibodies and cuorin antigen, does not contain the TPN37 syphilis specific total antibodies.The result judges identical with embodiment 1.
Similar to embodiment 1, its difference is that nitrocellulose membrane only is made up of TPN15, TPN37, TPN44.5, TPN47 syphilis specific total antibodies and cuorin antigen, does not contain the TPN17 syphilis specific total antibodies.The result judges identical with embodiment 1.
Similar to embodiment 1, its difference is that nitrocellulose membrane only is made up of TPN17, TPN37, TPN44.5, TPN47 syphilis specific total antibodies and cuorin antigen, does not contain the TPN15 syphilis specific total antibodies.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.
The performance verification test: the scheme by embodiment 1 prepares syphilis specific total antibodies and cuorin antibody mediated immunity trace kit, carries out performance verification then.
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: the positive control serum with the different titers of the total antibody positive of TP-and the TRUST positive adopts syphilis specific total antibodies and the calibrating of cuorin antibody mediated immunity trace kit for each 50 parts, calculates positive coincidence rate.Definite employing FTA-ABS of positive control serum (German Ou Meng company) method and the definite clinical samples of TRUST (Xiamen Ying Kexin wound).
3) negative sample coincidence rate:, calculate positive coincidence rate with 50 parts of negative control serum calibratings.The clinical samples that definite employing TPPA (Japanese fuji Co., Ltd.) method of negative control serum is determined.
4) criticize in difference: same batch of syphilis specific total antibodies and cuorin antibody mediated immunity trace kit, detect with characteristic serum, require the positive serum testing result to show the shade unanimity of colour band, the feminine gender as a result that negative serum detects.
5) differences between batches: different batches syphilis specific total antibodies and cuorin antibody mediated immunity trace 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 syphilis specific total antibodies and cuorin antibody mediated immunity trace 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, syphilis specific total antibodies and cuorin antibody mediated immunity trace kit are placed 37 ℃ to be detected 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 (9)
1. syphilis specific total antibodies and cuorin antibody mediated immunity trace kit, it is characterized in that being provided with carrier board, nitrocellulose membrane, syphilis specific total antibodies and cuorin antibody detection line, control line, syphilis specific total antibodies and cuorin antibody detection line and control line are located on the nitrocellulose membrane successively; Wrap by syphilis specific recombinant antigen and cuorin antigen at syphilis specific total antibodies and cuorin antibody detection line place, wrap at the control line place by people Ig antibody; The anti-people Ig of horseradish peroxidase-labeled antibody.
2. syphilis specific total antibodies as claimed in claim 1 and cuorin antibody mediated immunity trace kit is characterized in that described syphilis specific recombinant antigen is TPN15, TPN17, TPN37, TPN44.5, TPN47 syphilis specific recombinant antigen.
3. syphilis specific total antibodies as claimed in claim 1 and cuorin antibody mediated immunity trace kit is characterized in that described carrier board adopts the PVC plate.
4. the preparation method of syphilis specific total antibodies as claimed in claim 1 and cuorin antibody mediated immunity trace kit is characterized in that may further comprise the steps:
1) preparation syphilis specific recombinant antigen
Adopt gene clone technology, the DNA of pcr amplification coding treponemal antigen, and make its expression in the insertion Escherichia coli, get the syphilis specific recombinant antigen;
2) preparation cuorin antigen
The pig myocardium tissue is air-dry behind low-temperature quick-freezing, grinds to form behind the powdery with methyl alcohol and chloroform extracting, and extract is by silica gel column chromatography wash-out, the cuorin antigen of dna purity more than 95%;
3) point sample of nitrocellulose filter
Bag by people Ig antibody, is dried at control line place bag by syphilis specific recombinant antigen and cuorin antigen on the total antibody detection line of nitrocellulose filter;
4) the total specific fragment Ig of the anti-people of preparation chain monoclonal antibody
With the total specific fragment Ig of people is antigen immune Balb/c mouse, and by hybridoma technology, screening obtains the hybridoma cell strain of the anti-people Ig of stably excreting monoclonal antibody;
5) horseradish peroxidase-labeled of anti-people Ig monoclonal antibody
Adopt the sodium periodate method to carry out horseradish peroxidase-labeled;
6) preparation HP immunoblotting kit
Nitrocellulose membrane is sticked on the carrier board upper surface, and syphilis specific total antibodies and cuorin antibody detection line and control line are located on the nitrocellulose membrane successively; Wrap by syphilis specific recombinant antigen and cuorin antigen at syphilis specific total antibodies and cuorin antibody detection line place, wrap by people Ig antibody at the control line place, the anti-people Ig antibody and the substrate mutual group thereof that are cut into strip and enzyme labeling with cutting cutter are dressed up syphilis specific total antibodies and cuorin antibody mediated immunity trace kit.
5. the preparation method of syphilis specific total antibodies as claimed in claim 4 and cuorin antibody mediated immunity trace kit, it is characterized in that in step 1), 3), 6) in, described syphilis specific recombinant antigen is TPN15, TPN17, TPN37, TPN44.5, TPN47.
6. the preparation method of syphilis specific total antibodies as claimed in claim 4 and cuorin antibody mediated immunity trace kit is characterized in that in step 2) in, described methyl alcohol and chloroform, methyl alcohol by volume: chloroform is 2: 1.
7. the preparation method of syphilis specific total antibodies as claimed in claim 4 and cuorin antibody mediated immunity trace kit is characterized in that in step 4), adopts the method identification of M cAb of ELISA to tire at 1: 10
7More than.
8. the preparation method of syphilis specific total antibodies as claimed in claim 4 and cuorin antibody mediated immunity trace kit, it is characterized in that in step 5), the concentration of described syphilis specific recombinant antigen and the total antibody of people is 1～4mg/mL, and the point sample amount is 1 μ L/cm; The concentration of described cuorin antigen is 0.003%～0.03%, and the point sample amount is 1 μ L/cm.
9. the preparation method of syphilis specific total antibodies as claimed in claim 4 and cuorin antibody mediated immunity trace kit, it is characterized in that in step 6) 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).
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| CN107533060A (en) * | 2015-03-10 | 2018-01-02 | 生物辐射实验室股份有限公司 | Combine treponema and the test of non-treponema syphilis |
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| CN107533060A (en) * | 2015-03-10 | 2018-01-02 | 生物辐射实验室股份有限公司 | Combine treponema and the test of non-treponema syphilis |
| US10324091B2 (en) | 2015-03-10 | 2019-06-18 | Bio-Rad Laboratories, Inc. | Combination treponemal and non-treponemal syphilis test |
| CN107533060B (en) * | 2015-03-10 | 2019-11-05 | 生物辐射实验室股份有限公司 | Combine treponema and the test of non-treponema syphilis |
| US10648981B2 (en) | 2015-03-10 | 2020-05-12 | Bio-Rad Laboratories, Inc. | Combination treponemal and non-treponemal syphilis test |
| US10942182B2 (en) | 2015-03-10 | 2021-03-09 | Bio-Rad Laboratories, Inc. | Combination treponemal and non-treponemal syphilis test |
| US11913954B2 (en) | 2015-03-10 | 2024-02-27 | Bio-Rad Laboratories, Inc. | Combination treponemal and non-treponemal syphilis test |
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