CN101858914A - Reagent strip for testing syphilis specific total antibodies through gold immunochromatographic assay and preparation method thereof - Google Patents

Reagent strip for testing syphilis specific total antibodies through gold immunochromatographic assay and preparation method thereof Download PDF

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CN101858914A
CN101858914A CN201010179095A CN201010179095A CN101858914A CN 101858914 A CN101858914 A CN 101858914A CN 201010179095 A CN201010179095 A CN 201010179095A CN 201010179095 A CN201010179095 A CN 201010179095A CN 101858914 A CN101858914 A CN 101858914A
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syphilis
antigen
tpn17
tpn47
specific total
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CN101858914B (en
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林丽蓉
杨天赐
张忠英
张长弓
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Zhongshan Hospital Xiamen University
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Zhongshan Hospital Xiamen University
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Abstract

The invention provides a reagent strip for testing syphilis specific total antibodies through gold immunochromatographic assay and a preparation method thereof and relates to a reagent for testing the syphilis specific total antibodies. The reagent strip is provided with a carrier plate, a sample-adding pad, a colloidal gold pad, a nitrocellulose membrane, a syphilis specific total antibody testing line, a control line and an absorption pad. The method comprises the following steps: preparing the recombinant syphilis antigens TPN17 and TPN47; applying samples of the nitrocellulose membrane; preparing the colloidal gold; labeling the colloidal gold and TPN17 and TPN47; and preparing the strip for testing through immunochromatographic assay. The reagent strip can be used for testing the syphilis specific total antibodies in the specimens of whole blood, serum, blood plasma, cerebrospinal fluid and the like. The invention has the following advantages: during testing, the needed specimen quantity is small, special instruments are not needed, the results can be directly identified by naked eyes, testing is simple, convenient and rapid, the specificity is strong, the sensitivity is high, testing is accurate and reliable, the cost is low and the invention is widely applied.

Description

Syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar and preparation method thereof
Technical field
The present invention relates to a kind of syphilis specific total antibodies detectable, especially relate to syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar that a kind of employing colloidal gold immunochromatographimethod technology (immunochromatography) carries out 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, syphilis still is worldwide public health problem so far, lacks effective administrative control measure, annual global nearly 1,200 ten thousand patient, wherein 600,000 pregnant woman patients.(referring to: Health Protection Agency Centre for Infections.International Encyclopediaof Public Health-Syphilis[M] .London, UK:Health Protection Agency Centre, 2008,289-297) 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 do not disappear all the life (referring to: Luis J F, Felipe U S, Santa G C, et al.Evaluation of a rapid strip and a particle agglutinationtests for syphilis diagnosis[J] .Diagnostic Microbiology and Infectious Disease, 2007,59:123-126); And another kind of antibody materials reagin generation is later, generally produce in 5~7 weeks of infected back (referring to: Lin Yue circle .TPPA and value and the clinical relevant issues [J] of TRUST in controller used in syphilis diagnosis. the radioimmunology magazine, 2009,22 (3): 295-297.), and late syphilis, syphilis treatment later stage and latent syphilis may be negative.Therefore positive rate, the susceptibility of syphilis specific antibody are 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.Martina H etc. (referring to: Martina H, Daan W N, Mart M, et al.Comparison of a Treponema pallidum IgM immunoblot with a 19S fluorescenttreponemal antibody absorption test for the diagnosis of congenital syphilis[J] .DiagnosticMicrobiology and Infectious Disease, 2007,59:61-66.) think that TP-IgM is a syphilis early infection and a movable serologic marker, Li Burong etc. (referring to: 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 that once more syphilization is (referring to Rawstron SA, Mehta S, Bromberg K, et al.Evaluation of a Treponema pallidum2specific IgMenzyme immunoassay and Treponema pallidum western blot antibody detection in the diagnosisofmaternal 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.
Early stage serological method uses complete microspironema pallidum as antigen, the TP of research and diagnosis usefulness obtains with TP infected rabbits testis, the TP amount that the cost of this method is big, obtain less, impure (being mixed with host protein), have cross reaction with other pathogen, so false positive happens occasionally also.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 test is the syphilis confirmatory test, comprises TPHA, TPPA, and ELISA, FTA-ABS and Western-blot etc., its specificity is all higher.Yet the anti-system form in the face of severe not only needs special detection means accurately, also needs a kind of more simple and efficient reagent 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 syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar and preparation method thereof.
Syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar of the present invention is provided with carrier board, application of sample pad, collaurum pad, nitrocellulose membrane (NC film), syphilis specific total antibodies detection line, control line and absorption pad.
Application of sample pad, collaurum pad, nitrocellulose membrane and absorption pad stick on the carrier board upper surface successively, one end of application of sample pad is located on the end of collaurum pad, the other end of collaurum pad is located on the end of nitrocellulose membrane, one end of absorption pad is located on the other end of nitrocellulose membrane, and syphilis specific total antibodies detection line and control line are located on the nitrocellulose membrane successively; , wrapped by the IgG antibody of goat-anti syphilis antigen TPN17 and TPN47 by anti-people Ig monoclonal antibody or syphilis specific antigen TPN17 and/or syphilis specific antigen TPN47 at syphilis specific total antibodies detection line place bag at the control line place.
Described carrier board can adopt the PVC plate.
The preparation method of described syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar may further comprise the steps:
1) preparation syphilis recombinant antigen TPN17 and TPN47
Adopt gene clone technology, the DNA of pcr amplification coding treponemal antigen, and make its expression in the insertion Escherichia coli, get syphilis recombinant antigen TPN17 and TPN47
2) point sample of nitrocellulose filter
Bag by goat-anti syphilis antigen TPN17 and TPN47 IgG antibody, is dried at control line place bag by anti-people Ig monoclonal antibody on the syphilis specific total antibodies detection line;
3) preparation collaurum
Adopt the trisodium citrate method of reducing to prepare the 25nm collaurum, getting 1% gold chloride 1mL joins in the 100mL deionization distilled water, the gold chloride concentration that obtains is 0.01%, place the flask of band condensing unit to be heated to boiling, add 1% trisodium citrate aqueous solution 2.0mL under the magnetic force heated and stirred, continue heating till solution is vinicolor, cooling is placed in the brown bottle 4 ℃ of refrigerators and preserves standby;
4) mark of collaurum and TPN17, TPN47
The mark of collaurum and syphilis specific antigen TPN17: get collaurum 10ml, transfer to pH5.4, add 100 μ g TPN17 with 0.1mol/L NaOH, mixing, place 5min, add 5%BSA 1ml mixing, 4 ℃, the centrifugal 1h of 10000r/min, abandon supernatant, to precipitate with the TBS damping fluid and be dissolved to 10ml, 4 ℃, the centrifugal 1h of 10000r/min abandon supernatant, precipitation is diluted to 1ml with TBS, gets the TPN17 antigen of colloid gold label;
The same operation of mark of collaurum and syphilis specific antigen TPN47, the TPN47 antigen of colloid gold label;
After the TPN47 antigen of the TPN17 antigen of colloid gold label and colloid gold label mixed, be applied to equably on the glass fibre membrane, oven dry is prepared into the collaurum pad;
5) preparation immunochromatography detector bar
Application of sample pad, collaurum pad, nitrocellulose membrane and absorption pad are sticked on the carrier board upper surface successively, one end of application of sample pad is located on the end of collaurum pad, the other end of collaurum pad is located on the end of nitrocellulose membrane, one end of absorption pad is located on the other end of nitrocellulose membrane, and syphilis specific total antibodies detection line and control line are located on the nitrocellulose membrane successively; By anti-people Ig monoclonal antibody, by the IgG antibody of goat-anti syphilis antigen TPN17 and TPN47, be cut into strip at syphilis specific total antibodies detection line place bag, get syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar with cutting cutter at control line place bag.
In step 2) in, the concentration of described anti-people Ig monoclonal antibody is 1~4mg/mL, and the IgG antibody of goat-anti syphilis antigen TPN17 and TPN47 was mixed with anti-TPN47-IgG antibody by anti-TPN17-IgG antibody in 1: 1 by volume, and its final concentration is 1~4mg/mL; Three's point sample amount is 1 μ L/cm.
In step 3), the concentration of described trisodium citrate can be 2%.
In step 4), described with colloid gold label TPN17 antigen and the TPN47 antigen of colloid gold label mix, preferably the TPN47 antigen of the TPN17 antigen of colloid gold label and colloid gold label is with volume ratio 1: mix (0.2~5); The temperature of described oven dry can be 37 ℃.
The invention provides a kind of employing colloidal gold immunochromatographimethod technology and set up syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar, can be used for the detection of syphilis specific total antibodies in the samples such as whole blood, serum, blood plasma and cerebrospinal fluid.When detecting, required specimen amount 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 syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar embodiment of the present invention is formed synoptic diagram.
Fig. 2 is the experimental result pattern diagram.In Fig. 2, (1) is the synoptic diagram before using, and (2) are invalid test (product quality problem), (3) malicious specific total antibodies feminine gender, (4) malicious specific total antibodies positive.
Embodiment
Following examples will the present invention is further illustrated in conjunction with the accompanying drawings.
Referring to Fig. 1, syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar embodiment of the present invention is provided with carrier board 1, application of sample pad 2, collaurum pad 3, nitrocellulose membrane (NC film) 4, syphilis specific total antibodies detection line 5, control line 7 and absorption pad 8.
Application of sample pad 2, collaurum pad 3, nitrocellulose membrane 4 and absorption pad 8 stick on carrier board 1 upper surface successively, one end of application of sample pad 2 is located on the end of collaurum pad 3, the other end of collaurum pad 3 is located on the end of nitrocellulose membrane 4, one end of absorption pad 8 is located on the other end of nitrocellulose membrane 4, and syphilis specific total antibodies detection line 5 and control line 8 are located on the nitrocellulose membrane 4 successively; , wrapped by the IgG antibody of goat-anti syphilis antigen TPN17 and TPN47 by anti-people Ig monoclonal antibody or syphilis specific antigen TPN17 and/or syphilis specific antigen TPN47 at syphilis specific total antibodies detection line 5 places bag at control line 7 places.
Described carrier board 1 adopts the PVC plate.
The preparation method of described syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar may further comprise the steps:
1) preparation syphilis recombinant antigen TPN17 and TPN47
Adopt gene clone technology, the DNA of pcr amplification coding treponemal antigen, and make its expression in the insertion Escherichia coli, get syphilis recombinant antigen TPN17 and TPN47.
2) point sample of nitrocellulose filter
Bag is by anti-people Ig monoclonal antibody on the syphilis specific total antibodies detection line, wrap by goat-anti syphilis antigen TPN17 and TPN47 IgG antibody at the control line place, dry, the concentration of described anti-people Ig monoclonal antibody is 1~4mg/mL, the IgG antibody of goat-anti syphilis antigen TPN17 and TPN47 was mixed with anti-TPN47-IgG antibody by anti-TPN17-IgG antibody in 1: 1 by volume, and its final concentration is 1~4mg/mL; Three's point sample amount is 1 μ L/cm.
3) preparation collaurum
Adopt the trisodium citrate method of reducing to prepare the 25nm collaurum, getting 1% gold chloride 1mL joins in the 100mL deionization distilled water, the gold chloride concentration that obtains is 0.01%, place the flask of band condensing unit to be heated to boiling, add 1% trisodium citrate aqueous solution 2.0mL under the magnetic force heated and stirred, continue heating till solution is vinicolor, cooling is placed in the brown bottle 4 ℃ of refrigerators and preserves standbyly, and the concentration of described trisodium citrate can be 2%.
4) mark of collaurum and TPN17, TPN47
The mark of collaurum and syphilis specific antigen TPN17: get collaurum 10ml, transfer to pH5.4, add 100 μ g TPN17 with 0.1mol/L NaOH, mixing, place 5min, add 5%BSA 1ml mixing, 4 ℃, the centrifugal 1h of 10000r/min, abandon supernatant, to precipitate with the TBS damping fluid and be dissolved to 10ml, 4 ℃, the centrifugal 1h of 10000r/min abandon supernatant, precipitation is diluted to 1ml with TBS, gets the TPN17 antigen of colloid gold label.
The same operation of mark of collaurum and syphilis specific antigen TPN47, the TPN47 antigen of colloid gold label.
After the TPN47 antigen of the TPN17 antigen of colloid gold label and colloid gold label mixed, be applied to equably on the glass fibre membrane, oven dry is prepared into the collaurum pad.
Described with colloid gold label TPN17 antigen and the TPN47 antigen of colloid gold label mix, preferably the TPN47 antigen of the TPN17 antigen of colloid gold label and colloid gold label is with volume ratio 1: mix (0.2~5); The temperature of described oven dry can be 37 ℃.
5) preparation immunochromatography detector bar
Application of sample pad, collaurum pad, nitrocellulose membrane and absorption pad are sticked on the carrier board upper surface successively, one end of application of sample pad is located on the end of collaurum pad, the other end of collaurum pad is located on the end of nitrocellulose membrane, one end of absorption pad is located on the other end of nitrocellulose membrane, and syphilis specific total antibodies detection line and control line are located on the nitrocellulose membrane successively; By anti-people Ig monoclonal antibody, by the IgG antibody of goat-anti syphilis antigen TPN17 and TPN47, be cut into strip at syphilis specific total antibodies detection line place bag, get syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar with cutting cutter at control line place bag.
Below provide immunochromatographyassay assay patient's clinical samples:
Referring to Fig. 2, get sample to be checked (whole blood, serum, blood plasma, cerebrospinal fluid) 5~80 μ L, leave standstill the 20min observations.Only there is an aubergine band to occur, then is judged to feminine gender at detector bar check plot C; All there is an aubergine band to occur at detection zone T and check plot C, then is judged to the positive; After application of sample detected, the aubergine band did not all appear in detection zone T and check plot C, is null result.
Below provide the performance calibrating of syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar:
1) visual examination: white 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-adopts the calibrating of syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bars for each 50 parts, calculates positive coincidence rate.The clinical samples that definite employing TPPA (Japanese fuji Co., Ltd.) method of positive control serum is determined.
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) sensitivity detects: detect with the indoor quality controlled serum of the Ministry of Public Health, the minimum detectability degree should be less than or equal to 4NCU/mL, and is suitable with TPPA (Japanese fuji Co., Ltd.).
5) criticize interior difference: same batch of syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar, 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) differences between batches: different batches syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar, 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.
7) interference test: testing result is not subjected to the interference of sample hemolysis (n=50), piarhemia (n=50) and jaundice (n=50).Serum (or blood plasma) is from the applicant's clinical samples.
8) cross reaction: adopt syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar, 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.The serum of autoimmunity systemic disease is from the applicant's clinical definite patient.
9) Detection of Stability: use the Arrhenius rule, reagent strip 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
Bag is by anti-people Ig monoclonal antibody on the total detection line of nitrocellulose filter (NC film), and by goat-anti syphilis antigen (TPN17 and TPN47) IgG antibody, room temperature is dried at control line place bag, and the sealing room temperature preservation is standby.Wherein, the concentration of anti-people Ig monoclonal antibody is 1mg/mL, and goat-anti syphilis antigen (TPN17 and TPN47) IgG antibody was mixed with anti-TPN47-IgG antibody by anti-TPN17-IgG antibody in 1: 1 by volume, and its final concentration is 1mg/mL; The two point sample amount is 1 μ L/cm.
After the syphilis recombinant antigen TPN17 of the golden mark of purifying and TPN47 mixed with volume ratio at 1: 1, be applied to equably on the all-glass paper,, be prepared into the gold colloid pad, seal standby 37 ℃ of oven dry.The glass fibre that immobilised tunica fibrosa is combined with collaurum, thieving paper etc. are combined by PVC adhesive sticker base plate in certain sequence, are cut into the certain width detector bar with cutting cutter.Syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar is packed in the aluminium foil bag with drying agent, and machine seals, and sealing is preserved.
Get sample serum 50 μ L to be checked, application of sample leaves standstill the 20min observations in syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar sample application zone.Only there is an aubergine band to occur, then is judged to feminine gender in syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar check plot; All there is an aubergine band to occur at detection zone and check plot, then is judged to the positive; After application of sample detected, the aubergine band did not all appear in detection zone and check plot, is null result.
Embodiment 2
Similar to embodiment 1, difference is that the gold colloid pad only is made up of TPN17, does not contain TPN47.The result judges identical with embodiment 1.
Embodiment 3
Similar to embodiment 1, difference is that the gold colloid pad only is made up of TPN47, does not contain TPN17.The result judges identical with embodiment 1.
Embodiment 4
Similar to embodiment 1, difference is that sample to be checked is a samples of CSF, and the result judges identical with embodiment 1.
Embodiment 5
The performance verification test: the scheme by embodiment 1 prepares the syphilis specific total antibodies quick detection reagent, carries out performance verification then.
1) visual examination: white 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 the reagent strip width does not have and cuts oblique phenomenon at 3 ± 0.1mm.
2) positive sample coincidence rate: 50 parts of microspironema pallidum specific antibody GATs (TPPA) (Japanese fuji Co., Ltd.) detect the total antibody positive control serum of determining of TP-, adopt syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar to detect 50 parts of the total antibody positives of TP-, positive sample coincidence rate 100%.
3) negative sample coincidence rate: the negative control serum of 50 parts of microspironema pallidum specific antibody GATs (TPPA) (Japanese fuji Co., Ltd.), adopt syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar not detect positive sample, negative sample coincidence rate 100%.
4) sensitivity detects: detect with the indoor quality controlled serum of the Ministry of Public Health, the minimum detectability degree should be less than or equal to 4NCU/mL, and is suitable with TPPA (Japanese fuji Co., Ltd.).
5) criticize interior difference: same batch of syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar, detect with characteristic positive serum (TPPA (Japanese fuji Co., Ltd.) detects the positive TP-Headquarters of the General Staff of the clinical samples of determining than high, medium and low serum), identical titre testing result shows the shade unanimity of colour band, the feminine gender as a result that negative serum detects.
6) differences between batches: different batches syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar, detect with characteristic positive serum (TPPA (Japanese fuji Co., Ltd.) detects the positive TP-Headquarters of the General Staff of the clinical samples of determining than high, medium and low serum), identical titre testing result shows the shade unanimity of colour band, the feminine gender as a result that negative serum detects.
7) interference test: testing result is not subjected to the interference of sample hemolysis (n=50), piarhemia (n=50) and jaundice (n=50).
8) cross reaction: adopt syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar, carry out the detection of systemic loupus erythematosus (n=30), rheumatoid disease (n=38), autoallergic autoimmunity systemic diseases such as (n=40), do not find cross reaction.
9) Detection of Stability: syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar is placed 37 ℃ detects after 20 days, more than every index do not have marked change.
Detection of the present invention is carried out on a syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar, realizes the detection of syphilis specific total antibodies by dual mode.
Mode one: utilize the colloidal gold immunochromatographimethod technology, wrap respectively by anti-people Ig monoclonal antibody [or syphilis specific antigen (TPN17 and/or TPN47)] and goat-anti syphilis antigen (TPN17 and/or TPN47) IgG antibody at total antibody detection line and control line place on nitrocellulose filter.Be coated in advance on the all-glass paper after the syphilis recombinant antigen TPN17 of the golden mark of purifying and TPN47 mixed in certain proportion, dried is prepared into the gold colloid pad, is aided with appropriate sample preparation pad again, composite reagent bar (assembling mode is seen Fig. 1).When detecting positive sample, syphilis specific total antibodies combines the formation immune complex with the recombinant antigen TPN17 and/or the TPN47 of colloid gold label in the sample.Because the chromatography effect, compound moves forward along paper slip thieving paper direction.During through detection line, 1. the total antibody mediated immunity compound of TP combines formation " Au-TPN17 (and/or TPN47)-total antibody of the anti-syphilis of specificity-anti-people Ig monoclonal antibody-solid phase material " or " Au-TPN17 (and/or TPN47)-total antibody of the anti-syphilis of specificity-anti-people Ig monoclonal antibody-solid phase material " centre-fills with the anti-people Ig monoclonal antibody [or syphilis specific antigen (TPN17 and/or TPN47)] of pre-bag quilt and condenses colour developing; 2. free gold mark antigen is then in the antibodies of control line place and goat-anti syphilis antigen (TPN17 and TPN47) and enrichment develops the color.Negative sample then only develops the color at the control line place.
Mode two, envelope antigen, the antibody of mode one are exchanged: wrap respectively by syphilis recombinant antigen (TPN17/TPN47 combination) and human IgG antibody at total antibody detection line and control line place on nitrocellulose filter, and the anti-people Ig monoclonal antibody of golden mark is coated on the all-glass paper in advance.
The syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar that dual mode is prepared, all can be used for the detection of syphilis specific total antibodies in the samples such as whole blood, serum, blood plasma and cerebrospinal fluid, the sample consumption is small, 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.

Claims (8)

1. syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar is characterized in that being provided with carrier board, application of sample pad, collaurum pad, nitrocellulose membrane, syphilis specific total antibodies detection line, control line and absorption pad;
Application of sample pad, collaurum pad, nitrocellulose membrane and absorption pad stick on the carrier board upper surface successively, one end of application of sample pad is located on the end of collaurum pad, the other end of collaurum pad is located on the end of nitrocellulose membrane, one end of absorption pad is located on the other end of nitrocellulose membrane, and syphilis specific total antibodies detection line and control line are located on the nitrocellulose membrane successively; , wrapped by the IgG antibody of goat-anti syphilis antigen TPN17 and TPN47 by anti-people Ig monoclonal antibody or syphilis specific antigen TPN17 and/or syphilis specific antigen TPN47 at syphilis specific total antibodies detection line place bag at the control line place.
2. syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 1 is characterized in that described carrier board is the PVC plate.
3. the preparation method of syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 1 is characterized in that may further comprise the steps:
1) preparation syphilis recombinant antigen TPN17 and TPN47
Adopt gene clone technology, the DNA of pcr amplification coding treponemal antigen, and make its expression in the insertion Escherichia coli, get syphilis recombinant antigen TPN17 and TPN47;
2) point sample of nitrocellulose filter
Bag by goat-anti syphilis antigen TPN17 and TPN47 IgG antibody, is dried at control line place bag by anti-people Ig monoclonal antibody on the syphilis specific total antibodies detection line;
3) preparation collaurum
Adopt the trisodium citrate method of reducing to prepare the 25nm collaurum, getting 1% gold chloride 1mL joins in the 100mL deionization distilled water, the gold chloride concentration that obtains is 0.01%, place the flask of band condensing unit to be heated to boiling, add 1% trisodium citrate aqueous solution 2.0mL under the magnetic force heated and stirred, continue heating till solution is vinicolor, cooling is placed in the brown bottle 4 ℃ of refrigerators and preserves standby;
4) mark of collaurum and TPN17, TPN47
The mark of collaurum and syphilis specific antigen TPN17: get collaurum 10ml, transfer to pH5.4, add 100 μ g TPN17 with 0.1mol/L NaOH, mixing, place 5min, add 5%BSA 1ml mixing, 4 ℃, the centrifugal 1h of 10000r/min, abandon supernatant, to precipitate with the TBS damping fluid and be dissolved to 10ml, 4 ℃, the centrifugal 1h of 10000r/min abandon supernatant, precipitation is diluted to 1ml with TBS, gets the TPN17 antigen of colloid gold label;
The same operation of mark of collaurum and syphilis specific antigen TPN47, the TPN47 antigen of colloid gold label;
After the TPN47 antigen of the TPN17 antigen of colloid gold label and colloid gold label mixed, be applied to equably on the glass fibre membrane, oven dry is prepared into the collaurum pad;
5) preparation immunochromatography detector bar
Application of sample pad, collaurum pad, nitrocellulose membrane and absorption pad are sticked on the carrier board upper surface successively, one end of application of sample pad is located on the end of collaurum pad, the other end of collaurum pad is located on the end of nitrocellulose membrane, one end of absorption pad is located on the other end of nitrocellulose membrane, and syphilis specific total antibodies detection line and control line are located on the nitrocellulose membrane successively; By anti-people Ig monoclonal antibody, by the IgG antibody of goat-anti syphilis antigen TPN17 and TPN47, be cut into strip at syphilis specific total antibodies detection line place bag, get syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar with cutting cutter at control line place bag.
4. the preparation method of syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 3 is characterized in that in step 2) in, the concentration of described anti-people Ig monoclonal antibody is 1~4mg/mL.
5. the preparation method of syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 3, it is characterized in that in step 2) in, the IgG antibody of goat-anti syphilis antigen TPN17 and TPN47 was mixed with anti-TPN47-IgG antibody by anti-TPN17-IgG antibody in 1: 1 by volume, and its final concentration is 1~4mg/mL; Three's point sample amount is 1 μ L/cm.
6. the preparation method of syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 3 is characterized in that in step 3), and the concentration of described trisodium citrate is 2%.
7. the preparation method of syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 3, it is characterized in that in step 4), described with colloid gold label TPN17 antigen and the TPN47 antigen of colloid gold label mix, be that the TPN17 antigen of colloid gold label and the TPN47 antigen of colloid gold label mix with volume ratio 1: 0.2~5.
8. the preparation method of syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar as claimed in claim 3 is characterized in that in step 4), and the temperature of described oven dry is 37 ℃.
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CN102360012A (en) * 2011-09-19 2012-02-22 厦门大学附属中山医院 Immunochromatography detection reagent strip for combined detection of toxoplasmagondii IgG antibodies and total antibodies, and preparation method thereof
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CN102830229A (en) * 2012-08-27 2012-12-19 北京新兴四寰生物技术有限公司 Detection reagent of treponema pallidum IgM (Immunoglobulin M) antigen colloidal gold method and preparation method thereof
CN102830229B (en) * 2012-08-27 2014-12-17 北京新兴四寰生物技术有限公司 Detection reagent of treponema pallidum IgM (Immunoglobulin M) antigen colloidal gold method and preparation method thereof
CN105353125A (en) * 2015-11-13 2016-02-24 中国医学科学院输血研究所 A portable rapid test strip for syphilis for household use

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