CN101858914B - 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 PDFInfo
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- CN101858914B CN101858914B CN 201010179095 CN201010179095A CN101858914B CN 101858914 B CN101858914 B CN 101858914B CN 201010179095 CN201010179095 CN 201010179095 CN 201010179095 A CN201010179095 A CN 201010179095A CN 101858914 B CN101858914 B CN 101858914B
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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
Technical field
The present invention relates to a kind of syphilis specific total antibodies and detect reagent, 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 a kind of sexually transmitted disease that is caused by microspironema pallidum (Treponema pallidum, TP), and its pathogen is microspironema pallidum, belongs to Spirochaetaceae.The 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 the nearly all tissue of body and organ are got involved, and clinical manifestation is general, can be divided into different clinical stages, comprises first phase, second phase, three phases and latent period.The World Health Organization (WHO) (WHO) is prophesy optimistically once: " because high sensitivity detection method and efficient therapeutic scheme being arranged, syphilis be a kind of can be by the succeed sexually transmitted disease of control of 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 the people pessimistic in the imagination.
The investigation discovery, 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 large types.Syphilis specific antibody IgM (TP-IgM) and IgG (TP-IgG) antibody are respectively at 2 weeks and the rear 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 produces evening, generally produce in infected rear 5～7 weeks (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 syphilis early infection and a movable serologic marker, Li Burong etc. (referring to: Li Burong, He Juntao, Zhang Yi, Deng. the clinical meaning [J] that IgM antibody to treponema pallidum detects. Journal of the Fourth Military Medical University, 2007,28 (16): 1495-1497) think that TP-IgM is the same with TP-DNA, representing syphilis infectiousness index.Under the prerequisite of getting rid of recent anti-syphilis treatment, if TP-IgM does not turn out cloudy, the remaining microspironema pallidum of possibility or treatment are not thorough in the prompting body.It is positive that TP-IgM the moon person of turning turns when following up a case by regular visits to again, show that reinfect syphilis 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 large, obtain less, impure (being mixed with host protein), have cross reaction with other pathogen, so false positive happens occasionally also.Along with the in succession clone who reaches treponemal antigen that popularizes of Protocols in Molecular Biology, recombinant antigen is applied to the syphilis experiment more and more.The TP antigen that research and comparison is many at present has TPN17, TPN47, TPN15, TPN44.5, TPN36, TP0453, TP0684 and TPr family.The recombinant antigen that adopts recombinant DNA technology to prepare can overcome the shortcoming of complete TP antigen, can prepare fast, economically endless special restructuring TP antigen.
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 form anti-processed in the face of severe not only needs special accurately detection means, also needs a kind of more simple and efficient reagent to come examination, in order to provide countermeasure for clinical with control and prevention of disease.
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; Coated anti-human Ig monoclonal antibody or TPN17 and/or TPN47 at syphilis specific total antibodies detection line place, the IgG antibody of coated goat-anti syphilis antigen TPN17 and TPN47 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
In the coated anti-human Ig monoclonal antibody of syphilis specific total antibodies detection line, coated goat-anti syphilis antigen TPN17 and TPN47 IgG antibody dry at the control line place;
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 with condensing unit to be heated to boiling, magnetic force adds and adds 1% trisodium citrate aqueous solution 2.0mL under the thermal agitation, continue heating until solution is till the vinicolor, it is for subsequent use that cooling is placed in the brown bottle 4 ℃ of Refrigerator stores;
4) mark of collaurum and TPN17, TPN47
The mark of collaurum and TPN17: get collaurum 10ml, transfer to pH5.4 with 0.1mol/L NaOH, add 100 μ g TPN17, 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 the mark of collaurum and TPN47 gets 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; The coated anti-human Ig monoclonal antibody at syphilis specific total antibodies detection line place, the IgG antibody of coated goat-anti syphilis antigen TPN17 and TPN47 is cut into strip with cutting cutter at the control line place, gets syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar.
In step 2) in, the concentration of described anti-human 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) in, the concentration of described trisodium citrate can be 2%.
In step 4) in, 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 forms schematic diagram.
Fig. 2 is the experimental result pattern diagram.In Fig. 2, (1) is the schematic diagram before using, and (2) are invalid test (product quality problem), and (3) malicious specific total antibodies is negative, and (4) malicious specific total antibodies is positive.
Embodiment
The present invention is further illustrated in connection with accompanying drawing for following examples.
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; Coated anti-human Ig monoclonal antibody or TPN17 and/or TPN47 at syphilis specific total antibodies detection line 5 places, the IgG antibody of coated goat-anti syphilis antigen TPN17 and TPN47 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
In the coated anti-human Ig monoclonal antibody of syphilis specific total antibodies detection line, coated goat-anti syphilis antigen TPN17 and TPN47 IgG antibody at the control line place, dry, the concentration of described anti-human 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 with condensing unit to be heated to boiling, magnetic force adds and adds 1% trisodium citrate aqueous solution 2.0mL under the thermal agitation, continue heating until solution is till the vinicolor, it is for subsequent use that cooling is placed in the brown bottle 4 ℃ of Refrigerator stores, and the concentration of described trisodium citrate can be 2%.
4) mark of collaurum and TPN17, TPN47
The mark of collaurum and TPN17: get collaurum 10ml, transfer to pH5.4 with 0.1mol/L NaOH, add 100 μ g TPN17, 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 the mark of collaurum and TPN47 gets 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; The coated anti-human Ig monoclonal antibody at syphilis specific total antibodies detection line place, the IgG antibody of coated goat-anti syphilis antigen TPN17 and TPN47 is cut into strip with cutting cutter at the control line place, gets syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar.
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 at detector bar check plot C, then is judged to feminine gender; 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 detecting of syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar:
1) visual examination: coated smooth, the clean pollution-free spot of reaction film of white, free from flaw, adhesive tape is without coming unglued, without cutting oblique phenomenon.
2) positive sample coincidence rate: adopt the calibrating of syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bars with each 50 parts of the positive control serums of the different titers of the total antibody positive of TP-, calculate positive coincidence rate.The clinical samples that definite employing TPPA (Japanese fuji Co., Ltd.) method of positive control serum is determined.
3) ' negative ' specimens coincidence rate: with 50 parts of negative control serum calibratings, calculate positive coincidence rate.The clinical samples that definite employing TPPA (Japanese fuji Co., Ltd.) method of negative control serum is determined.
4) sensitivity detects: detect with Ministry of Public Health's Internal Quality Control serum, 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 positive serum testing result to show that the shade of colour band is consistent, result's feminine gender 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 positive serum testing result to show that the shade of colour band is consistent, the result that negative serum detects is negative.
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 the autoimmunity systemic diseases such as systemic loupus erythematosus (n=30), rheumatoid disease (n=30), autoallergic (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 is placed 37 ℃ detected afterwards in 20 days, above indices is without 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.
In the coated anti-human Ig monoclonal antibody of the total detection line of nitrocellulose filter (NC film), coated goat-anti syphilis antigen (TPN17 and TPN47) the IgG antibody at the control line place, room temperature is dried, and the sealing room temperature saves backup.Wherein, the concentration of anti-human 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, 37 ℃ of oven dry, be prepared into the gold colloid pad, seal for subsequent use.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, the machine sealing, 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 in syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar check plot, then is judged to feminine gender; 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.
Similar to embodiment 1, difference is that the gold colloid pad only is comprised of TPN17, does not contain TPN47.The result judges identical with embodiment 1.
Similar to embodiment 1, difference is that the gold colloid pad only is comprised of TPN47, does not contain TPN17.The result judges identical with embodiment 1.
Similar to embodiment 1, difference is that sample to be checked is samples of CSF, and the result judges identical with embodiment 1.
The performance verification test: the scheme by embodiment 1 prepares the syphilis specific total antibodies quick detection reagent, then carries out performance verification.
1) visual examination: coated smooth, the clean pollution-free spot of reaction film of white, free from flaw, adhesive tape is without coming unglued, and the reagent strip width is at 3 ± 0.1mm, without cutting oblique phenomenon.
2) positive sample coincidence rate: 50 parts of Treponema pallidum specific antibody GATs (TPPA) (Japanese fuji Co., Ltd.) detect the total antibody positive control serum of TP-of determining, 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 ' specimens coincidence rate: the negative control serum of 50 parts of Treponema 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 ' specimens coincidence rate 100%.
4) sensitivity detects: detect with Ministry of Public Health's Internal Quality Control serum, 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 that the shade of colour band is consistent, and the result that negative serum detects is negative.
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 that the shade of colour band is consistent, and the result that negative serum detects is negative.
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 the autoimmunity systemic diseases such as systemic loupus erythematosus (n=30), rheumatoid disease (n=38), autoallergic (n=40), do not find cross reaction.
9) Detection of Stability: syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar is placed 37 ℃ of afterwards detections in 20 days, and above indices is without marked change.
Detection of the present invention is carried out at 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, total antibody detection line and control line place are coated with respectively anti-human Ig monoclonal antibody [or syphilis specific antigen (TPN17 and/or TPN47)] and goat-anti syphilis antigen (TPN17 and/or TPN47) IgG antibody on nitrocellulose filter.With syphilis recombinant antigen TPN17 and the TPN47 of the golden mark of purifying mix rear pre-coated on all-glass paper in certain proportion, the dry processing, be prepared into the gold colloid pad, be aided with again appropriate sample preparation pad, composite reagent bar (assembling mode is seen Fig. 1).When detecting positive sample, the recombinant antigen TPN17 of syphilis specific total antibodies and colloid gold label and/or TPN47 are combined the formation immune complex in the sample.Because the chromatography effect, compound moves forward along paper slip thieving paper direction.When the detection line, 1. the total antibody mediated immunity compound of TP and pre-coated anti-human Ig monoclonal antibody [or syphilis specific antigen (TPN17 and/or TPN47)] are condensed colour developing in conjunction with formation " Au-TPN17 (and/or TPN47)-total antibody of the anti-syphilis of specificity-anti-human Ig monoclonal antibody-solid phase material " or " Au-TPN17 (and/or TPN47)-total antibody of the anti-syphilis of specificity-anti-human Ig monoclonal antibody-solid phase material " centre-fills; 2. free gold mark antigen then is combined and the enrichment colour developing with the antibody of goat-anti syphilis antigen (TPN17 and TPN47) at the control line place.' negative ' specimens then only develops the color at the control line place.
Mode two, envelope antigen, the antibody of mode one are exchanged: total antibody detection line and control line place are coated with respectively syphilis recombinant antigen (TPN17/TPN47 combination) and human IgG antibody on nitrocellulose filter, and the anti-human Ig monoclonal antibody of golden mark is pre-coated on all-glass paper.
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, coated colloid gold label TPN17 antigen and TPN47 antigen on the described collaurum pad, 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; Coated anti-human Ig monoclonal antibody or TPN17 and/or TPN47 at syphilis specific total antibodies detection line place, the IgG antibody of coated goat-anti syphilis antigen TPN17 and TPN47 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
In the coated anti-human Ig monoclonal antibody of syphilis specific total antibodies detection line, coated goat-anti syphilis antigen TPN17 and TPN47IgG antibody dry at the control line place;
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 with condensing unit to be heated to boiling, magnetic force adds and adds 1% trisodium citrate aqueous solution 2.0mL under the thermal agitation, continue heating until solution is till the vinicolor, it is for subsequent use that cooling is placed in the brown bottle 4 ℃ of Refrigerator stores;
4) mark of collaurum and TPN17, TPN47
The mark of collaurum and TPN17: get collaurum 10ml, transfer to pH5.4 with 0.1mol/LNaOH, add 100 μ g TPN17, 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 the mark of collaurum and TPN47 gets 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; The coated anti-human Ig monoclonal antibody at syphilis specific total antibodies detection line place, the IgG antibody of coated goat-anti syphilis antigen TPN17 and TPN47 is cut into strip with cutting cutter at the control line place, gets syphilis specific total antibodies colloidal gold immunochromatographiassay assay reagent bar.
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-human 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; The two 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) in, 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) in, 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) in, the temperature of described oven dry is 37 ℃.
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| 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 |
| CN111638331A (en) * | 2020-06-15 | 2020-09-08 | 郑州方欣生物科技有限责任公司 | Detection kit for treponema pallidum antibody in urine, preparation method and use method |
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