CN201697920U - Syphilis specificity IgM antibody and specificity total antibody combined testing reagent strip - Google Patents
Syphilis specificity IgM antibody and specificity total antibody combined testing reagent strip Download PDFInfo
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- CN201697920U CN201697920U CN 201020198882 CN201020198882U CN201697920U CN 201697920 U CN201697920 U CN 201697920U CN 201020198882 CN201020198882 CN 201020198882 CN 201020198882 U CN201020198882 U CN 201020198882U CN 201697920 U CN201697920 U CN 201697920U
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Abstract
A syphilis specificity IgM antibody and specificity total antibody combined testing reagent strip relates to a syphilis specificity IgM antibody and specificity total antibody combined testing reagent strip and comprises two reagent strips. Each reagent strip is provided with a carrier plate, a sample loading pad, a colloid metal pad, a nitrocellulose membrane, comparison lines and an absorbing pad, and the first reagent strip and the second reagent strip are respectively provided with a syphilis specificity IgM antibody detection line and a specificity total antibody detection line. The syphilis specificity IgM antibody and specificity total antibody combined testing reagent strip is characterized by syphilis recombinant antigen TPN17 and TPN47 preparation, point sampling of the nitrocellulose membrane, colloid metal preparation, marking of colloid metal, the TPN17 and the TPN47 and immuno-chromatographic assay strip preparation. The combined testing reagent strip can be used for testing syphilis specificity IgM antibodies and specificity total antibodies in whole blood samples, blood serum samples, blood plasma samples, cerebrospinal fluid samples and the like, is extremely small in sample quantity, requires no special instruments during testing, and has the advantages of simple, convenient and speedy testing, strong specificity, high sensibility, accuracy, reliability, low cost and wide application range, and further, results can be directly determined and read by bare eyes.
Description
Technical field
The utility model relates to a kind of syphilis specific IgM antibodies and specific total antibodies combined detection reagent, especially relates to syphilis specific IgM antibodies that a kind of employing colloidal gold immunochromatographimethod technology (immunochromatography) carries out and specific total antibodies reagent strip for joint detection 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 utility model provides a kind of syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection.
The utility model is provided with the 1st reagent strip and the 2nd reagent strip;
The 1st reagent strip is provided with the 1st carrier board, the 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane (NC film), syphilis specific IgM antibodies detection line, the 1st control line and the 1st absorption pad; The 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane and the 1st absorption pad stick on the 1st carrier board upper surface successively, one end of the 1st application of sample pad is located on the end of the 1st collaurum pad, the other end of the 1st collaurum pad is located on the end of the 1st nitrocellulose membrane, one end of the 1st absorption pad is located on the other end of the 1st nitrocellulose membrane, and syphilis specific IgM antibodies detection line and the 1st control line are located on the 1st nitrocellulose membrane successively; , wrapped by the IgG antibody of goat-anti syphilis antigen TPN17 and TPN47 by anti-people IgM specific fragment μ chain monoclonal antibody at syphilis specific IgM antibodies detection line place bag at the 1st control line place.
The 2nd reagent strip is provided with the 2nd carrier board, the 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane (NC film), specific total antibodies detection line, the 2nd control line and the 2nd absorption pad; The 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane and the 2nd absorption pad stick on the 2nd carrier board upper surface successively, one end of the 2nd application of sample pad is located on the end of the 2nd collaurum pad, the other end of the 2nd collaurum pad is located on the end of the 2nd nitrocellulose membrane, one end of the 2nd absorption pad is located on the other end of the 2nd nitrocellulose membrane, and specific total antibodies detection line and the 2nd control line are located on the 2nd nitrocellulose membrane successively; , wrapped by goat-anti syphilis antigen TPN17 and TPN47IgG antibody by anti-people Ig monoclonal antibody or syphilis specific antigen TPN17 and/or syphilis specific antigen TPN47 at specific total antibodies detection line place bag at the 2nd control line place.
The useable glass tunica fibrosa is connected between the 1st application of sample pad of the 1st reagent strip and the 2nd application of sample pad of the 2nd reagent strip, puts into kit (claiming the single hole application of sample) again, also can connect or puts up a bridge (claiming the diplopore application of sample) without glass fibre membrane.
Described the 1st carrier board and the 2nd carrier board can adopt the PVC plate.
Below provide preparation method of the present utility model, may further comprise the steps:
1) preparation syphilis recombinant antigen TPN17, 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 IgM specific fragment μ chain monoclonal antibody on the syphilis specific IgM antibodies detection line, wrap by anti-people Ig monoclonal antibody or syphilis specific antigen TPN17 and/or syphilis specific antigen TPN47 at specific total antibodies detection line place, dry, the concentration of described anti-people Ig monoclonal antibody or syphilis specific antigen TPN17 and/or syphilis specific antigen TPN47 can be 1～4mg/mL, the concentration of anti-people IgM specific fragment μ chain monoclonal antibody can be 1～4mg/mL, goat-anti syphilis antigen TPN17 and TPN47IgG antibody were 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
The 1st reagent strip is provided with the 1st carrier board, the 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane (NC film), syphilis specific IgM antibodies detection line, the 1st control line and the 1st absorption pad; The 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane and the 1st absorption pad stick on the 1st carrier board upper surface successively, one end of the 1st application of sample pad is located on the end of the 1st collaurum pad, the other end of the 1st collaurum pad is located on the end of the 1st nitrocellulose membrane, one end of the 1st absorption pad is located on the other end of the 1st nitrocellulose membrane, and syphilis specific IgM antibodies detection line and the 1st control line are located on the 1st nitrocellulose membrane successively; Wrap by anti-people IgM specific fragment μ chain monoclonal antibody at syphilis specific IgM antibodies detection line place, wrap by the IgG antibody of goat-anti syphilis antigen TPN17 and TPN47 at the 1st control line place, be cut into strip with cutting cutter, get the 1st reagent strip of syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection;
The 2nd reagent strip is provided with the 2nd carrier board, the 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane (NC film), specific total antibodies detection line, the 2nd control line and the 2nd absorption pad; The 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane and the 2nd absorption pad stick on the 2nd carrier board upper surface successively, one end of the 2nd application of sample pad is located on the end of the 2nd collaurum pad, the other end of the 2nd collaurum pad is located on the end of the 2nd nitrocellulose membrane, one end of the 2nd absorption pad is located on the other end of the 2nd nitrocellulose membrane, and specific total antibodies detection line and the 2nd control line are located on the 2nd nitrocellulose membrane successively; Wrap by anti-people Ig monoclonal antibody or syphilis specific antigen TPN17 and/or syphilis specific antigen TPN47 at specific total antibodies detection line place, wrap by goat-anti syphilis antigen TPN17 and TPN47IgG antibody at the 2nd control line place, be cut into strip with cutting cutter, get the 2nd reagent strip of syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection.
Available junctional membrane (can adopt glass fibre membrane) is connected the 1st reagent strip (claiming the single hole application of sample) with the 2nd reagent strip on the 1st application of sample pad and the 2nd application of sample pad, also can connect or put up a bridge (title diplopore application of sample) without junctional membrane, syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection can be put into box, make test card, pack in the aluminium foil bag with drying agent, machine seals, and sealing is preserved.
The utility model provides a kind of employing colloidal gold immunochromatographimethod technology to set up syphilis specific IgM antibodies and specific total antibodies associating quick detection reagent, can be used for the detection of syphilis specific IgM antibodies and specific total antibodies in the samples such as whole blood, serum, blood plasma and cerebrospinal fluid.Specimen amount required during detection is minimum, does not need specific apparatus, the direct sentence read result of naked eyes, and detect easy fast, high specificity, highly sensitive, accurately and reliably, cost is low, is widely used.
Description of drawings
Fig. 1 is that the structure of the utility model embodiment is formed synoptic diagram; A is the 1st reagent strip, and B is the 2nd reagent strip, and 1 is junctional membrane (can adopt glass fibre membrane).
Fig. 2 is the assembling synoptic diagram of the utility model embodiment.In Fig. 2, (1) is single hole application of sample assembly drawing, and (2) are diplopore application of sample assembly drawing; A is the 1st reagent strip, and B is the 2nd reagent strip.
Fig. 3 is the experimental result pattern diagram.In Fig. 3, (1) is the synoptic diagram before using, and (2)～(4) are invalid test (product quality problem), and (5) are syphilis specific total antibodies, the IgM antibody positive, and (6) are the syphilis specific total antibodies positive, the IgM negative antibody; A is the 1st reagent strip, and B is the 2nd reagent strip; T is a detection line, and C is a control line.
Embodiment
Following examples will be further described the utility model in conjunction with the accompanying drawings.
Referring to Fig. 1 and 2, the utility model embodiment is provided with the 1st reagent strip A and the 2nd reagent strip B;
The 1st reagent strip A is provided with the 1st carrier board A1, the 1st application of sample pad A2, the 1st collaurum pad A3, the 1st nitrocellulose membrane (NC film) A4, syphilis specific IgM antibodies detection line A6, the 1st control line A7 and the 1st absorption pad A8; The 1st application of sample pad A2, the 1st collaurum pad A3, the 1st nitrocellulose membrane A4 and the 1st absorption pad A8 stick on the 1st carrier board A1 upper surface successively, the end of the 1st application of sample pad A2 is located on the end of the 1st collaurum pad A3, the other end of the 1st collaurum pad A3 is located on the end of the 1st nitrocellulose membrane A4, the end of the 1st absorption pad A8 is located on the other end of the 1st nitrocellulose membrane A4, and syphilis specific IgM antibodies detection line A6 and the 1st control line A7 are located on the 1st nitrocellulose membrane A4 successively; , wrapped by the IgG antibody of goat-anti syphilis antigen TPN17 and TPN47 by anti-people IgM specific fragment μ chain monoclonal antibody at syphilis specific IgM antibodies detection line A6 place bag at the 1st control line A7 place.
The 2nd reagent strip B is provided with the 2nd carrier board B1, the 2nd application of sample pad B2, the 2nd collaurum pad B3, the 2nd nitrocellulose membrane (NC film) B4, specific total antibodies detection line B6, the 2nd control line B7 and the 2nd absorption pad B8; The 2nd application of sample pad B2, the 2nd collaurum pad B3, the 2nd nitrocellulose membrane B4 and the 2nd absorption pad B8 stick on the 2nd carrier board B1 upper surface successively, the end of the 2nd application of sample pad B2 is located on the end of the 2nd collaurum pad B3, the other end of the 2nd collaurum pad B3 is located on the end of the 2nd nitrocellulose membrane B4, the end of the 2nd absorption pad B8 is located on the other end of the 2nd nitrocellulose membrane B4, and specific total antibodies detection line B6 and the 2nd control line B7 are located on the 2nd nitrocellulose membrane B4 successively; , wrapped by goat-anti syphilis antigen TPN17 and TPN47IgG antibody by anti-people Ig monoclonal antibody or syphilis specific antigen TPN17 and/or syphilis specific antigen TPN47 at specific total antibodies detection line B6 place bag at the 2nd control line B7 place.
Available junctional membrane (can adopt glass fibre membrane) is connected between the 1st application of sample pad A2 of the 1st reagent strip A and the 2nd application of sample pad B2 of the 2nd reagent strip B, puts into kit (claiming the single hole application of sample) again, also can connect or puts up a bridge (claiming the diplopore application of sample) without junctional membrane.
Described the 1st carrier board A1 and the 2nd carrier board B1 can adopt the PVC plate.
Below provide the preparation method of the utility model embodiment, may further comprise the steps:
1) preparation syphilis recombinant antigen TPN17, 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 IgM specific fragment μ chain monoclonal antibody on the syphilis specific IgM antibodies detection line, wrap by anti-people Ig monoclonal antibody or syphilis specific antigen TPN17 and/or syphilis specific antigen TPN47 at specific total antibodies detection line place, dry, the concentration of described anti-people Ig monoclonal antibody or syphilis specific antigen TPN17 and/or syphilis specific antigen TPN47 can be 1～4mg/mL, the concentration of anti-people IgM specific fragment μ chain monoclonal antibody can be 1～4mg/mL, goat-anti syphilis antigen TPN17 and TPN47IgG antibody were 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
The 1st reagent strip is provided with the 1st carrier board, the 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane (NC film), syphilis specific IgM antibodies detection line, the 1st control line and the 1st absorption pad; The 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane and the 1st absorption pad stick on the 1st carrier board upper surface successively, one end of the 1st application of sample pad is located on the end of the 1st collaurum pad, the other end of the 1st collaurum pad is located on the end of the 1st nitrocellulose membrane, one end of the 1st absorption pad is located on the other end of the 1st nitrocellulose membrane, and syphilis specific IgM antibodies detection line and the 1st control line are located on the 1st nitrocellulose membrane successively; Wrap by anti-people IgM specific fragment μ chain monoclonal antibody at syphilis specific IgM antibodies detection line place, wrap by the IgG antibody of goat-anti syphilis antigen TPN17 and TPN47 at the 1st control line place, be cut into strip with cutting cutter, get the 1st reagent strip of syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection;
The 2nd reagent strip is provided with the 2nd carrier board, the 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane (NC film), specific total antibodies detection line, the 2nd control line and the 2nd absorption pad; The 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane and the 2nd absorption pad stick on the 2nd carrier board upper surface successively, one end of the 2nd application of sample pad is located on the end of the 2nd collaurum pad, the other end of the 2nd collaurum pad is located on the end of the 2nd nitrocellulose membrane, one end of the 2nd absorption pad is located on the other end of the 2nd nitrocellulose membrane, and specific total antibodies detection line and the 2nd control line are located on the 2nd nitrocellulose membrane successively; Wrap by anti-people Ig monoclonal antibody or syphilis specific antigen TPN17 and/or syphilis specific antigen TPN47 at specific total antibodies detection line place, wrap by goat-anti syphilis antigen TPN17 and TPN47IgG antibody at the 2nd control line place, be cut into strip with cutting cutter, get the 2nd reagent strip of syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection.
Use junctional membrane (can adopt glass fibre membrane) with the 2nd application of sample pad place the 1st reagent strip to be connected (claiming the single hole application of sample) with the 2nd reagent strip at the 1st application of sample pad again, also can connect or put up a bridge (title diplopore application of sample) without junctional membrane, syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection can be put into box, make test card, pack in the aluminium foil bag with drying agent, machine seals, and sealing is preserved.
Below provide immunochromatographyassay assay patient's clinical samples:
Get dilution sample to be checked (whole blood, serum, blood plasma, cerebrospinal fluid) 120 μ L, application of sample leaves standstill the 20min observations in immunochromatography detector bar sample place (single hole application of sample); Or respectively get dilution sample to be checked (whole blood, serum, blood plasma, cerebrospinal fluid) 60 μ L, application of sample leaves standstill the 20min observations in syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection sample place (diplopore application of sample).Only respectively there is an aubergine band to occur, then is judged to feminine gender at two syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection check plot; Detection zone T and check plot C at two syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection all have an aubergine band to occur, and then are judged to the positive; After application of sample detected, the aubergine band did not all appear in detection zone and check plot, is the null result (see figure 3).Wherein, sample to be checked (whole blood, serum, blood plasma, cerebrospinal fluid) dilution adopts physiological saline, 0～50 times of extension rate.
Below provide the calibrating of syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection performance:
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: use the positive control serum of the different titers of TP-IgM and syphilis specific total antibodies to adopt syphilis specific IgM antibodies and the calibrating of specific total antibodies reagent strip for joint detection for each 50 parts, calculate positive coincidence rate.The positive control serum of syphilis specific total antibodies adopts TPPA (Japanese fuji Co., Ltd.) method to determine, the positive control serum of TP-IgM antibody adopts FTA-ABS (German Ou Meng company) method to determine.
3) negative sample coincidence rate:, calculate positive coincidence rate with 50 parts of negative control serum calibratings.Definite employing TPPA (Japanese fuji Co., Ltd.) method of the negative control serum of syphilis specific total antibodies, the negative control serum of TP-IgM antibody adopt FTA-ABS (German Ou Meng company) method to determine.
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 in difference: same batch of syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection, detect with characteristic serum, require the positive serum testing result to show the shade unanimity of colour band, the feminine gender as a result of negative serum detection.
6) differences between batches: different batches syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection, 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 IgM antibodies and specific total antibodies reagent strip for joint detection, 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, syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection 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.
The 1st reagent strip A wraps on nitrocellulose filter (NC film) IgM detection line by anti-people IgM specific fragment μ chain monoclonal antibody, bag is by anti-people Ig monoclonal antibody on the specific total antibodies detection line of the 2nd reagent strip B, wrap by goat-anti syphilis antigen TPN17 and TPN47 antibody at the C of control line place, room temperature is dried, and the sealing room temperature preservation is standby.Wherein, the concentration of anti-people IgM specific fragment μ chain monoclonal antibody, anti-people Ig monoclonal antibody is 1mg/mL, 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; Three's 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.The 1st reagent strip is connected with the 2nd reagent strip at application of sample pad place with all-glass paper again.Or the 1st reagent strip and the 2nd reagent strip put into the plastic casing of corresponding specification, make test card.Syphilis specific IgM antibodies is packed in the aluminium foil bag with specific total antibodies reagent strip for joint detection or detection kit and drying agent, and machine seals, and sealing is preserved.
Get the sample serum to be checked 120 μ L of dilution in 1: 10, application of sample leaves standstill the 20min observations in syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection sample application zone.Only there is an aubergine band to occur, then is judged to feminine gender at syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection 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 T and check plot C, is null result.(referring to Fig. 3)
Similar to embodiment 1, difference is that gold colloid pad, syphilis specific total antibodies detection line only be made up of TPN17, does not contain TPN47.The result judges identical with embodiment 1.
Similar to embodiment 1, difference is that gold colloid pad, syphilis specific total antibodies detection line only be made up 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 a samples of CSF, and the result judges identical with embodiment 1.
The performance verification test: the scheme by embodiment 1 prepares syphilis specific IgM antibodies and specific total antibodies associating quick detection reagent bar, 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 syphilis specific IgM antibodies and specific total antibodies associating quick detection reagent bar width are at 3 ± 0.1mm, and nothing is cut oblique phenomenon.
2) positive sample coincidence rate: 50 parts are detected the positive control serum of the TP-IgM that determines through FTA-ABS (German Ou Meng company), adopt syphilis specific IgM antibodies and specific total antibodies associating quick detection reagent bar to detect positive 50 parts of TP-IgM, positive sample coincidence rate 100%; And 50 parts of positive control serums of syphilis specific total antibodies of determining through microspironema pallidum specific antibody GAT (TPPA) (Japanese fuji Co., Ltd.) detection, adopt syphilis specific IgM antibodies and specific total antibodies associating quick detection reagent bar to detect 49 parts of syphilis specific antibody, positive sample coincidence rate 98%.
3) negative sample coincidence rate: 50 parts are detected the negative control serum of the TP-IgM that determines through FTA-ABS (German Ou Meng company), adopt syphilis specific IgM antibodies and specific total antibodies associating quick detection reagent bar to detect negative 50 parts of TP-IgM, negative sample coincidence rate 100%; And 50 parts of negative control serums of syphilis specific total antibodies of determining through microspironema pallidum specific antibody GAT (TPPA) (Japanese fuji Co., Ltd.) detection, adopt syphilis specific IgM antibodies and specific total antibodies associating quick detection reagent bar to detect 50 parts of syphilis specific antibody, negative sample coincidence rate 100%.
4) sensitivity detects: with the indoor quality controlled serum of the Ministry of Public Health (lot number: 200902001) detect minimum detectability degree 4NCU/mL.
5) criticize in difference: same batch of syphilis specific IgM antibodies united the quick detection reagent bar with specific total antibodies, detect with characteristic positive serum (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 IgM antibodies and specific total antibodies associating quick detection reagent bar, detect with characteristic positive serum (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 IgM antibodies and specific total antibodies associating quick detection reagent bar or detect box, 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 IgM antibodies and specific total antibodies associating quick detection reagent bar is placed 37 ℃ detects after 20 days, more than every index do not have marked change.
Claims (3)
1. syphilis specific IgM antibodies and specific total antibodies reagent strip for joint detection is characterized in that being provided with the 1st reagent strip and the 2nd reagent strip;
The 1st reagent strip is provided with the 1st carrier board, the 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane, syphilis specific IgM antibodies detection line, the 1st control line and the 1st absorption pad; The 1st application of sample pad, the 1st collaurum pad, the 1st nitrocellulose membrane and the 1st absorption pad stick on the 1st carrier board upper surface successively, one end of the 1st application of sample pad is located on the end of the 1st collaurum pad, the other end of the 1st collaurum pad is located on the end of the 1st nitrocellulose membrane, one end of the 1st absorption pad is located on the other end of the 1st nitrocellulose membrane, and syphilis specific IgM antibodies detection line and the 1st control line are located on the 1st nitrocellulose membrane successively; , wrapped by the IgG antibody of goat-anti syphilis antigen TPN17 and TPN47 by anti-people IgM specific fragment μ chain monoclonal antibody at syphilis specific IgM antibodies detection line place bag at the 1st control line place;
The 2nd reagent strip is provided with the 2nd carrier board, the 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane, specific total antibodies detection line, the 2nd control line and the 2nd absorption pad; The 2nd application of sample pad, the 2nd collaurum pad, the 2nd nitrocellulose membrane and the 2nd absorption pad stick on the 2nd carrier board upper surface successively, one end of the 2nd application of sample pad is located on the end of the 2nd collaurum pad, the other end of the 2nd collaurum pad is located on the end of the 2nd nitrocellulose membrane, one end of the 2nd absorption pad is located on the other end of the 2nd nitrocellulose membrane, and specific total antibodies detection line and the 2nd control line are located on the 2nd nitrocellulose membrane successively; , wrapped by goat-anti syphilis antigen TPN17 and TPN47IgG antibody by anti-people Ig monoclonal antibody or syphilis specific antigen TPN17 and/or syphilis specific antigen TPN47 at specific total antibodies detection line place bag at the 2nd control line place.
2. be connected with glass fibre membrane between syphilis specific IgM antibodies as claimed in claim 1 and the specific total antibodies reagent strip for joint detection, the 1st application of sample pad that it is characterized in that the 1st reagent strip and the 2nd application of sample pad of the 2nd reagent strip.
3. syphilis specific IgM antibodies as claimed in claim 1 and specific total antibodies reagent strip for joint detection is characterized in that described the 1st carrier board and the 2nd carrier board are the PVC plate.
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| CN 201020198882 CN201697920U (en) | 2010-05-19 | 2010-05-19 | Syphilis specificity IgM antibody and specificity total antibody combined testing reagent strip |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102095861A (en) * | 2011-01-25 | 2011-06-15 | 厦门大学附属中山医院 | Western blot kit for syphilis specific total antibodies and anticardiolipin antibodies and preparation method of kit |
| CN103163298A (en) * | 2011-12-19 | 2013-06-19 | 天津中新科炬生物制药有限公司 | Rapid detective reagent strip of treponema pallidum immunoglobulin m (IgM) antibody and preparation method thereof |
-
2010
- 2010-05-19 CN CN 201020198882 patent/CN201697920U/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102095861A (en) * | 2011-01-25 | 2011-06-15 | 厦门大学附属中山医院 | Western blot kit for syphilis specific total antibodies and anticardiolipin antibodies and preparation method of kit |
| CN103163298A (en) * | 2011-12-19 | 2013-06-19 | 天津中新科炬生物制药有限公司 | Rapid detective reagent strip of treponema pallidum immunoglobulin m (IgM) antibody and preparation method thereof |
| CN103163298B (en) * | 2011-12-19 | 2015-04-08 | 天津中新科炬生物制药有限公司 | Rapid detective reagent strip of treponema pallidum immunoglobulin m (IgM) antibody and preparation method thereof |
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