CN104330566A - Syphilis test strip and preparation method thereof, and syphilis detection card and preparation method thereof - Google Patents
Syphilis test strip and preparation method thereof, and syphilis detection card and preparation method thereof Download PDFInfo
- Publication number
- CN104330566A CN104330566A CN201310478624.XA CN201310478624A CN104330566A CN 104330566 A CN104330566 A CN 104330566A CN 201310478624 A CN201310478624 A CN 201310478624A CN 104330566 A CN104330566 A CN 104330566A
- Authority
- CN
- China
- Prior art keywords
- binding protein
- syphilis
- cuorin
- cardiolipin binding
- monoclonal antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 208000006379 syphilis Diseases 0.000 title claims abstract description 122
- 238000001514 detection method Methods 0.000 title claims abstract description 117
- 238000002360 preparation method Methods 0.000 title claims abstract description 53
- 238000009600 syphilis test Methods 0.000 title claims abstract description 34
- 108091008324 binding proteins Proteins 0.000 claims abstract description 90
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 69
- 102000025851 cardiolipin binding proteins Human genes 0.000 claims abstract description 58
- 108091009148 cardiolipin binding proteins Proteins 0.000 claims abstract description 58
- 241000589884 Treponema pallidum Species 0.000 claims abstract description 54
- 239000000427 antigen Substances 0.000 claims abstract description 49
- 102000036639 antigens Human genes 0.000 claims abstract description 49
- 108091007433 antigens Proteins 0.000 claims abstract description 49
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 40
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 40
- 238000011534 incubation Methods 0.000 claims abstract description 25
- 238000002156 mixing Methods 0.000 claims abstract description 16
- 102000023732 binding proteins Human genes 0.000 claims abstract 21
- 238000003908 quality control method Methods 0.000 claims description 93
- 239000010931 gold Substances 0.000 claims description 60
- 229910052737 gold Inorganic materials 0.000 claims description 60
- 239000000084 colloidal system Substances 0.000 claims description 59
- 238000012360 testing method Methods 0.000 claims description 53
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 39
- 230000003429 anti-cardiolipin effect Effects 0.000 claims description 30
- 241000589970 Spirochaetales Species 0.000 claims description 29
- 239000003365 glass fiber Substances 0.000 claims description 25
- 238000003556 assay Methods 0.000 claims description 21
- 239000012467 final product Substances 0.000 claims description 19
- 208000015181 infectious disease Diseases 0.000 abstract description 23
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 239000012528 membrane Substances 0.000 abstract 3
- 229920002678 cellulose Polymers 0.000 abstract 2
- 239000001913 cellulose Substances 0.000 abstract 2
- 239000011521 glass Substances 0.000 abstract 2
- 230000002745 absorbent Effects 0.000 abstract 1
- 239000002250 absorbent Substances 0.000 abstract 1
- ZGSPNIOCEDOHGS-UHFFFAOYSA-L disodium [3-[2,3-di(octadeca-9,12-dienoyloxy)propoxy-oxidophosphoryl]oxy-2-hydroxypropyl] 2,3-di(octadeca-9,12-dienoyloxy)propyl phosphate Chemical compound [Na+].[Na+].CCCCCC=CCC=CCCCCCCCC(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COP([O-])(=O)OCC(O)COP([O-])(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COC(=O)CCCCCCCC=CCC=CCCCCC ZGSPNIOCEDOHGS-UHFFFAOYSA-L 0.000 abstract 1
- 102000036213 phospholipid binding proteins Human genes 0.000 abstract 1
- 108091011000 phospholipid binding proteins Proteins 0.000 abstract 1
- 238000007789 sealing Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 72
- 102000014914 Carrier Proteins Human genes 0.000 description 69
- 210000002966 serum Anatomy 0.000 description 25
- 238000000034 method Methods 0.000 description 23
- 239000007864 aqueous solution Substances 0.000 description 19
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 11
- 229940098773 bovine serum albumin Drugs 0.000 description 11
- 238000004587 chromatography analysis Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 230000000890 antigenic effect Effects 0.000 description 6
- 230000036039 immunity Effects 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- 239000007921 spray Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000004140 cleaning Methods 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 238000013016 damping Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 239000012460 protein solution Substances 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000003760 magnetic stirring Methods 0.000 description 3
- 230000000405 serological effect Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 208000019802 Sexually transmitted disease Diseases 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 201000006901 congenital syphilis Diseases 0.000 description 2
- 208000018631 connective tissue disease Diseases 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 206010042880 Syphilis genital Diseases 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000001446 dark-field microscopy Methods 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012128 rapid plasma reagin Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 230000009329 sexual behaviour Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of immunodetection, and particularly relates to a syphilis test strip and a preparation method thereof, and a syphilis detection card and a preparation method thereof. The syphilis test strip comprises a bottom plate (1). The bottom plate (1) is provided with a sample pad (2), a glass cellulose membrane (3), a nitrocellulose membrane (4) and absorbent paper (5) in sequence. The glass cellulose membrane (3) is provided with a colloidal gold-marked treponema pallidum specific antigen and a colloidal gold-marked cardiolipin-phospholipid binding protein complex. A preparation method of the colloidal gold-marked cardiolipin-phospholipid binding protein complex comprises the steps of mixing cardiolipin and phospholipid binding protein; carrying out first incubation to obtain the cardiolipin-phospholipid binding protein complex; mixing the cardiolipin-phospholipid binding protein complex and colloidal gold; carrying out second incubation; and sealing to obtain the colloidal gold-marked cardiolipin-phospholipid binding protein complex. The detection card provided by the invention is simple and convenient to operate, has high sensitivity, high specificity and low false positive rate, and prevents interference of previous infection in current infection.
Description
Technical field
The present invention relates to technical field of immunoassay, particularly syphilis test strips and preparation method thereof, Lues Assay card and preparation method thereof.
Background technology
Syphilis is the Common STD occurred by infection by Treponema pallidum human body, appearance centuries, and all having distribution at world wide at present, is very important sexually transmitted disease, is divided into acquired syphilis and congenital syphilis according to source of infection.Wherein, acquired syphilis refers to that adult is by sexual behaviour, blood transfusion, operation and infected syphilis, is divided into for three phases according to clinical manifestation, and cardinal symptom comprises to be invaded mucocutaneous and involves internal organs, is a kind of comparatively serious sexually transmitted disease; Congenital syphilis is that microspironema pallidum directly enters the syphilis caused in fetus body by placenta because syphilitic pregnant woman is without treating in time or malpractice.
At present, the test in laboratory method of syphilis mainly comprises dark-field microscopy, directly fluorescence antibody inspection, Serological testing and examination of cerebrospinal fluid, and modal detection method is Serological testing.Serological testing is divided into again detection of specific antibody method and non-specific antibody detection method, and detection of specific antibody method mainly comprises enzyme linked immunosorbent assay (ELISA) and colloidal gold method; Non-specific antibody detection method mainly comprises rapid plasma reagin card test (RPR) and tolulized red unheated serum test (TRUST).
The advantage of detection of specific antibody method is high specificity, highly sensitive, but the defect of depositing both ways, on the one hand because its detection is IgG antibody, and after human infection microspironema pallidum, need the time of more than 2 weeks just can produce IgG antibody, therefore initial infection cannot detect syphilis; Syphilis specific IgG antibodies can carry after producing all the life on the other hand, even if syphilis treats rear human body still can carry syphilis specific IgG antibodies, therefore detection syphilis specific IgG antibodies cannot distinguish syphilis is merely Current Infection or previous infection, previous infection will produce certain interference to Current Infection, also needs to do non-specific antibody further and detects.
The advantage of non-specific antibody detection method is highly sensitive, but specificity is poor, and false positive rate is higher.In population, the false positive rate of this type of test is 1% ~ 2%, but false positive rate can up to 10% in drug addict, in the special populations such as tuberculosis, connective tissue disease (CTD) and pregnant woman, false positive rate all has rising in various degree in addition, therefore also needs to do specific antibody detection and makes a definite diagnosis.
All there is certain defect based on detection of specific antibody method and non-specific antibody detection method, therefore a kind of highly sensitive, high specificity is provided, not by the Lues Assay card of previous infection interference, has important practical significance.
Summary of the invention
In view of this, the invention provides syphilis test strips and preparation method thereof, Lues Assay card and preparation method thereof.This syphilis test strips is passed through colloid gold label cuorin, thus achieve syphilis specific antibody test and syphilis non-specific antibody are detected the object combined, both there is highly sensitive, that high specificity, false positive rate are low advantage, avoid the interference of previous infection to Current Infection, and simple to operation.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of syphilis test strips, syphilis test strips comprises base plate 1, and base plate 1 is disposed with sample pad 2, glass fibre element film 3, nitrocellulose filter 4, thieving paper 5;
Glass fibre element film 3 is provided with the microspironema pallidum specific antigen of colloid gold label and the cuorin-cardiolipin binding protein complex of colloid gold label;
The preparation method of the cuorin-cardiolipin binding protein complex of colloid gold label is: core phosphatide and cardiolipin binding protein mixing, through the first incubation, obtain cuorin-cardiolipin binding protein complex, core phosphatide-cardiolipin binding protein complex and collaurum mixing, through the second incubation, close, obtain the cuorin-cardiolipin binding protein complex of colloid gold label;
Nitrocellulose filter 4 is provided with the first detection zone 7, first, detection zone 6, second quality control region 8 and the second quality control region 9;
First detection zone 6 is provided with recombinant syphilis spirochete antigen, second detection zone 7 is provided with mouse-anti human IgG monoclonal antibody, first quality control region 8 is provided with mouse-anti microspironema pallidum IgG monoclonal antibody, and the second quality control region 9 is provided with the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit;
Or the first detection zone 6 is provided with mouse-anti human IgG monoclonal antibody, second detection zone 7 is provided with recombinant syphilis spirochete antigen, first quality control region 8 is provided with the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit, and the second quality control region 9 is provided with mouse-anti microspironema pallidum IgG monoclonal antibody.
At present, the detection of syphilis specific antibody adopts colloidal gold immunity chromatography to detect, the Cleaning Principle of the method is: be fixed on film by specific antigen or antibody with ribbon, colloid gold label reagent (antibody or monoclonal antibody) is adsorbed on pad, when sample to be checked is added to after in test strips sample pad, moved forward by capillary action, react to each other after dissolving the colloid gold label reagent on pad, when moving to the region of fixing antigen or antibody, there is specific binding with it again and be trapped in the bond of thing to be checked and gold marked reagent, be gathered in detection zone, by being observed visually colour developing result, use very convenient.When utilizing the method to make test strips, the antibody of colloid gold label is generally protein and other, and syphilis non-specific antibody---cuorin is hydrophobic Small molecular, up to the present also do not have the report how using colloid gold label cuorin, the method therefore utilizing colloidal gold-labeled method to detect syphilis non-specific antibody does not also realize.The present invention, by the key bridge effect of cardiolipin binding protein, achieves the combination of cuorin and collaurum, reaches the object detecting syphilis non-specific antibody with colloidal gold-labeled method.
The detection of syphilis specific antibody and syphilis non-specific antibody is incorporated in a test card by the present invention, thus achieves the synchronous detection to syphilis specific antibody and syphilis non-specific antibody, makes testing result more accurate, reduces false positive rate.
Be provided with two detection zones in the present invention, comprise the first detection zone and the second detection zone.In embodiments more provided by the invention, the first detection zone is for being coated with recombinant syphilis spirochete antigen (can detection specificity antibody), and the second detection zone is coated with mouse-anti human IgG monoclonal antibody (can detect non-specific antibody); In other embodiments provided by the invention, the first detection zone is for being coated with mouse-anti human IgG monoclonal antibody, and the second detection zone is coated with recombinant syphilis spirochete antigen.The Cleaning Principle of syphilis specific antibody and syphilis non-specific antibody is as follows:
The Cleaning Principle of syphilis specific antibody is: if containing syphilis specific antibody in serum sample, serum sample is added in sample pad, glass fibre element film is entered along chromatography direction, the microspironema pallidum specific antigen of the colloid gold label of the syphilis specific antibody in serum sample on glass fibre element film is combined, and forms the microspironema pallidum specific antigen complex of syphilis specific antibody-colloidal gold mark; Then the microspironema pallidum specific antigen complex of syphilis specific antibody-colloidal gold mark continues to enter nitrocellulose filter along chromatography direction, be combined with the recombinant syphilis spirochete antigen of position, detection zone, form the microspironema pallidum specific antigen complex of recombinant syphilis spirochete antigen-syphilis specific antibody-colloidal gold mark, red in detection zone position display, be syphilis specific antibody positive, do not develop the color in position, detection zone, be syphilis specific negative antibody.
The Cleaning Principle of syphilis non-characteristic antibody is: if containing the non-characteristic antibody of syphilis in serum sample, serum sample is added in sample pad, glass fibre element film is entered along chromatography direction, cuorin-cardiolipin binding protein the complex of the colloid gold label of the non-characteristic antibody of the syphilis in serum sample on glass fibre element film is combined, and forms the cuorin-cardiolipin binding protein complex of the non-characteristic antibody-colloidal gold mark of syphilis; Then cuorin-cardiolipin binding protein the complex of the non-characteristic antibody-colloidal gold mark of syphilis continues to enter nitrocellulose filter along chromatography direction, be combined with the mouse-anti human IgG monoclonal antibody of position, detection zone, form the cuorin-cardiolipin binding protein complex of the non-characteristic antibody-colloidal gold mark of mouse-anti human IgG monoclonal antibody-syphilis, red in detection zone position display, be the non-characteristic antibody positive of syphilis, do not develop the color in position, detection zone, be syphilis non-specific antibody negative.
When using test card provided by the invention to detect serum sample, result decision method is: when the detection zone detecting syphilis specific antibody and the detection zone detecting syphilis non-specific antibody all show redness, can be judged to be syphilis; The detection zone display detecting syphilis specific antibody is red, and when the detection zone detecting syphilis non-specific antibody does not show redness, can be judged to be syphilis previous infection; The detection zone display detecting syphilis non-specific antibody is red, and when the detection zone detecting syphilis specific antibody does not show redness, can be judged to be doubtful syphilis, to cross after certain hour (thoughtful month) repetition measurement again; When the detection zone detecting syphilis specific antibody and the detection zone detecting syphilis non-specific antibody all do not show redness, can be judged to be that syphilis is negative.
Quality control region is used to the region whether test strip lost efficacy, and quality control region display redness then shows that test strips is effective; Quality control region does not show redness, then show that test strips lost efficacy.Be provided with two quality control region in the present invention, comprise the first quality control region and the second quality control region.In embodiments more provided by the invention, the first quality control region is for being coated with mouse-anti microspironema pallidum IgG monoclonal antibody (i.e. specific antibody), and the second quality control region is coated with the anti-cardiolipin binding protein IgG monoclonal antibody (i.e. non-specific antibody) of rabbit; In other embodiments provided by the invention, the first quality control region is for being coated with the anti-cardiolipin binding protein IgG monoclonal antibody (i.e. non-specific antibody) of rabbit, and the second quality control region is coated with mouse-anti microspironema pallidum IgG monoclonal antibody (i.e. specific antibody).Quality control region Cleaning Principle is as follows:
The microspironema pallidum specific antigen of the colloid gold label on glass fibre element film enters nitrocellulose filter with serum sample along chromatography method, be fixed with mouse-anti microspironema pallidum IgG monoclonal antibody to be combined on nitrocellulose filter, be coated with the quality control region position display redness of specific antibody or do not showing redness;
Cuorin-cardiolipin binding protein the complex of the colloid gold label on glass fibre element film enters nitrocellulose filter with serum sample along chromatography method, be fixed with the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit to be combined on nitrocellulose filter, be coated with the quality control region position display redness of non-specific antibody or do not showing redness;
When the quality control region position being coated with specific antibody shows redness with the quality control region position being coated with non-specific antibody simultaneously, then test strips is effective;
When the quality control region position display being coated with specific antibody is red, and the quality control region position being coated with non-specific antibody does not show redness, then test strips lost efficacy;
When the quality control region position display being coated with non-specific antibody is red, and the quality control region position being coated with specific antibody does not show redness, then test strips lost efficacy;
All do not show redness in the quality control region position being coated with specific antibody with the quality control region position being coated with non-specific antibody, then test strips lost efficacy.
In embodiments more provided by the invention, first detection zone 6 is provided with recombinant syphilis spirochete antigen (being used for detecting syphilis specific antibody), second detection zone 7 is provided with mouse-anti human IgG monoclonal antibody (being used for detecting syphilis non-specific antibody), first quality control region 8 is provided with mouse-anti microspironema pallidum IgG monoclonal antibody, when second quality control region 9 is provided with rabbit anti-cardiolipin binding protein IgG monoclonal antibody, Results of Syphilis is as follows:
(1) first detection zone 7, first, detection zone 6, second quality control region 8, second quality control region 9 all develops the color, be then judged to be that syphilis is positive, i.e. syphilization;
(2) first detection zone 6, first quality control region 8, second quality control region 9 develop the color, and not developing the color in the second detection zone 7, is then judged to be syphilis previous infection;
(3) second detection zone 7, first quality control region 8, second quality control region 9 develop the color, and not developing the color in the first detection zone 6, is then judged to be doubtful syphilis;
Do not develop the color in (4) first detection zone 7, detection zones 6, second, the first quality control region 8, second quality control region 9 develops the color, be then judged to be that syphilis is negative, i.e. non-syphilization;
(5) first detection zone 7, first, detection zone 6, second quality control region 8, second quality control region 9 all do not develop the color, then test strips lost efficacy.
In other embodiments provided by the invention, first detection zone 6 is provided with mouse-anti human IgG monoclonal antibody (being used for detecting syphilis non-specific antibody), second detection zone 7 is provided with recombinant syphilis spirochete antigen (being used for detecting syphilis specific antibody), first quality control region 8 is provided with the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit, when second quality control region 9 is provided with mouse-anti microspironema pallidum IgG monoclonal antibody, Results of Syphilis is as follows:
(1) first detection zone 7, first, detection zone 6, second quality control region 8, second quality control region 9 all develops the color, be then judged to be that syphilis is positive, i.e. syphilization;
(2) first detection zone 7, first quality control region 8, second quality control region 9 develop the color, and not developing the color in the second detection zone 7, is then judged to be syphilis previous infection;
(3) second detection zone 6, first quality control region 8, second quality control region 9 develop the color, and not developing the color in the first detection zone 6, is then judged to be doubtful syphilis;
Do not develop the color in (4) first detection zone 7, detection zones 6, second, the first quality control region 8, second quality control region 9 develops the color, be then judged to be that syphilis is negative, i.e. non-syphilization;
(5) first detection zone 7, first, detection zone 6, second quality control region 8, second quality control region 9 all do not develop the color, then test strips lost efficacy.
By research, the combination of cuorin and cardiolipin binding protein is similar to the combination of antigen-antibody, namely combined by electrostatic attraction, Van der Waals force, hydrogen bond or hydrophobic forces, in order to ensure that cuorin is combined fully with cardiolipin binding protein, association reaction needs incubation under uniform temperature condition.In embodiments more provided by the invention, the first incubation is hatch 1 ~ 4h under the condition of 37 DEG C, then places 8 ~ 12h under the condition of 2 ~ 8 DEG C.
In embodiments more provided by the invention, in the preparation method of the cuorin-cardiolipin binding protein complex of colloid gold label, the mass ratio of cuorin and cardiolipin binding protein is (1 ~ 5): (1 ~ 5).
As preferably, the mass ratio of cuorin and cardiolipin binding protein is 1:1.
In embodiments more provided by the invention, when preparing the cuorin-cardiolipin binding protein complex of colloid gold label, first cuorin and cardiolipin binding protein are made cuorin ethanolic solution and cardiolipin binding protein aqueous solution respectively, again by cuorin ethanolic solution and cardiolipin binding protein aqueous solution, carry out the first incubation.
In embodiments more provided by the invention, the preparation method of cuorin ethanolic solution is: be dissolved in by cuorin in absolute ethyl alcohol, obtain final product.
In embodiments more provided by the invention, the mass body volume concentrations of cuorin ethanolic solution is preferably 0.5 ~ 5mg/mL.
Preferably, the mass body volume concentrations of cuorin ethanolic solution is preferably 1mg/mL.
In embodiments more provided by the invention, the preparation method of cardiolipin binding protein aqueous solution is: be dissolved in the water by cardiolipin binding protein, obtain final product.
In embodiments more provided by the invention, the mass body volume concentrations of cardiolipin binding protein aqueous solution is preferably 0.5 ~ 5mg/mL.
Preferably, the mass body volume concentrations of cardiolipin binding protein aqueous solution is 1mg/mL.
In order to ensure that colloid gold particle is fully combined with cuorin-cardiolipin binding protein complex, association reaction needs incubation under uniform temperature condition.In embodiments more provided by the invention, in the cuorin-cardiolipin binding protein complex process preparing colloid gold label, the second incubation is react 2h under the condition of 2 ~ 8 DEG C.
In embodiments more provided by the invention, in order to close unnecessary protein binding site, the present invention adopts the cuorin-cardiolipin binding protein complex of bovine serum albumin solution to colloid gold label to close.
As preferably, the mass percentage concentration of bovine serum albumin solution is 1%.
In embodiments more provided by the invention, the temperature closed is 37 DEG C.
In embodiments more provided by the invention, the time closed is 1h.
In embodiments more provided by the invention, the preparation method of the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit, for getting cardiolipin binding protein immunize rabbit, to obtain final product.
Present invention also offers the preparation method of syphilis test strips provided by the invention, comprise the steps:
Steps A: core phosphatide and cardiolipin binding protein mixing, through the first incubation, obtain cuorin-cardiolipin binding protein complex, phosphatide of coring-cardiolipin binding protein complex and collaurum mixing, through the second incubation, close, obtain the cuorin-cardiolipin binding protein complex of colloid gold label;
Obtain the microspironema pallidum specific antigen of recombinant syphilis spirochete antigen, mouse-anti microspironema pallidum IgG monoclonal antibody, mouse-anti human IgG monoclonal antibody, the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit and colloid gold label;
Step B: the microspironema pallidum specific antigen of the cuorin-cardiolipin binding protein complex and colloid gold label of getting colloid gold label is coated in glass fibre element film 3;
Get recombinant syphilis spirochete antigen coated in the first detection zone 6 of nitrocellulose filter 4, get the second detection zone 7 that mouse-anti human IgG monoclonal antibody is coated in nitrocellulose filter 4, get the first quality control region 8 that mouse-anti microspironema pallidum IgG monoclonal antibody is coated in nitrocellulose filter 4, get the second quality control region 9 that the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit is coated in nitrocellulose filter 4;
Or get the first detection zone 6 that mouse-anti human IgG monoclonal antibody is coated in nitrocellulose filter 4, get recombinant syphilis spirochete antigen coated in the second detection zone 7 of nitrocellulose filter 4, get the first quality control region 8 that the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit is coated in nitrocellulose filter 4, get the second quality control region 9 that mouse-anti microspironema pallidum IgG monoclonal antibody is coated in nitrocellulose filter 4;
Step C: sample thief pad 2, glass fibre element film 3, nitrocellulose filter 4, thieving paper 5 are sequentially fixed on base plate 1, to obtain final product.
In the preparation method of syphilis test strips, as preferably, the first incubation is hatch 1 ~ 4h under the condition of 37 DEG C, then places 8 ~ 12h under the condition of 2 ~ 8 DEG C.
In the preparation method of syphilis test strips, when preparing the cuorin-cardiolipin binding protein complex of colloid gold label, the mass ratio of cuorin and cardiolipin binding protein is (1 ~ 5): (1 ~ 5).
As preferably, the mass ratio of cuorin and cardiolipin binding protein is 1:1.
When preparing the cuorin-cardiolipin binding protein complex of colloid gold label, first cuorin and cardiolipin binding protein are made cuorin ethanolic solution and cardiolipin binding protein aqueous solution respectively, again by cuorin ethanolic solution and cardiolipin binding protein aqueous solution, carry out the first incubation.
In embodiments more provided by the invention, the preparation method of cuorin ethanolic solution is: be dissolved in by cuorin in absolute ethyl alcohol, obtain final product.
In embodiments more provided by the invention, the mass body volume concentrations of cuorin ethanolic solution is preferably 0.5 ~ 5mg/mL.
Preferably, the mass body volume concentrations of cuorin ethanolic solution is preferably 1mg/mL.
In embodiments more provided by the invention, the preparation method of cardiolipin binding protein aqueous solution is: be dissolved in the water by cardiolipin binding protein, obtain final product.
In embodiments more provided by the invention, the mass body volume concentrations of cardiolipin binding protein aqueous solution is preferably 0.5 ~ 5mg/mL.
Preferably, the mass body volume concentrations of cardiolipin binding protein aqueous solution is 1mg/mL.
In embodiments more provided by the invention, in the cuorin-cardiolipin binding protein complex process preparing colloid gold label, the second incubation is react 2h under the condition of 2 ~ 8 DEG C.
In embodiments more provided by the invention, closing the reagent adopted is bovine serum albumin solution.
As preferably, the mass percentage concentration of bovine serum albumin solution is 1%.
In embodiments more provided by the invention, the temperature closed is 37 DEG C.
In embodiments more provided by the invention, the time closed is 1h.
In the preparation method of syphilis test strips, the preparation method of the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit, for getting cardiolipin binding protein immunize rabbit, to obtain final product.
Present invention also offers a kind of Lues Assay card, comprise test card housing and syphilis test strips provided by the invention;
In this Lues Assay card, syphilis test strips wherein comprises base plate 1, and base plate 1 is disposed with sample pad 2, glass fibre element film 3, nitrocellulose filter 4, thieving paper 5; Glass fibre element film 3 is provided with the microspironema pallidum specific antigen of colloid gold label and the cuorin-cardiolipin binding protein complex of colloid gold label; The preparation method of the cuorin-cardiolipin binding protein complex of colloid gold label is: core phosphatide and cardiolipin binding protein mixing, through the first incubation, obtain cuorin-cardiolipin binding protein complex, core phosphatide-cardiolipin binding protein complex and collaurum mixing, through the second incubation, close, obtain the cuorin-cardiolipin binding protein complex of colloid gold label; Nitrocellulose filter 4 is provided with the first detection zone 7, first, detection zone 6, second quality control region 8 and the second quality control region 9; First detection zone 6 is provided with recombinant syphilis spirochete antigen, second detection zone 7 is provided with mouse-anti human IgG monoclonal antibody, first quality control region 8 is provided with mouse-anti microspironema pallidum IgG monoclonal antibody, and the second quality control region 9 is provided with the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit; Or the first detection zone 6 is provided with mouse-anti human IgG monoclonal antibody, second detection zone 7 is provided with recombinant syphilis spirochete antigen, first quality control region 8 is provided with the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit, and the second quality control region 9 is provided with mouse-anti microspironema pallidum IgG monoclonal antibody; The preparation method of cuorin ethanolic solution is: be dissolved in by cuorin in absolute ethyl alcohol, obtain final product; The mass body volume concentrations of cuorin ethanolic solution is preferably 0.5 ~ 5mg/mL; More preferably, the mass body volume concentrations of cuorin ethanolic solution is preferably 1mg/mL; The preparation method of cardiolipin binding protein aqueous solution is: be dissolved in the water by cardiolipin binding protein, obtain final product; The mass body volume concentrations of cardiolipin binding protein aqueous solution is preferably 0.5 ~ 5mg/mL; More preferably, the mass body volume concentrations of cardiolipin binding protein aqueous solution is 1mg/mL; Second incubation is react 2h under the condition of 2 ~ 8 DEG C; Closing the reagent adopted is bovine serum albumin solution; In bovine serum albumin solution, bovine serum albumin(BSA) accounts for the mass percentage concentration of bovine serum albumin solution is 1%; The temperature closed is 37 DEG C; The time closed is 1h; The preparation method of the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit, for getting cardiolipin binding protein immunize rabbit, to obtain final product.
Test card housing is provided with sample application zone 10 and detection zone 11.
Present invention also offers the preparation method of Lues Assay card provided by the invention, get test card housing and syphilis test strips provided by the invention assembling, to obtain final product.
The invention provides syphilis test strips and preparation method thereof, Lues Assay card and preparation method thereof.This this syphilis test strips comprises base plate 1, and base plate 1 is disposed with sample pad 2, glass fibre element film 3, nitrocellulose filter 4, thieving paper 5; Glass fibre element film 3 is provided with the microspironema pallidum specific antigen of colloid gold label and the cuorin-cardiolipin binding protein complex of colloid gold label; The preparation method of the cuorin-cardiolipin binding protein complex of colloid gold label is: core phosphatide and cardiolipin binding protein mixing, through the first incubation, obtain cuorin-cardiolipin binding protein complex, core phosphatide-cardiolipin binding protein complex and collaurum mixing, through the second incubation, close, obtain the cuorin-cardiolipin binding protein complex of colloid gold label; Nitrocellulose filter 4 is provided with the first detection zone 7, first, detection zone 6, second quality control region 8 and the second quality control region 9; First detection zone 6 is provided with recombinant syphilis spirochete antigen, second detection zone 7 is provided with mouse-anti human IgG monoclonal antibody, first quality control region 8 is provided with mouse-anti microspironema pallidum IgG monoclonal antibody, and the second quality control region 9 is provided with the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit; Or the first detection zone 6 is provided with mouse-anti human IgG monoclonal antibody, second detection zone 7 is provided with recombinant syphilis spirochete antigen, first quality control region 8 is provided with the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit, and the second quality control region 9 is provided with mouse-anti microspironema pallidum IgG monoclonal antibody.Known by the test findings detected a large amount of serum specimen, the sensitivity of test card provided by the invention reaches 98.23%, specificity reaches 93.50%, false positive rate only 6.50%, and the sensitivity adopting RPR method to carry out the test card A detected is 93.11%, specificity is 89.25%, false positive rate is 10.75%, the sensitivity adopting TRUST method to carry out the test card B detected is 94.29%, and specificity is 90.78%, and false positive rate is 9.22%.As can be seen here, test card provided by the invention is highly sensitive, high specificity, and false positive rate is low, without the interference of previous infection, and simple to operation.
Accompanying drawing explanation
Fig. 1 shows the structural representation of the test strips that embodiment 5 provides; Wherein, 1 shows base plate, and 2 show sample pad, and 3 show glass fibre element film, and 4 show nitrocellulose filter, and 5 show thieving paper, and 6 show the first detection zone, and 7 show the second detection zone, and 8 show the first quality control region, and 9 show the second quality control region;
Fig. 2 shows the structural representation of the test card housing that embodiment 8 provides; Wherein, 10 show sample application zone, and 11 show detection zone.
Embodiment
The invention discloses syphilis test strips and preparation method thereof, Lues Assay card and preparation method thereof, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
In syphilis test strips provided by the invention and preparation method thereof, Lues Assay card and preparation method thereof, agents useful for same and consumptive material all can be buied by market.Wherein, Freund's complete adjuvant, not formula Freund's incomplete adjuvant, cuorin and cardiolipin binding protein equal purchased from American SIGMA company, the syphilis specific antigen of colloidal gold solution, mouse-anti human IgG antibody, colloid gold label, syphilis recombinant antigen, mouse-anti syphilis IgG antibody all inspire confidence in company purchased from Guangzhou ten thousand.
Below in conjunction with embodiment, set forth the present invention further:
The preparation of the cuorin-cardiolipin binding protein complex of embodiment 1 colloid gold label
Get 1g cuorin to be dissolved in 500mL absolute ethyl alcohol, then be settled to 1L, be mixed with the cuorin ethanolic solution of 1mg/mL; Get 1g cardiolipin binding protein to be dissolved in 500mL water, then be settled to 1L, be mixed with the cardiolipin binding protein aqueous solution of 1mg/mL.
Get 100mL cuorin ethanolic solution and 100mL cardiolipin binding protein aqueous solution, after 37 DEG C of 2h again 4 DEG C place 10h, obtain cuorin-cardiolipin binding protein complex solution; Get the colloidal gold solution of 50mL5mg/mL, magnetic stirring apparatus slowly stirs, slowly add above-mentioned cuorin-cardiolipin binding protein complex solution again, 2h is stirred at 4 DEG C, add 50mL1% bovine serum albumin solution again, the centrifugal 30min of 1h, 12000rpm is closed at 37 DEG C, stand-by under being placed on the condition of 2 ~ 8 DEG C with the phosphate buffer redissolution of 50mL50mM, pH7.4.
The preparation of the cuorin-cardiolipin binding protein complex of embodiment 2 colloid gold label
Get 0.5g cuorin to be dissolved in 500mL absolute ethyl alcohol, then be settled to 1L, be mixed with the cuorin ethanolic solution of 0.5mg/mL; Get 5g cardiolipin binding protein to be dissolved in 500mL water, then be settled to 1L, be mixed with the cardiolipin binding protein aqueous solution of 5mg/mL.
Get 200mL cuorin ethanolic solution and 100mL cardiolipin binding protein aqueous solution, after 37 DEG C of 1h again 2 DEG C place 12h, obtain cuorin-cardiolipin binding protein complex solution; Get the colloidal gold solution of 50mL5mg/mL, magnetic stirring apparatus slowly stirs, slowly add above-mentioned cuorin-cardiolipin binding protein complex solution again, 2h is stirred at 2 DEG C, add 70mL1% bovine serum albumin solution again, the centrifugal 30min of 1h, 12000rpm is closed at 37 DEG C, stand-by under being placed on the condition of 2 ~ 8 DEG C with the phosphate buffer redissolution of 50mL50mM, pH7.4.
The preparation of the cuorin-cardiolipin binding protein complex of embodiment 3 colloid gold label
Get 5g cuorin to be dissolved in 500mL absolute ethyl alcohol, then be settled to 1L, be mixed with the cuorin ethanolic solution of 5mg/mL; Get 0.5g cardiolipin binding protein to be dissolved in 500mL water, then be settled to 1L, be mixed with the cardiolipin binding protein aqueous solution of 0.5mg/mL.
Get 100mL cuorin ethanolic solution and 200mL cardiolipin binding protein aqueous solution, after 37 DEG C of 4h again 8 DEG C place 8h, obtain cuorin-cardiolipin binding protein complex solution; Get the colloidal gold solution of 50mL5mg/mL, magnetic stirring apparatus slowly stirs, slowly add above-mentioned cuorin-cardiolipin binding protein complex solution again, 2h is stirred at 8 DEG C, add 70mL1% bovine serum albumin solution again, the centrifugal 30min of 1h, 12000rpm is closed at 37 DEG C, stand-by under being placed on the condition of 2 ~ 8 DEG C with the phosphate buffer redissolution of 50mL50mM, pH7.4.
The preparation of the anti-cardiolipin binding protein IgG monoclonal antibody of embodiment 4 rabbit
By cardiolipin binding protein physiological saline solution, be mixed with the cardiolipin binding protein solution of 0.5mg/mL, 10mL cardiolipin binding protein solution is mixed with isodose Freund's complete adjuvant, fully obtained first mixed liquor after mixing.Get healthy adult New Zealand white big ear rabbit, with syringe, above-mentioned for 5mL the first mixed liquor is injected into two metapedes pads, completes first immunisation.
By cardiolipin binding protein physiological saline solution, be mixed with the cardiolipin binding protein solution of 0.5mg/mL, 10mL cardiolipin binding protein solution is mixed with isodose incomplete Freund's adjuvant, fully obtained second mixed liquor after mixing.10-14 days after first immunisation, is injected into rabbit back with syringe by above-mentioned second mixed liquor subcutaneous, completes second time immunity.
10-14 days after second time immunity, adopts the dosage of second time immunity and method to carry out third time immunity.
5-7 days after third time immunity, rabbit heart is taken a blood sample, separation of serum, adopts the anti-cardiolipin binding protein IgG monoclonal antibody of ammonium sulfate precipitation method separation and purification rabbit, adds equivalent glycerine frozen stand-by after dialysis.
The preparation of embodiment 5 syphilis test strips
The phosphate buffer of the microspironema pallidum specific antigen of 100mg colloid gold label with 100mL50mM, pH7.4 is dissolved, is mixed with the microspironema pallidum specific antigen solution of the colloid gold label of 1mg/mL.Glass fibre element film sprays the obtained cuorin-cardiolipin binding protein complex solution of colloid gold label of embodiment 1 and the microspironema pallidum specific antigen solution of above-mentioned colloid gold label, and quantity for spray is 8.5 μ L/cm, dry 2h at 37 DEG C.
Get the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit that recombinant syphilis spirochete antigen, mouse-anti microspironema pallidum IgG monoclonal antibody, mouse-anti human IgG monoclonal antibody, embodiment 4 are obtained, be dissolved in the Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid (Tris-HCl damping fluid) of 50mM, pH8.0 respectively, be mixed with 1mg/mL recombinant syphilis spirochete antigenic solution, 1mg/mL mouse-anti microspironema pallidum IgG monoclonal antibody solution, 1mg/mL mouse-anti human IgG monoclonal antibody solution, 1mg/mL rabbit anti-cardiolipin binding protein IgG monoclonal antibody solution.
On nitrocellulose filter, by recombinant syphilis spirochete antigenic solution point film in the first detection zone 6, by mouse-anti human IgG monoclonal antibody solution point film in the second detection zone 7, by mouse-anti microspironema pallidum IgG monoclonal antibody solution point film in the first quality control region 8, by anti-for rabbit cardiolipin binding protein IgG monoclonal antibody solution point film in the second quality control region 9, dry 2h under 37 DEG C of conditions.
Sample pad 2, glass fibre element film 3, nitrocellulose filter 4, thieving paper 5 are sequentially fixed on base plate 1, obtain final product.Test strips structure as shown in Figure 1.
The preparation of embodiment 6 syphilis test strips
The phosphate buffer of the microspironema pallidum specific antigen of 100mg colloid gold label with 100mL50mM, pH7.4 is dissolved, is mixed with the microspironema pallidum specific antigen solution of the colloid gold label of 1mg/mL.Glass fibre element film sprays the obtained cuorin-cardiolipin binding protein complex solution of colloid gold label of embodiment 2 and the microspironema pallidum specific antigen solution of above-mentioned colloid gold label, and quantity for spray is 8.5 μ L/cm, dry 2h at 37 DEG C.
Get the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit that recombinant syphilis spirochete antigen, mouse-anti microspironema pallidum IgG monoclonal antibody, mouse-anti human IgG monoclonal antibody, embodiment 4 are obtained, be dissolved in the Tris-HCl damping fluid of 50mM, pH8.0 respectively, be mixed with 1mg/mL recombinant syphilis spirochete antigenic solution, 1mg/mL mouse-anti microspironema pallidum IgG monoclonal antibody solution, 1mg/mL mouse-anti human IgG monoclonal antibody solution, 1mg/mL rabbit anti-cardiolipin binding protein IgG monoclonal antibody solution.
On nitrocellulose filter, by mouse-anti human IgG monoclonal antibody solution point film in the first detection zone 6, by recombinant syphilis spirochete antigenic solution point film in the second detection zone 7, by anti-for rabbit cardiolipin binding protein IgG monoclonal antibody solution point film in the first quality control region 8, by mouse-anti microspironema pallidum IgG monoclonal antibody solution point film in the second quality control region 9, dry 2h under 37 DEG C of conditions.
Sample pad 2, glass fibre element film 3, nitrocellulose filter 4, thieving paper 5 are fixed on base plate 1, obtain final product.
The preparation of embodiment 7 syphilis test strips
The phosphate buffer of the microspironema pallidum specific antigen of 100mg colloid gold label with 100mL50mM, pH7.4 is dissolved, is mixed with the microspironema pallidum specific antigen solution of the colloid gold label of 1mg/mL.Glass fibre element film sprays the obtained cuorin-cardiolipin binding protein complex solution of colloid gold label of embodiment 3 and the microspironema pallidum specific antigen solution of above-mentioned colloid gold label, and quantity for spray is 8.5 μ L/cm, dry 2h at 37 DEG C.
Get the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit that recombinant syphilis spirochete antigen, mouse-anti microspironema pallidum IgG monoclonal antibody, mouse-anti human IgG monoclonal antibody, embodiment 4 are obtained, be dissolved in the Tris-HCl damping fluid of 50mM, pH8.0 respectively, be mixed with 1mg/mL recombinant syphilis spirochete antigenic solution, 1mg/mL mouse-anti microspironema pallidum IgG monoclonal antibody solution, 1mg/mL mouse-anti human IgG monoclonal antibody solution, 1mg/mL rabbit anti-cardiolipin binding protein IgG monoclonal antibody solution.
On nitrocellulose filter, by recombinant syphilis spirochete antigenic solution point film in the first detection zone 6, by mouse-anti human IgG monoclonal antibody solution point film in the second detection zone 7, by mouse-anti microspironema pallidum IgG monoclonal antibody solution point film in the first quality control region 8, by anti-for rabbit cardiolipin binding protein IgG monoclonal antibody solution point film in the second quality control region 9, dry 2h under 37 DEG C of conditions.
Sample pad 2, glass fibre element film 3, nitrocellulose filter 4, thieving paper 5 are fixed on base plate 1, obtain final product.
The preparation of embodiment 8 Lues Assay card
The syphilis test strips that Example 5 provides, is assembled in test card housing, obtains final product.The structure of test card housing as shown in Figure 2.
The preparation of embodiment 9 Lues Assay card
The syphilis test strips that Example 6 provides, is assembled in test card housing, obtains final product.
The preparation of embodiment 10 Lues Assay card
The syphilis test strips that Example 7 provides, is assembled in test card housing, obtains final product.
Embodiment 11 clinical testing
Obtain serum specimen totally 5812 parts in disease prevention and control center of Henan Province and Henan Province's Blood Center, wherein, make a definite diagnosis syphilization patients serum sample 372 parts, previous infection syphilitic serum specimen 191 parts, healthy normal human serum sample 5249 parts.Utilize the embodiment of the present invention 8 obtained Lues Assay card, commercially available test card A(RPR method) and commercially available test card B(TRUST method) carry out sensitivity, specificity and false positive rate detection respectively.
When the test card adopting embodiment 8 to provide detects serum specimen, test card is kept flat, by 100 μ L serum specimen instillation sample application zone, observations after 10min.All there is red stripes in the first detection zone, the second detection zone, the first quality control region and the second quality control region place, then judges that serum specimen is as making a definite diagnosis syphilization patients serum sample; There is red stripes in the first detection zone, the first quality control region and the second quality control region place, the second detection zone place not Show Color, then judge that serum specimen is as previous infection syphilitic serum specimen; There is red stripes in the first quality control region and the second quality control region place, the first detection zone and the second detection zone place not Show Color, then judge that serum specimen is as healthy normal human serum sample; There is red stripes in the first quality control region and the second quality control region place, the first detection zone place not Show Color, and red stripes appears in the second detection zone place, then judge that serum specimen is as the suspicious sample of syphilis.Commercially available test card A and the operation of test card B and result detect and perform with reference to instructions.Testing result is as shown in table 1.
Table 1 sensitivity, specificity and false positive rate testing result
From above-mentioned test findings, the sensitivity of the syphilis non-specific antibody item of the Lues Assay card that the embodiment of the present invention 8 provides is 98.23%, specificity is 93.50%, false positive rate is 6.50%, sensitivity and specificity are obviously better than commercially available test card A and test card B, and false positive rate is low, without the interference of previous infection.As can be seen here, the Lues Assay card that the embodiment of the present invention 8 provides is highly sensitive, high specificity, and false positive rate is low, without the interference of previous infection.
The Lues Assay card that Example 9, embodiment 10 provide carries out clinical testing, and similarly, show embodiment 9, Lues Assay card that embodiment 10 provides is highly sensitive, high specificity, false positive rate is low, without the interference of previous infection for result.
As can be seen here, Lues Assay card provided by the invention is highly sensitive, high specificity, and false positive rate is low, without the interference of previous infection, simple to operation.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a syphilis test strips, is characterized in that, described syphilis test strips comprises base plate (1), and described base plate (1) is disposed with sample pad (2), glass fibre element film (3), nitrocellulose filter (4), thieving paper (5);
Described glass fibre element film (3) is provided with the microspironema pallidum specific antigen of colloid gold label and the cuorin-cardiolipin binding protein complex of colloid gold label;
The preparation method of the cuorin-cardiolipin binding protein complex of described colloid gold label is: core phosphatide and cardiolipin binding protein mixing, through the first incubation, obtain cuorin-cardiolipin binding protein complex, get described cuorin-cardiolipin binding protein complex and collaurum mixing, through the second incubation, close, obtain the cuorin-cardiolipin binding protein complex of described colloid gold label;
Described nitrocellulose filter (4) is provided with the first detection zone (6), the second detection zone (7), the first quality control region (8) and the second quality control region (9);
Described first detection zone (6) is provided with recombinant syphilis spirochete antigen, described second detection zone (7) is provided with mouse-anti human IgG monoclonal antibody, described first quality control region (8) is provided with mouse-anti microspironema pallidum IgG monoclonal antibody, and described second quality control region (9) is provided with the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit;
Or described first detection zone (6) is provided with mouse-anti human IgG monoclonal antibody, described second detection zone (7) is provided with recombinant syphilis spirochete antigen, described first quality control region (8) is provided with the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit, and described second quality control region (9) is provided with mouse-anti microspironema pallidum IgG monoclonal antibody.
2. syphilis test strips according to claim 1, is characterized in that, described first incubation is hatch 1 ~ 4h under the condition of 37 DEG C, then places 8 ~ 12h under the condition of 2 ~ 8 DEG C.
3. syphilis test strips according to claim 1, is characterized in that, in the preparation method of the cuorin-cardiolipin binding protein complex of described colloid gold label, the mass ratio of described cuorin and described cardiolipin binding protein is (1 ~ 5): (1 ~ 5).
4. syphilis test strips according to claim 3, is characterized in that, the mass ratio of described cuorin and described cardiolipin binding protein is 1:1.
5. the preparation method of the syphilis test strips according to any one of Claims 1-4, is characterized in that, comprise the steps:
Steps A: core phosphatide and cardiolipin binding protein mixing, through the first incubation, obtain cuorin-cardiolipin binding protein complex, get described cuorin-cardiolipin binding protein complex and collaurum mixing, through the second incubation, close, obtain the cuorin-cardiolipin binding protein complex of colloid gold label;
Obtain the microspironema pallidum specific antigen of recombinant syphilis spirochete antigen, mouse-anti microspironema pallidum IgG monoclonal antibody, mouse-anti human IgG monoclonal antibody, the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit and colloid gold label;
Step B: the microspironema pallidum specific antigen of the cuorin-cardiolipin binding protein complex and described colloid gold label of getting described colloid gold label is coated in glass fibre element film (3);
Get described recombinant syphilis spirochete antigen coated in first detection zone (6) of nitrocellulose filter (4), get the second detection zone (7) that described mouse-anti human IgG monoclonal antibody is coated in nitrocellulose filter (4), get the first quality control region (8) that described mouse-anti microspironema pallidum IgG monoclonal antibody is coated in nitrocellulose filter (4), get the second quality control region (9) that the anti-cardiolipin binding protein IgG monoclonal antibody of described rabbit is coated in nitrocellulose filter (4);
Or get the first detection zone (6) that described mouse-anti human IgG monoclonal antibody is coated in nitrocellulose filter (4), get described recombinant syphilis spirochete antigen coated in second detection zone (7) of nitrocellulose filter (4), get the first quality control region (8) that the anti-cardiolipin binding protein IgG monoclonal antibody of described rabbit is coated in nitrocellulose filter (4), get the second quality control region (9) that described mouse-anti microspironema pallidum IgG monoclonal antibody is coated in nitrocellulose filter (4);
Step C: sample thief pad (2), described glass fibre element film (3), described nitrocellulose filter (4), thieving paper (5) are sequentially fixed on base plate (1), to obtain final product.
6. preparation method according to claim 5, is characterized in that, described first incubation is hatch 1 ~ 4h under the condition of 37 DEG C, then places 8 ~ 12h under the condition of 2 ~ 8 DEG C.
7. preparation method according to claim 5, is characterized in that, in the preparation method of the cuorin-cardiolipin binding protein complex of described colloid gold label, the mass ratio of described cuorin and described cardiolipin binding protein is (1 ~ 5): (1 ~ 5).
8. preparation method according to claim 7, is characterized in that, the mass ratio of described cuorin and described cardiolipin binding protein is 1:1.
9. a Lues Assay card, is characterized in that, comprises test card housing and the syphilis test strips according to any one of Claims 1-4;
Described test card housing is provided with sample application zone (10) and detection zone (11).
10. the preparation method of Lues Assay card as claimed in claim 9, is characterized in that, gets test card housing and the assembling of the syphilis test strips according to any one of Claims 1-4, to obtain final product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310478624.XA CN104330566B (en) | 2013-10-14 | 2013-10-14 | Syphilis test strips and preparation method thereof, Lues Assay card and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310478624.XA CN104330566B (en) | 2013-10-14 | 2013-10-14 | Syphilis test strips and preparation method thereof, Lues Assay card and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104330566A true CN104330566A (en) | 2015-02-04 |
| CN104330566B CN104330566B (en) | 2016-07-06 |
Family
ID=52405332
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310478624.XA Active CN104330566B (en) | 2013-10-14 | 2013-10-14 | Syphilis test strips and preparation method thereof, Lues Assay card and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104330566B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198957A (en) * | 2016-06-30 | 2016-12-07 | 深圳市亚辉龙生物科技股份有限公司 | Modified coated nanometer magnetic bead of cuorin and preparation method thereof |
| CN107037211A (en) * | 2016-11-04 | 2017-08-11 | 上海海茉生物科技有限公司 | A kind of test-strips of quantitative detection compound and the detection method of lateral immunochromatography |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101243321A (en) * | 2005-06-21 | 2008-08-13 | 美国政府健康及人类服务部，疾病控制和预防中心 | Methods, immunoassays and devices for detection of anti-lipoidal antibodies |
| WO2009003177A1 (en) * | 2007-06-27 | 2008-12-31 | Inbios International, Inc. | Lateral flow assay system and methods for its use |
| CN101360997A (en) * | 2005-11-18 | 2009-02-04 | 美国政府健康及人类服务部，疾病控制和预防中心 | Modified cardiolipin and uses therefor |
| CN201673157U (en) * | 2010-05-19 | 2010-12-15 | 厦门大学附属中山医院 | Colloidal gold immunochromatographic test reagent strip for syphilis specific total antibody |
| CN201955343U (en) * | 2011-01-25 | 2011-08-31 | 厦门大学附属中山医院 | Syphilis cardiolipin and specific antibody IgM western-blot kit |
-
2013
- 2013-10-14 CN CN201310478624.XA patent/CN104330566B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101243321A (en) * | 2005-06-21 | 2008-08-13 | 美国政府健康及人类服务部，疾病控制和预防中心 | Methods, immunoassays and devices for detection of anti-lipoidal antibodies |
| CN101360997A (en) * | 2005-11-18 | 2009-02-04 | 美国政府健康及人类服务部，疾病控制和预防中心 | Modified cardiolipin and uses therefor |
| WO2009003177A1 (en) * | 2007-06-27 | 2008-12-31 | Inbios International, Inc. | Lateral flow assay system and methods for its use |
| CN201673157U (en) * | 2010-05-19 | 2010-12-15 | 厦门大学附属中山医院 | Colloidal gold immunochromatographic test reagent strip for syphilis specific total antibody |
| CN201955343U (en) * | 2011-01-25 | 2011-08-31 | 厦门大学附属中山医院 | Syphilis cardiolipin and specific antibody IgM western-blot kit |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198957A (en) * | 2016-06-30 | 2016-12-07 | 深圳市亚辉龙生物科技股份有限公司 | Modified coated nanometer magnetic bead of cuorin and preparation method thereof |
| CN106198957B (en) * | 2016-06-30 | 2018-10-02 | 深圳市亚辉龙生物科技股份有限公司 | Coated nanometer magnetic bead of modified cuorin and preparation method thereof |
| CN107037211A (en) * | 2016-11-04 | 2017-08-11 | 上海海茉生物科技有限公司 | A kind of test-strips of quantitative detection compound and the detection method of lateral immunochromatography |
| CN107037211B (en) * | 2016-11-04 | 2019-03-01 | 苏州海苗生物科技有限公司 | A kind of test-strips of quantitative detection compound and the detection method of lateral immunochromatography |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104330566B (en) | 2016-07-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102209489B1 (en) | Method and device for combined detection of viral and bacterial infections | |
| US5187065A (en) | Method and materials for detecting lyme disease | |
| CN102072957B (en) | Hepatitis C virus antibody diagnostic kit and preparation method thereof | |
| CN101738473B (en) | Treponema pallidum antibody diagnostic kit and preparation method thereof | |
| BRPI0611866A2 (en) | immunoassay device and method for immobilizing immunoreactive cardiolipin | |
| CN101196518A (en) | Hepatitis virus type C immune body chemiluminescence method diagnostic reagent kit and its producing method | |
| CN101165491A (en) | Method for detecting double-chain DNA resistant antibody using gold magnetic particle as carrier | |
| CN101393217B (en) | Detection kit for schistosomiasis japonica blood serum designated object | |
| CN109188000A (en) | A kind of test strips and preparation method thereof of portable inspectiont human parathyroid hormone | |
| KR101612095B1 (en) | Biomaker detecting probe possible to early detection and precise quantification and use thereof | |
| EP3349010B1 (en) | Anti-vla-4 related assays | |
| TW201814291A (en) | Method for removing inhibitory components | |
| EP1185872B1 (en) | Method for detecting syphilis using synthetic antigens | |
| CN105753981B (en) | The immune chromatography reagent kit of anti-human Respiratory Syncytial Virus(RSV) N protein antibody and the application antibody | |
| US20230341392A1 (en) | Detection of antibodies to sars-cov2 | |
| GB1590524A (en) | Assay of immune complexes | |
| CN104330566B (en) | Syphilis test strips and preparation method thereof, Lues Assay card and preparation method thereof | |
| JP2022505934A (en) | Lateral flow assay for differential isotype detection | |
| CN107870244A (en) | A kind of immunoliposome LAMP method for the super quick detection of protein | |
| JP5969777B2 (en) | Tumor marker and diagnostic kit for lung adenosquamous cell carcinoma | |
| CN107402300A (en) | A kind of method of quick discriminating human parathyroid | |
| Chen et al. | Determination of echinococcosis IgG antibodies using magnetic bead-based chemiluminescence immunoassay | |
| EP4261541A1 (en) | Sensitizer for immunochromatographic assays, and assay | |
| CN102368068A (en) | Kit for detecting chlamydia pneumoniae IgM antibody | |
| USRE43914E1 (en) | Method for detecting syphilis using synthetic antigens |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210402 Address after: 464000 intersection of Gongwu road and Donghuan Road, high tech Zone, Xinyang City, Henan Province Patentee after: Henan Taiyu Gaogao Medical Equipment Co.,Ltd. Patentee after: Henan Taifu Medical Laboratory Center Co.,Ltd. Address before: 450000 No.6, building 7, District 2, Luoman Weisen, No.168, Guoji Road, Jinshui District, Zhengzhou City, Henan Province Patentee before: Zhao Lei |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211022 Address after: 464000 intersection of Gongwu road and Donghuan Road, high tech Zone, Xinyang City, Henan Province Patentee after: Henan Taiyu Gaogao Medical Equipment Co.,Ltd. Patentee after: Henan Taifu Medical Laboratory Center Co.,Ltd. Patentee after: Lu Mingxia Address before: 464000 intersection of Gongwu road and Donghuan Road, high tech Zone, Xinyang City, Henan Province Patentee before: Henan Taiyu Gaogao Medical Equipment Co.,Ltd. Patentee before: Henan Taifu Medical Laboratory Center Co.,Ltd. |