CN104330566B - Syphilis test strips and preparation method thereof, Lues Assay card and preparation method thereof - Google Patents
Syphilis test strips and preparation method thereof, Lues Assay card and preparation method thereof Download PDFInfo
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- CN104330566B CN104330566B CN201310478624.XA CN201310478624A CN104330566B CN 104330566 B CN104330566 B CN 104330566B CN 201310478624 A CN201310478624 A CN 201310478624A CN 104330566 B CN104330566 B CN 104330566B
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- 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
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- 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
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- 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
Abstract
The present invention relates to technical field of immunoassay, particularly to syphilis test strips and preparation method thereof, Lues Assay card and preparation method thereof.This syphilis test strips includes base plate 1, and base plate 1 is disposed with sample pad 2, glass fibre element film 3, nitrocellulose filter 4, absorbent paper 5；Glass fibre element film 3 is provided with the treponema pallidum specific antigen of colloid gold label and the cuorin-cardiolipin binding protein complex of colloid gold label；The preparation method of the cuorin of colloid gold label-cardiolipin binding protein complex is: core phospholipid and cardiolipin binding protein mixing, through the first incubation, obtain cuorin-cardiolipin binding protein complex, core phospholipid-cardiolipin binding protein complex and gold colloidal mixing, through the second incubation, closing, obtain the cuorin-cardiolipin binding protein complex of colloid gold label.Detection card provided by the invention is simple to operation, highly sensitive, and high specificity, false positive rate is low, it is to avoid the previous infection interference to Current Infection.
Description
Technical field
The present invention relates to technical field of immunoassay, particularly to 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, and appearance centuries all has distribution at world wide at present, is highly important sexually transmitted disease (STD), is divided into acquired syphilis and congenital syphilis according to source of infection.Wherein, it is thus achieved that property syphilis refers to that adult is by sexual behaviour, blood transfusion, operation and infected syphilis, it is divided into three phases, cardinal symptom to include invading mucocutaneous and involving internal organs according to clinical manifestation, is a kind of comparatively serious sexually transmitted disease (STD)；Congenital syphilis is that treponema pallidum is directly entered in fetus body the syphilis caused by Placenta Hominis owing to syphilitic anemia of pregnant woman is without treatment or malpractice in time.
At present, the test in laboratory method of syphilis mainly includes dark-field microscopy, direct fluorescent 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 includes enzyme linked immunosorbent assay (ELISA) and colloidal gold method；Non-specific antibody detection method mainly includes 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 deposit defect both ways, on the one hand owing to its detection is IgG antibody, and after human infection treponema pallidum, the time needing more than 2 weeks just can produce IgG antibody, and therefore initial infection cannot detect syphilis；Syphilis specific IgG antibodies can carry after producing all the life on the other hand, even if human body still can carry syphilis specific IgG antibodies after syphilis is treated, therefore detection syphilis specific IgG antibodies cannot be distinguished by syphilis merely is Current Infection or previous infection, Current Infection will be produced certain interference by previous infection, in addition it is also necessary to do non-specific antibody detection further.
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 may be up to 10% in junkie, additionally in the special populations such as tuberculosis, connective tissue disease and anemia of pregnant woman, false positive rate all has rising in various degree, 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 provide a kind of highly sensitive, high specificity, not by the Lues Assay card of previous infection interference, have 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 by by colloid gold label cuorin, it is achieved thereby that syphilis specific antibody test and syphilis non-specific antibody are detected the purpose combined, both there is advantage highly sensitive, that high specificity, false positive rate are low, avoid the previous infection interference to Current Infection, and simple to operation.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides a kind of syphilis test strips, syphilis test strips includes base plate 1, and base plate 1 is disposed with sample pad 2, glass fibre element film 3, nitrocellulose filter 4, absorbent paper 5；
Glass fibre element film 3 is provided with the treponema pallidum specific antigen of colloid gold label and the cuorin-cardiolipin binding protein complex of colloid gold label；
The preparation method of the cuorin of colloid gold label-cardiolipin binding protein complex is: core phospholipid and cardiolipin binding protein mixing, through the first incubation, obtain cuorin-cardiolipin binding protein complex, core phospholipid-cardiolipin binding protein complex and gold colloidal mixing, through the second incubation, closing, obtain the cuorin-cardiolipin binding protein complex of colloid gold label；
Nitrocellulose filter 4 is provided with the first detection zone the 6, second detection zone the 7, first 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 treponema 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 treponema 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, after sample to be checked is added in test strips sample pad, moved forward by capillarity, react to each other after dissolving the colloid gold label reagent on pad, when the region of mobile extremely fixing antigen or antibody, the conjugate of thing to be checked and gold marked reagent occurs with it again specific binding and is trapped, it is gathered on detection band, can pass through to be 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 little molecule, up to the present how also do not have with the report of colloid gold label cuorin, detect the method for syphilis non-specific antibody but without realization hence with colloidal gold-labeled method.The present invention key bridge effect by cardiolipin binding protein, it is achieved that the combination of cuorin and gold colloidal, has reached to detect the purpose of syphilis non-specific antibody with colloidal gold-labeled method.
The detection of syphilis specific antibody and syphilis non-specific antibody is incorporated on a detection card by the present invention, it is achieved thereby that the synchronous detecting to syphilis specific antibody and syphilis non-specific antibody so that testing result is more accurate, reduces false positive rate.
It is provided with two detection zones in the present invention, including 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 detect specific 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 treponema pallidum specific antigen of the colloid gold label on glass fibre element film of the syphilis specific antibody in serum sample is combined, and forms the treponema pallidum specific antigen complex of syphilis specific antibody-colloidal gold labelling；Then the treponema pallidum specific antigen complex of syphilis specific antibody-colloidal gold labelling continues to enter nitrocellulose filter along chromatography direction, be combined with the recombinant syphilis spirochete antigen of detection zone position, form the treponema pallidum specific antigen complex of recombinant syphilis spirochete antigen-syphilis specific antibody-colloidal gold labelling, red in detection zone position display, it is syphilis specific antibody positive, do not develop the color in detection zone position, 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 on glass fibre element film of the non-characteristic antibody of syphilis in serum sample is combined, and forms the cuorin-cardiolipin binding protein complex of the non-characteristic antibody-colloidal gold labelling of syphilis；Then the cuorin of the non-characteristic antibody-colloidal gold labelling of syphilis-cardiolipin binding protein complex continues to enter nitrocellulose filter along chromatography direction, be combined with the mouse-anti human IgG monoclonal antibody of detection zone position, form the cuorin-cardiolipin binding protein complex of the non-characteristic antibody-colloidal gold labelling of mouse-anti human IgG monoclonal antibody-syphilis, red in detection zone position display, it is the non-characteristic antibody positive of syphilis, do not develop the color in detection zone position, be syphilis non-specific antibody negative.
When using detection card detection serum sample provided by the invention, result decision method is: when the detection zone of detection syphilis specific antibody and the detection zone of detection syphilis non-specific antibody all show redness, can determine that as syphilis；The detection zone display redness of detection syphilis specific antibody, and when the detection zone detecting syphilis non-specific antibody does not show redness, can determine that as syphilis previous infection；The detection zone display redness of detection syphilis non-specific antibody, and when the detection zone detecting syphilis specific antibody does not show redness, can determine that as doubtful syphilis, repetition measurement again after spending certain time (one thoughtful one month)；When the detection zone of detection syphilis specific antibody and the detection zone of detection syphilis non-specific antibody all do not show redness, can determine that as syphilis 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.It is provided with two quality control region in the present invention, including 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 treponema 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 treponema pallidum IgG monoclonal antibody (i.e. specific antibody).Quality control region Cleaning Principle is as follows:
The treponema pallidum specific antigen of the colloid gold label on glass fibre element film enters nitrocellulose filter with serum sample along chromatography method, it is fixed with mouse-anti treponema pallidum IgG monoclonal antibody on nitrocellulose filter to be combined, is being coated with the quality control region position display redness of specific antibody or is not showing redness；
The cuorin of the colloid gold label on glass fibre element film-cardiolipin binding protein complex enters nitrocellulose filter with serum sample along chromatography method, it is fixed with the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit on nitrocellulose filter to be combined, is being coated with the quality control region position display redness of non-specific antibody or is 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 treponema 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 the 6, second detection zone the 7, first quality control region the 8, second quality control region 9 all develops the color, then be judged to that syphilis is positive, i.e. syphilization；
(2) first detection zone the 6, first quality control region the 8, second quality control region 9 develop the color, and the second detection zone 7 does not develop the color, then be judged to syphilis previous infection；
(3) second detection zone the 7, first quality control region the 8, second quality control region 9 develop the color, and the first detection zone 6 does not develop the color, then be judged to doubtful syphilis；
(4) first detection zone the 6, second detection zones 7 do not develop the color, and first quality control region the 8, second quality control region 9 develops the color, then be judged to that syphilis is negative, be namely uninfected by syphilis；
(5) first detection zone the 6, second detection zone the 7, first quality control region the 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 treponema pallidum IgG monoclonal antibody, Results of Syphilis is as follows:
(1) first detection zone the 6, second detection zone the 7, first quality control region the 8, second quality control region 9 all develops the color, then be judged to that syphilis is positive, i.e. syphilization；
(2) first detection zone the 7, first quality control region the 8, second quality control region 9 develop the color, and the second detection zone 7 does not develop the color, then be judged to syphilis previous infection；
(3) second detection zone the 6, first quality control region the 8, second quality control region 9 develop the color, and the first detection zone 6 does not develop the color, then be judged to doubtful syphilis；
(4) first detection zone the 6, second detection zones 7 do not develop the color, and first quality control region the 8, second quality control region 9 develops the color, then be judged to that syphilis is negative, be namely uninfected by syphilis；
(5) first detection zone the 6, second detection zone the 7, first quality control region the 8, second quality control region 9 all do not develop the color, then test strips lost efficacy.
By studying, 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 the incubation when uniform temperature.In embodiments more provided by the invention, the first incubation is hatch 1～4h when 37 DEG C, then places 8～12h when 2～8 DEG C.
In embodiments more provided by the invention, in the preparation method of the cuorin of colloid gold label-cardiolipin binding protein complex, 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 respectively prepared cuorin alcoholic solution and cardiolipin binding protein aqueous solution, again by cuorin alcoholic solution and cardiolipin binding protein aqueous solution, carry out the first incubation.
In embodiments more provided by the invention, the preparation method of cuorin alcoholic solution is: is dissolved in dehydrated alcohol by cuorin, to obtain final product.
In embodiments more provided by the invention, the mass body volume concentrations of cuorin alcoholic solution is preferably 0.5～5mg/mL.
Preferably, the mass body volume concentrations of cuorin alcoholic solution is preferably 1mg/mL.
In embodiments more provided by the invention, the preparation method of cardiolipin binding protein aqueous solution is: is dissolved in the water by cardiolipin binding protein, to 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 the incubation when uniform temperature.In embodiments more provided by the invention, in the cuorin-cardiolipin binding protein complex process preparing colloid gold label, the second incubation is the reaction 2h when 2～8 DEG C.
In embodiments more provided by the invention, in order to close unnecessary protein binding site, the present invention adopts bovine serum albumin solution that the cuorin-cardiolipin binding protein complex of colloid gold label is closed.
As preferably, the mass percentage concentration of bovine serum albumin solution is 1%.
In embodiments more provided by the invention, the temperature of closing is 37 DEG C.
In embodiments more provided by the invention, the time of closing is 1h.
In embodiments more provided by the invention, the preparation method of the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit, for taking cardiolipin binding protein immunity rabbit, to obtain final product.
The preparation method that present invention also offers syphilis test strips provided by the invention, comprises the steps:
Step A: core phospholipid and cardiolipin binding protein mixing, through the first incubation, obtaining cuorin-cardiolipin binding protein complex, phospholipid of coring-cardiolipin binding protein complex and gold colloidal mix, through the second incubation, closing, obtain the cuorin-cardiolipin binding protein complex of colloid gold label；
Obtain the treponema pallidum specific antigen of recombinant syphilis spirochete antigen, mouse-anti treponema 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 treponema pallidum specific antigen of the cuorin-cardiolipin binding protein complex and colloid gold label that take colloid gold label is coated in glass fibre element film 3；
Take recombinant syphilis spirochete antigen coated in the first detection zone 6 of nitrocellulose filter 4, take mouse-anti human IgG monoclonal antibody and be coated in the second detection zone 7 of nitrocellulose filter 4, take mouse-anti treponema pallidum IgG monoclonal antibody and be coated in the first quality control region 8 of nitrocellulose filter 4, take the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit and be coated in the second quality control region 9 of nitrocellulose filter 4；
Or take mouse-anti human IgG monoclonal antibody and be coated in the first detection zone 6 of nitrocellulose filter 4, take recombinant syphilis spirochete antigen coated in the second detection zone 7 of nitrocellulose filter 4, take the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit and be coated in the first quality control region 8 of nitrocellulose filter 4, take mouse-anti treponema pallidum IgG monoclonal antibody and be coated in the second quality control region 9 of nitrocellulose filter 4；
Step C: take sample pad 2, glass fibre element film 3, nitrocellulose filter 4, absorbent 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 when 37 DEG C, then places 8～12h when 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 respectively prepared cuorin alcoholic solution and cardiolipin binding protein aqueous solution, again by cuorin alcoholic solution and cardiolipin binding protein aqueous solution, carry out the first incubation.
In embodiments more provided by the invention, the preparation method of cuorin alcoholic solution is: is dissolved in dehydrated alcohol by cuorin, to obtain final product.
In embodiments more provided by the invention, the mass body volume concentrations of cuorin alcoholic solution is preferably 0.5～5mg/mL.
Preferably, the mass body volume concentrations of cuorin alcoholic solution is preferably 1mg/mL.
In embodiments more provided by the invention, the preparation method of cardiolipin binding protein aqueous solution is: is dissolved in the water by cardiolipin binding protein, to 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 the reaction 2h when 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 of closing is 37 DEG C.
In embodiments more provided by the invention, the time of closing 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 taking cardiolipin binding protein immunity rabbit, to obtain final product.
Present invention also offers a kind of Lues Assay card, including detection casing clamping body and syphilis test strips provided by the invention；
In this Lues Assay card, syphilis test strips therein includes base plate 1, and base plate 1 is disposed with sample pad 2, glass fibre element film 3, nitrocellulose filter 4, absorbent paper 5；Glass fibre element film 3 is provided with the treponema pallidum specific antigen of colloid gold label and the cuorin-cardiolipin binding protein complex of colloid gold label；The preparation method of the cuorin of colloid gold label-cardiolipin binding protein complex is: core phospholipid and cardiolipin binding protein mixing, through the first incubation, obtain cuorin-cardiolipin binding protein complex, core phospholipid-cardiolipin binding protein complex and gold colloidal mixing, through the second incubation, closing, obtain the cuorin-cardiolipin binding protein complex of colloid gold label；Nitrocellulose filter 4 is provided with the first detection zone the 6, second detection zone the 7, first 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 treponema 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 treponema pallidum IgG monoclonal antibody；The preparation method of cuorin alcoholic solution is: is dissolved in dehydrated alcohol by cuorin, to obtain final product；The mass body volume concentrations of cuorin alcoholic solution is preferably 0.5～5mg/mL；It is highly preferred that the mass body volume concentrations of cuorin alcoholic solution is preferably 1mg/mL；The preparation method of cardiolipin binding protein aqueous solution is: is dissolved in the water by cardiolipin binding protein, to obtain final product；The mass body volume concentrations of cardiolipin binding protein aqueous solution is preferably 0.5～5mg/mL；It is highly preferred that the mass body volume concentrations of cardiolipin binding protein aqueous solution is 1mg/mL；Second incubation is the reaction 2h when 2～8 DEG C；Closing the reagent adopted is bovine serum albumin solution；In bovine serum albumin solution, the mass percentage concentration of bovine serum albumin white beeswax 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 taking cardiolipin binding protein immunity rabbit, to obtain final product.
Detection casing clamping body is provided with sample application zone 10 and detection zone 11.
The preparation method that present invention also offers Lues Assay card provided by the invention, takes detection casing clamping body and syphilis test strips provided by the invention assembles, and 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 includes base plate 1, and base plate 1 is disposed with sample pad 2, glass fibre element film 3, nitrocellulose filter 4, absorbent paper 5；Glass fibre element film 3 is provided with the treponema pallidum specific antigen of colloid gold label and the cuorin-cardiolipin binding protein complex of colloid gold label；The preparation method of the cuorin of colloid gold label-cardiolipin binding protein complex is: core phospholipid and cardiolipin binding protein mixing, through the first incubation, obtain cuorin-cardiolipin binding protein complex, core phospholipid-cardiolipin binding protein complex and gold colloidal mixing, through the second incubation, closing, obtain the cuorin-cardiolipin binding protein complex of colloid gold label；Nitrocellulose filter 4 is provided with the first detection zone the 6, second detection zone the 7, first 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 treponema 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 treponema pallidum IgG monoclonal antibody.By the result of the test that a large amount of serum specimens are detected, the sensitivity of detection card provided by the invention reaches 98.23%, specificity reaches 93.50%, false positive rate only 6.50%, and adopting the sensitivity of detection card A that RPR method carries out detecting is 93.11%, specificity is 89.25%, false positive rate is 10.75%, adopting the sensitivity that the detection that TRUST method carries out detecting blocks B is 94.29%, and specificity is 90.78%, and false positive rate is 9.22%.As can be seen here, detection 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 absorbent 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 detecting casing clamping body that embodiment 8 provides；Wherein, 10 show sample application zone, and 11 show detection zone.
Detailed description of the invention
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, is suitably modified technological parameter and realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are considered as including in the present invention.Method and the application of the present invention are described already by preferred embodiment, method described herein and application substantially can be modified or suitably change and combination by related personnel in without departing from present invention, spirit and scope, 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, colloidal gold solution, mouse-anti human IgG antibody, the syphilis specific antigen of 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, the present invention is expanded on further:
The preparation of the cuorin of embodiment 1 colloid gold label-cardiolipin binding protein complex
Take 1g cuorin to be dissolved in 500mL dehydrated alcohol, then be settled to 1L, be configured to the cuorin alcoholic solution of 1mg/mL；Take 1g cardiolipin binding protein to be dissolved in 500mL water, then be settled to 1L, be configured to the cardiolipin binding protein aqueous solution of 1mg/mL.
Take 100mL cuorin alcoholic 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；Take the colloidal gold solution of 50mL5mg/mL, magnetic stirring apparatus is slowly stirred, it is slow added into above-mentioned cuorin-cardiolipin binding protein complex solution, 2h is stirred at 4 DEG C, add 50mL1% bovine serum albumin solution, at 37 DEG C, close the centrifugal 30min of 1h, 12000rpm, redissolve when being placed on 2～8 DEG C with the phosphate buffer of 50mL50mM, pH7.4 stand-by.
The preparation of the cuorin of embodiment 2 colloid gold label-cardiolipin binding protein complex
Take 0.5g cuorin to be dissolved in 500mL dehydrated alcohol, then be settled to 1L, be configured to the cuorin alcoholic solution of 0.5mg/mL；Take 5g cardiolipin binding protein to be dissolved in 500mL water, then be settled to 1L, be configured to the cardiolipin binding protein aqueous solution of 5mg/mL.
Take 200mL cuorin alcoholic 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；Take the colloidal gold solution of 50mL5mg/mL, magnetic stirring apparatus is slowly stirred, it is slow added into above-mentioned cuorin-cardiolipin binding protein complex solution, 2h is stirred at 2 DEG C, add 70mL1% bovine serum albumin solution, at 37 DEG C, close the centrifugal 30min of 1h, 12000rpm, redissolve when being placed on 2～8 DEG C with the phosphate buffer of 50mL50mM, pH7.4 stand-by.
The preparation of the cuorin of embodiment 3 colloid gold label-cardiolipin binding protein complex
Take 5g cuorin to be dissolved in 500mL dehydrated alcohol, then be settled to 1L, be configured to the cuorin alcoholic solution of 5mg/mL；Take 0.5g cardiolipin binding protein to be dissolved in 500mL water, then be settled to 1L, be configured to the cardiolipin binding protein aqueous solution of 0.5mg/mL.
Take 100mL cuorin alcoholic 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；Take the colloidal gold solution of 50mL5mg/mL, magnetic stirring apparatus is slowly stirred, it is slow added into above-mentioned cuorin-cardiolipin binding protein complex solution, 2h is stirred at 8 DEG C, add 70mL1% bovine serum albumin solution, at 37 DEG C, close the centrifugal 30min of 1h, 12000rpm, redissolve when being placed on 2～8 DEG C with the phosphate buffer of 50mL50mM, pH7.4 stand-by.
The preparation of the anti-cardiolipin binding protein IgG monoclonal antibody of embodiment 4 rabbit
By cardiolipin binding protein physiological saline solution, it is configured to the cardiolipin binding protein solution of 0.5mg/mL, 10mL cardiolipin binding protein solution is mixed with isodose Freund's complete adjuvant, fully prepare the first mixed liquor after mixing.Take healthy adult New Zealand white big ear rabbit, foot pad after above-mentioned for 5mL the first mixed liquor being injected into two with syringe, complete first immunisation.
By cardiolipin binding protein physiological saline solution, it is configured to the cardiolipin binding protein solution of 0.5mg/mL, 10mL cardiolipin binding protein solution is mixed with isodose incomplete Freund's adjuvant, fully prepare the second mixed liquor after mixing.After first immunisation 10-14 days, with syringe, above-mentioned second mixed liquor is injected into rabbit back subcutaneous, completes second time immunity.
After second time immunity 10-14 days, the dosage of second time immunity and method is adopted to carry out third time immunity.
After third time immunity 5-7 days, rabbit heart was taken a blood sample, and separated serum, adopts the anti-cardiolipin binding protein IgG monoclonal antibody of ammonium sulfate precipitation method separation purified rabbit, adds equivalent glycerol frozen stand-by after dialysis.
The preparation of embodiment 5 syphilis test strips
The treponema pallidum specific antigen of 100mg colloid gold label is dissolved with the phosphate buffer of 100mL50mM, pH7.4, is configured to the treponema pallidum specific antigen solution of the colloid gold label of 1mg/mL.Spraying the cuorin-cardiolipin binding protein complex solution of the colloid gold label that embodiment 1 prepares and the treponema pallidum specific antigen solution of above-mentioned colloid gold label on glass fibre element film, quantity for spray is 8.5 μ L/cm, dry 2h at 37 DEG C.
Take recombinant syphilis spirochete antigen, the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit that mouse-anti treponema pallidum IgG monoclonal antibody, mouse-anti human IgG monoclonal antibody, embodiment 4 prepare, it is dissolved separately in the Tri(Hydroxymethyl) Amino Methane Hydrochloride buffer (Tris-HCl buffer) of 50mM, pH8.0, is configured to 1mg/mL recombinant syphilis spirochete antigenic solution, 1mg/mL mouse-anti treponema pallidum IgG monoclonal antibody solution, 1mg/mL mouse-anti human IgG monoclonal antibody solution, the anti-cardiolipin binding protein IgG monoclonal antibody solution of 1mg/mL rabbit.
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 treponema 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, absorbent paper 5 are sequentially fixed on base plate 1, to obtain final product.Test strips structure is as shown in Figure 1.
The preparation of embodiment 6 syphilis test strips
The treponema pallidum specific antigen of 100mg colloid gold label is dissolved with the phosphate buffer of 100mL50mM, pH7.4, is configured to the treponema pallidum specific antigen solution of the colloid gold label of 1mg/mL.Spraying the cuorin-cardiolipin binding protein complex solution of the colloid gold label that embodiment 2 prepares and the treponema pallidum specific antigen solution of above-mentioned colloid gold label on glass fibre element film, quantity for spray is 8.5 μ L/cm, dry 2h at 37 DEG C.
Take recombinant syphilis spirochete antigen, the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit that mouse-anti treponema pallidum IgG monoclonal antibody, mouse-anti human IgG monoclonal antibody, embodiment 4 prepare, it is dissolved separately in the Tris-HCl buffer of 50mM, pH8.0, is configured to 1mg/mL recombinant syphilis spirochete antigenic solution, 1mg/mL mouse-anti treponema pallidum IgG monoclonal antibody solution, 1mg/mL mouse-anti human IgG monoclonal antibody solution, the anti-cardiolipin binding protein IgG monoclonal antibody solution of 1mg/mL rabbit.
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 treponema 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, absorbent paper 5 are fixed on base plate 1, to obtain final product.
The preparation of embodiment 7 syphilis test strips
The treponema pallidum specific antigen of 100mg colloid gold label is dissolved with the phosphate buffer of 100mL50mM, pH7.4, is configured to the treponema pallidum specific antigen solution of the colloid gold label of 1mg/mL.Spraying the cuorin-cardiolipin binding protein complex solution of the colloid gold label that embodiment 3 prepares and the treponema pallidum specific antigen solution of above-mentioned colloid gold label on glass fibre element film, quantity for spray is 8.5 μ L/cm, dry 2h at 37 DEG C.
Take recombinant syphilis spirochete antigen, the anti-cardiolipin binding protein IgG monoclonal antibody of rabbit that mouse-anti treponema pallidum IgG monoclonal antibody, mouse-anti human IgG monoclonal antibody, embodiment 4 prepare, it is dissolved separately in the Tris-HCl buffer of 50mM, pH8.0, is configured to 1mg/mL recombinant syphilis spirochete antigenic solution, 1mg/mL mouse-anti treponema pallidum IgG monoclonal antibody solution, 1mg/mL mouse-anti human IgG monoclonal antibody solution, the anti-cardiolipin binding protein IgG monoclonal antibody solution of 1mg/mL rabbit.
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 treponema 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, absorbent paper 5 are fixed on base plate 1, to obtain final product.
The preparation of embodiment 8 Lues Assay card
The syphilis test strips that Example 5 provides, is assembled in detection casing clamping body, to obtain final product.The structure of detection casing clamping body is as shown in Figure 2.
The preparation of embodiment 9 Lues Assay card
The syphilis test strips that Example 6 provides, is assembled in detection casing clamping body, to obtain final product.
The preparation of embodiment 10 Lues Assay card
The syphilis test strips that Example 7 provides, is assembled in detection casing clamping body, to obtain final product.
Embodiment 11 clinical trial
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's specimen 372 parts, previous infection syphilitic's serum specimen 191 parts, healthy normal human serum specimen 5249 parts.Utilize the Lues Assay card that the embodiment of the present invention 8 prepares, commercially available detection card A(RPR method) and commercially available detection card B(TRUST method) carry out sensitivity, specificity and false positive rate respectively and detect.
When adopting the detection card that embodiment 8 provides that serum specimen is detected, detection card is kept flat, 100 μ L serum specimens are instilled sample application zone, observed result after 10min.All there is red stripes in first detection zone, the second detection zone, the first quality control region and the second quality control region place, then judge that serum specimen is as making a definite diagnosis syphilization patients serum's specimen；There is red stripes in 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's serum specimen；There is red stripes in 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 specimen；There is red stripes in first quality control region and the second quality control region place, the first detection zone place not Show Color, and red stripes occurs in the second detection zone place, then judge that serum specimen is as the suspicious specimen of syphilis.Operation and the result detection of commercially available detection card A and detection card B perform with reference to description.Testing result is as shown in table 1.
Table 1 sensitivity, specificity and false positive rate testing result
From above-mentioned result of the test, 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 substantially better than commercially available detection card A and detection 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, and high specificity, false positive rate is low, without the interference of previous infection.
The Lues Assay card that Example 9, embodiment 10 provide carries out clinical trial, and result is similarly, it was shown that the Lues Assay card that embodiment 9, embodiment 10 provide is highly sensitive, and high specificity, false positive rate is low, without the interference of previous infection.
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 making some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. a syphilis test strips, including base plate (1), described base plate (1) is disposed with sample pad (2), glass fibre element film (3), nitrocellulose filter (4), absorbent paper (5), it is characterised in that
Described glass fibre element film (3) is provided with the treponema pallidum specific antigen of colloid gold label and the cuorin-cardiolipin binding protein complex of colloid gold label；
The preparation method of the cuorin of described colloid gold label-cardiolipin binding protein complex is: core phospholipid and cardiolipin binding protein mixing, through the first incubation, obtain cuorin-cardiolipin binding protein complex, take described cuorin-cardiolipin binding protein complex and gold colloidal mixing, through the second incubation, closing, 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 treponema 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 treponema pallidum IgG monoclonal antibody.
2. syphilis test strips according to claim 1, it is characterised in that described first incubation is hatch 1～4h when 37 DEG C, then places 8～12h when 2～8 DEG C.
3. syphilis test strips according to claim 1, it is characterized in that, in the preparation method of the cuorin of described colloid gold label-cardiolipin binding protein complex, the mass ratio of described cuorin and described cardiolipin binding protein is (1～5): (1～5).
4. syphilis test strips according to claim 3, it is characterised 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 as according to any one of Claims 1-4, it is characterised in that comprise the steps:
Step A: core phospholipid and cardiolipin binding protein mixing, through the first incubation, obtain cuorin-cardiolipin binding protein complex, take described cuorin-cardiolipin binding protein complex and gold colloidal mixing, through the second incubation, closing, obtain the cuorin-cardiolipin binding protein complex of colloid gold label；
Obtain the treponema pallidum specific antigen of recombinant syphilis spirochete antigen, mouse-anti treponema 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 treponema pallidum specific antigen of the cuorin-cardiolipin binding protein complex and described colloid gold label that take described colloid gold label is coated in glass fibre element film (3)；
Take described recombinant syphilis spirochete antigen coated in first detection zone (6) of nitrocellulose filter (4), take described mouse-anti human IgG monoclonal antibody and be coated in second detection zone (7) of nitrocellulose filter (4), take described mouse-anti treponema pallidum IgG monoclonal antibody and be coated in first quality control region (8) of nitrocellulose filter (4), take the anti-cardiolipin binding protein IgG monoclonal antibody of described rabbit and be coated in second quality control region (9) of nitrocellulose filter (4)；
Or take described mouse-anti human IgG monoclonal antibody and be coated in first detection zone (6) of nitrocellulose filter (4), take described recombinant syphilis spirochete antigen coated in second detection zone (7) of nitrocellulose filter (4), take the anti-cardiolipin binding protein IgG monoclonal antibody of described rabbit and be coated in first quality control region (8) of nitrocellulose filter (4), take described mouse-anti treponema pallidum IgG monoclonal antibody and be coated in second quality control region (9) of nitrocellulose filter (4)；
Step C: take sample pad (2), described glass fibre element film (3), described nitrocellulose filter (4), absorbent paper (5) are sequentially fixed on base plate (1), to obtain final product.
6. preparation method according to claim 5, it is characterised in that described first incubation is hatch 1～4h when 37 DEG C, then places 8～12h when 2～8 DEG C.
7. preparation method according to claim 5, it 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, it is characterised in that the mass ratio of described cuorin and described cardiolipin binding protein is 1:1.
9. a Lues Assay card, it is characterised in that include detection casing clamping body and the syphilis test strips as according to any one of Claims 1-4；
Described detection casing clamping body is provided with sample application zone (10) and detection zone (11).
10. the preparation method of Lues Assay card as claimed in claim 9, it is characterised in that take detection casing clamping body and the syphilis test strips as according to any one of Claims 1-4 assembles, to obtain final product.
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| EP1949108B1 (en) * | 2005-11-18 | 2013-09-25 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA as represented by THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES | 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 |
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