CN102830229B - Detection reagent of treponema pallidum IgM (Immunoglobulin M) antigen colloidal gold method and preparation method thereof - Google Patents
Detection reagent of treponema pallidum IgM (Immunoglobulin M) antigen colloidal gold method and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a detection reagent of a treponema pallidum IgM (Immunoglobulin M) antigen colloidal gold method and a preparation method thereof. The reagent comprises a gold conjugate pad (3), a cellulose nitrate reaction membrane (4), a sample pad (2), water absorbing paper (5) and a PVC (Polyvinyl Chloride) back lining (1), wherein the sample pad, the gold conjugate pad, the cellulose nitrate reaction membrane and the water absorbing paper are sequentially and mutually laminated and adhered on the PVC back lining; the sample pad is laminated on the gold conjugate pad for 2-3 mm, the gold conjugate pad is laminated on the cellulose nitrate reaction membrane for 2-3 mm, and the water absorbing paper is laminated on the cellulose nitrate reaction membrane for 2-3 mm; the gold conjugate pad is coated with an anti-human IgM monoclonal antibody-colloidal gold conjugate; and positions of a quality control region (7) and a detection region (6) of the cellulose nitrate reaction membrane are coated with goat anti-mouse IgG antibody or rabbit anti-mouse IgG antibody and specific gene recombinant treponema pallidum antigen respectively. A production process of a product provided by the invention is simple and easy to control; the detection reagent is easy, convenient and quick in detection; and a result can be read in 25 minutes and is not affected by the IgG antibody and is accurate and reliable.
Description
Technical field
The invention belongs to field of medical examination, particularly relate to a kind of colloidal gold immunity chromatography and detect reagent of IgM antibody to treponema pallidum and preparation method thereof.
Background technology
Syphilis infects by microspironema pallidum (Treponema pallidum, TP) chronic disease caused, and is the disease that in venereal disease, harmfulness is more serious, and passability, blood and maternal route are propagated.Microspironema pallidum harm cardiovascular system, can cause aortitis, aortic insuffciency, aortic aneurysm etc., also can damage skeletal system, cause tissue and organ damage, afunction, cause deformity or death; After placental infection fetus, can cause spontaneous abortion, stillbirth or congenital syphilis etc., female pregnant early infection person fetus 40%-50% infects, and usually causes stillborn foetus, stillbirth, miscarriage, premature labor or FGR.Therefore, syphilis its harmfulness and fatal rate in sexually transmitted disease are only second to acquired immune deficiency syndrome (AIDS), and it also can vertical transmission, has a strong impact on infantile health, have become a large severe social public health problem.
Serologic detection is the important test in laboratory method of current diagnosis syphilis.It is non-treponemal antigen serum test that the Serology test of current syphilis mainly contains two kinds: one class, as Rapid plasma reagintest, tolulized red unheated serum test etc., is commonly used for the Screening tests of syphilis.Because this type of test is by various factors, often occur biologic fale-positive and false negative result, to one, the positive rate of tertiary syphilis is not high, is only applicable to the diagnosis of mesosyphilis.Another kind of is treponemal antigen Serum experiments, as treponema pallidum haemagglutination assay, GAT, FTA-ABS test and enzyme linked immunosorbent assay etc., the susceptibility of this kind of test is high, high specificity, the clinical validation test be commonly used to as syphilis.But this type of testing inspection is microspironema pallidum IgG antibody and IgM antibody, and IgG antibody long-term existence, seroreaction sustainable existence is positive, and can pass through placenta, passive immunity fetus, and sustainable even lifelong for many years; Thus whether can not distinguish Neonatal antibody response from mother or fetus itself, the observation of curative effect of syphilis treatment can not be used as.
After syphilis, IgM antibody is the humoral immunoresponse(HI) that body occurs at first, and nonspecific infection can detect for 2 weeks from serum, as long as and have microspironema pallidum alive to exist, IgM antibody will maintain certain level.Report that IgM antibody 100% is turned out cloudy, primary syphilis be 3 months, mesosyphilis is 9 months, early latent syphilis is 2 years.Therefore, TP-IgM can be regarded as syphilis early infection and a movable serologic marker.
IgM antibody test is the recommended confirmatory test as early congenital syphilis already abroad, although and domesticly at present have on a small quantity to the research of the diagnosis of early syphilis, the confirmation of syphilis and curative effect method for supervising, but first need be separated IgG antibody, to prevent IgG and IgM antibody from competing specific antigen binding site, cause the false negative of IgM antibody.Owing to still not grasping completely IgG antibody, IgM antibody isolation technics, do not form commercial prod, clinical related kit used is essentially import, brings larger financial burden, be not easy to the early diagnosis to syphilis, confirmation and control to patient and society.
Collaurum is used to have no corresponding commercialization reagent as the colloidal gold immunity chromatography detection IgM antibody to treponema pallidum of tracer, the method is easy and simple to handle, quick, good stability, be easy to carry out single part of pattern detection, and do not need specific apparatus to detect, because tracer is that solid phase is easy to preservation, good stability.
Summary of the invention
The object of the invention is to carry out in order to what overcome that current detection IgM antibody to treponema pallidum technology exists in promoting the use of the defect that IgG is separated with IgM antibody in advance, there is provided a kind of easy and simple to handle, do not need particular instrument equipment to assist, do not need carry out the quick detection reagent of Special Training to testing staff and effectively reduce testing cost, the preparation method of this reagent is provided simultaneously.
The present invention for solving the problems of the technologies described above adopted technical scheme is: a kind of IgM antibody to treponema pallidum colloidal gold method detects reagent, and this reagent comprises golden bond pad (3), cellulose nitrate reaction film (4), sample pad (2), thieving paper (5) and PVC backing (1); Sample pad, golden bond pad, cellulose nitrate reaction film and thieving paper mutually being laminated in turn adheres on PVC backing; Sample pad laminates golden bond pad 2-3mm, and golden bond pad laminates cellulose nitrate reaction film 2-3mm, and thieving paper laminates cellulose nitrate reaction film 2-3mm; On gold bond pad, bag is by the bond of anti-human IgM monoclonal antibody-collaurum, bag quilt sheep anti-mouse igg antibody or rabbit anti-mouse igg antibody and specific genetic recombination treponemal antigen respectively on cellulose nitrate reaction film quality control region (7) and detection zone (6) position.
Described sample pad is that bag is by the glass fibre of staphylococcal protein A (SPA) element film or polyester cellulose film.
The treponemal antigen of described detection zone bag quilt is the specific gene recombinant antigen of purifying.
The preparation method of this reagent comprises the following steps:
A: prepare anti-human IgM monoclonal antibody: get many portions of normal human serums, mixing, the IgM antibody that purity reaches 95% is obtained through dialysis, centrifugal, purifying, using people IgM as antigen, through intrasplenic injection immunity 6-8 female BAl BIc/c mouse in age in week, get immune spleen cell and the myeloma cell fusion of mouse, selectivity is cultivated, screen and cloning acquisition hybridoma, hybridoma is inoculated in BALB/c mouse abdominal cavity and prepares ascites, with affinity chromatography purification mouse-anti people IgM monoclonal antibody;
B: prepare polyclonal antibody: with the mouse-anti people IgM monoclonal antibody immune goat prepared by steps A or rabbit, extracts goat anti-mouse igg polyclonal antibody or rabbit anti-mouse IgG polyclonal antibody after getting serum purifying;
C: prepare collaurum: adopt trisodium citrate reduction method to obtain the colloid gold particle of particle diameter 20 ~ 40nm;
D: prepare mouse-anti people IgM monoclonal antibody-colloidal gold conjugate: regulate colloidal gold solution pH value to optimum mark pH value with 0.1M sal tartari, mouse-anti people IgM monoclonal antibody is added according to the ratio of 0.010 ~ 0.030 mg albumen/mL collaurum, mixing leaves standstill, high speed centrifugation, abandon supernatant, gained precipitation is dissolved with the golden bond conserving liquid of 1/10th initial colloid gold volumes, obtained mouse-anti people IgM monoclonal antibody-colloidal gold conjugate; Wherein, golden bond conserving liquid be containing 2 ~ 5% the pH value of bovine serum albumin(BSA) be 8.0 ~ 8.2 0.01 ~ 0.02M Tris-HCl damping fluid, and containing 1 ~ 4% sucrose, the casein-sodium of 0.5% ~ 1%, the Tween-20 of 1 ~ 2% and 5/10000ths Sodium azide;
E: preparation sample pad: by staphylococcal protein A (SPA) with diluted to 0.5mg ~ 2 mg/mL, even application is on glass fibre element film or polyester cellulose film, abundant drying, wherein, dilution be containing the pH value of 1 ~ 2% trehalose be 7.2 ~ 7.6 0.02 ~ 0.05M PBS solution, and containing 1 ~ 2% bovine serum albumin(BSA);
F: prepare golden bond pad: evenly wrapped by the mouse-anti people IgM monoclonal antibody-colloidal gold conjugate prepared by step D by glass fibre membrane is fully dry;
G: prepare nitrocellulose filter reaction film: the quality control region and the position, detection zone that goat anti-mouse igg polyclonal antibody or rabbit anti-mouse IgG polyclonal antibody and recombinant syphilis spirochete antigen are coated on respectively nitrocellulose filter is fully dry;
H: sample pad, golden bond pad, cellulose nitrate reaction film and thieving paper are laminated in turn mutually and adheres on PVC backing; Sample pad laminates golden bond pad 2-3mm, and golden bond pad laminates cellulose nitrate reaction film 2-3mm, and thieving paper laminates cellulose nitrate reaction film 2-3mm; Obtain test paper plate, test paper plate is cut into the test strips of different in width according to demand.
Effect of the present invention is:
(1) production run is simple and easy to control;
(2) detection speed is fast, can obtain testing result in 25 minutes, as long as and do not need professional training operate to specifications can teaching display stand detection;
(3) owing to adding IgG antibody disposal system in sample pad, employing solid phase associated methods catches the IgG antibody in sample, thus do not need to carry out being separated to serum before detection to remove the process of IgG antibody, detection highly sensitive, specificity good, result is accurately and reliably;
(4) single part detection can be carried out, and product stability is good, does not need special storage condition (normal temperature preservation).
Accompanying drawing explanation
Fig. 1 is the structural representation of reagent of the present invention.
Embodiment
(1) get 3 ~ 5 portions of normal human serums, mixing, through cold distilled water (4 DEG C) dialysis, centrifuging and taking precipitates, and crosses AcA3 post purifying and obtains the IgM antibody that purity reaches 95%; Using extract, the people IgM of purifying as antigen, through intrasplenic injection immunity 6-8 female BAl BIc/c mouse in age in week; Get immune spleen cell and the myeloma cell fusion of mouse, selectivity cultivation, screening and cloning obtain the hybridoma of the anti-human IgM monoclonal antibody of secretion; The hybridoma of the anti-IgM of secretion is inoculated in BALB/c mouse abdominal cavity and prepares ascites, with affinity chromatography purification mouse-anti people IgM monoclonal antibody;
(2) with mouse-anti people IgM monoclonal antibody, immune goat or new zealand rabbit, get serum and obtain goat anti-mouse igg polyclonal antibody or rabbit anti-mouse IgG polyclonal antibody with the extraction of saturated ammonium sulfate method;
(3) collaurum is prepared: the citric acid three sodium solution that adds 1.2mL ~ 1.8mL 1% to boiling of being heated while stirring by the chlorauric acid solution of 0.01% of 100mL continues to be heated to solution and to become after claret continuous heating 5 minutes again; After stopping heating, continue to stir, add purified water after being cooled to room temperature and revert to original volume, 4 DEG C keep in Dark Place; Obtained colloid gold particle measures absorption peak at 520 ~ 530nm through visible spectrophotometer, and particle size is about 20 ~ 40nm;
(4) mouse-anti people IgM monoclonal antibody colloid gold label thing is prepared: with sodium carbonate (potassium) solution of 0.1M, colloidal gold solution pH value is adjusted to 8.0 ~ 8.5; Slowly add mouse-anti people IgM monoclonal antibody (every 1mL collaurum adds 10 ~ 30 μ g mouse-anti people IgM monoclonal antibody) while stirring in proportion, leave standstill more than 15 minutes, add a certain proportion of bovine serum albumin(BSA) (every 100ml adds 10% bovine serum albumin solution 10mL) while stirring, after mixing, leave standstill 10 minutes; By high speed centrifugation, abandon supernatant, gained precipitation is dissolved with the golden bond conserving liquid of 1/10th initial colloid gold volumes, obtained mouse-anti people IgM monoclonal antibody-colloidal gold conjugate; Gold bond conserving liquid be containing the pH value of the bovine serum albumin(BSA) of 2 ~ 5% be 8.0 ~ 8.2 0.01 ~ 0.02M Tris-HCl damping fluid, and containing 1 ~ 4% sucrose, the casein-sodium of 0.5% ~ 1%, the Tween-20 of 1 ~ 2% and 5/10000ths Sodium azide;
(5) sample pad is prepared: by staphylococcal protein A (SPA) 0.02 ~ 0.05M PBS(pH 7.2 ~ 7.6, containing 1 ~ 2% trehalose and 1 ~ 2% bovine serum albumin(BSA)) be diluted to 0.5 ~ 2.0mg/mL, obtained staphylococcal protein A (SPA) coating buffer, by coating buffer with being sprayed onto on glass fibre element film or polyester cellulose film every stream metal spraying pen machine, fully dry;
(6) cellulose nitrate reaction film is prepared: by goat-anti (or rabbit resist) mouse IgG polyclonal antibody and recombinant syphilis spirochete antigen respectively with 0.02 ~ 0.05M PBS(pH 7.2 ~ 7.6, containing 1 ~ 2% trehalose) be all diluted to finite concentration obtained C, T line coating buffer respectively; By the coating buffer control zone and the position, detection zone that are coated on nitrocellulose filter every stream metal spraying pen machine respectively, fully dry;
(7) golden bond pad is prepared: by the mouse-anti people IgM monoclonal antibody colloid gold label thing solution obtained by step (4) with every flowing metal spraying pen machine even application on golden bond pad, fully dry;
(8) sample pad, golden bond pad, cellulose nitrate reaction film and thieving paper are laminated in turn mutually adhere on PVC backing; Sample pad laminates golden bond pad 2-3mm, and golden bond pad laminates cellulose nitrate reaction film 2-3mm, and thieving paper laminates cellulose nitrate reaction film 2-3mm; Obtain test paper plate;
(9) PVC board pasted is cut into the bar of one fixed width (2.5 ~ 4mm), namely obtained IgM antibody to treponema pallidum colloidal gold method detects reagent.
Before detection, first by sample with detect reagent balance to room temperature, take out from aluminium foil bag and detect reagent, add 5 ~ 15 μ L sample to be checked (serum) at sample pad place, then add 10 ~ 30 μ L physiological saline or Sample dilution makes sample fully spread out; Add 50 μ L physiological saline or Sample dilution after 5 minutes, within 15 ~ 20 minutes, get final product sentence read result; After 20 minutes, interpretation is invalid.
Result judges: if there is IgM antibody to treponema pallidum in sample, then nature controlling line, detection line place all occur red stripes, and result is positive; If there is not IgM antibody to treponema pallidum in sample, then only there is a red stripes in nature controlling line place; As nature controlling line, place has no band, then reagent is invalid.
Claims (3)
1. IgM antibody to treponema pallidum colloidal gold method detects a preparation method for reagent, it is characterized in that comprising the following steps:
A: prepare anti-human IgM monoclonal antibody: get many portions of normal human serums, mixing, the IgM antibody that purity reaches 95% is obtained through dialysis, centrifugal, purifying, using people IgM as antigen, through intrasplenic injection immunity 6-8 female BAl BIc/c mouse in age in week, get immune spleen cell and the myeloma cell fusion of mouse, selectivity is cultivated, screen and cloning acquisition hybridoma, hybridoma is inoculated in BALB/c mouse abdominal cavity and prepares ascites, with affinity chromatography purification mouse-anti people IgM monoclonal antibody;
B: prepare polyclonal antibody: with the mouse-anti people IgM monoclonal antibody immune goat prepared by steps A or rabbit, extracts goat anti-mouse igg polyclonal antibody or rabbit anti-mouse IgG polyclonal antibody after getting serum purifying;
C: prepare collaurum: adopt trisodium citrate reduction method to obtain the colloid gold particle of particle diameter 20 ~ 40nm;
D: prepare mouse-anti people IgM monoclonal antibody-colloidal gold conjugate: regulate colloidal gold solution pH value to optimum mark pH value with 0.1M sal tartari, mouse-anti people IgM monoclonal antibody is added according to the ratio of 0.010 ~ 0.030mg albumen/mL collaurum, mixing leaves standstill, high speed centrifugation, abandon supernatant, gained precipitation is dissolved with the golden bond conserving liquid of 1/10th initial colloid gold volumes, obtained mouse-anti people IgM monoclonal antibody-colloidal gold conjugate; Wherein, gold bond conserving liquid be containing the pH value of the bovine serum albumin(BSA) of 2 ~ 5% be 8.0 ~ 8.2 0.01 ~ 0.02M Tris-HCl damping fluid, and containing 1 ~ 4% sucrose, the casein-sodium of 0.5% ~ 1%, the Tween-20 of 1 ~ 2% and 5/10000ths Sodium azide;
E: preparation sample pad: by staphylococcal protein A (SPA) with diluted to 0.5mg ~ 2mg/mL, even application is on glass fibre element film or dacron sorrel, abundant drying, wherein, dilution be containing the pH value of 1 ~ 2% trehalose be 7.2 ~ 7.6 0.02 ~ 0.05M PBS solution, and containing 1 ~ 2% bovine serum albumin(BSA);
F: prepare golden bond pad: evenly wrapped by the mouse-anti people IgM monoclonal antibody-colloidal gold conjugate prepared by step D by glass fibre membrane is fully dry;
G: prepare nitrocellulose filter reaction film: the quality control region and the position, detection zone that goat anti-mouse igg polyclonal antibody or rabbit anti-mouse IgG polyclonal antibody and recombinant syphilis spirochete antigen are coated on respectively nitrocellulose filter is fully dry;
H: sample pad, golden bond pad, cellulose nitrate reaction film and thieving paper are laminated in turn mutually and adheres on PVC backing; Sample pad laminates golden bond pad 2-3mm, and golden bond pad laminates cellulose nitrate reaction film 2-3mm, and thieving paper laminates cellulose nitrate reaction film 2-3mm; Obtain test paper plate, test paper plate is cut into the test strips of different in width according to demand.
2. the IgM antibody to treponema pallidum colloidal gold method that method according to claim 1 prepares detects a reagent, it is characterized in that this reagent comprises golden bond pad (3), cellulose nitrate reaction film (4), sample pad (2), thieving paper (5) and PVC backing (1); Sample pad, golden bond pad, cellulose nitrate reaction film and thieving paper mutually being laminated in turn adheres on PVC backing; Sample pad laminates golden bond pad 2-3mm, and golden bond pad laminates cellulose nitrate reaction film 2-3mm, and thieving paper laminates cellulose nitrate reaction film 2-3mm; On gold bond pad, bag is by the bond of anti-human IgM monoclonal antibody-collaurum, on cellulose nitrate reaction film quality control region (7) and detection zone (6) position, bag is by sheep anti-mouse igg antibody or rabbit anti-mouse igg antibody and specific genetic recombination treponemal antigen respectively, and described sample pad has been that bag is by the glass fibre of staphylococcal protein A (SPA) element film or dacron film.
3. IgM antibody to treponema pallidum colloidal gold method according to claim 2 detects reagent, it is characterized in that: the treponemal antigen of described detection zone bag quilt is the specific gene recombinant antigen of purifying.
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| CN105353125A (en) * | 2015-11-13 | 2016-02-24 | 中国医学科学院输血研究所 | A portable rapid test strip for syphilis for household use |
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