CN105067809A - ELISA percolation method for rapidly detecting pathogen antibody, kit for detection, and preparation method of kit - Google Patents
ELISA percolation method for rapidly detecting pathogen antibody, kit for detection, and preparation method of kit Download PDFInfo
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- CN105067809A CN105067809A CN201510445471.8A CN201510445471A CN105067809A CN 105067809 A CN105067809 A CN 105067809A CN 201510445471 A CN201510445471 A CN 201510445471A CN 105067809 A CN105067809 A CN 105067809A
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Abstract
The present invention provides an ELISA percolation method for rapidly detecting pathogen antibody, a kit for detection, and a preparation method of the kit. According to the present invention, an antigen-antibody reaction is used to make antigen or second antibody be immobilized on a nitrocellulose membrane, the pathogen IgM (IgG) antibody in a serum sample and the corresponding solid-phase antigen or second antibody on the membrane produce specific binding so as to form a complex when the serum sample passes through the nitrocellulose membrane due to a percolation effect while other unrelated substances are filtered, enzyme-labeled antibody or antigen is added and is combined with the antigen-antibody complex on the membrane during the filtering, and a coloration liquid is added to carry out coloration so as to produce the purple blot conveniently observed by naked eyes; and the time consuming of the detection process is short, the sensitivity is high, the accuracy is strong, the shelf life of the kit can achieve more than or equal to 12 months with the stable dilution buffer solution and the coloration prepared through the method, and the whole detection has characteristics of simple and rapid operation process, no requirement of special equipment, high sensitivity, and high accuracy.
Description
Technical field
The present invention relates to biology techniques field, being specifically related to the kit that a kind of enzyme for detecting pathogen antigen is fast exempted from percolation, detected, with and preparation method thereof.
Background technology
Method at present for antibody test mainly contains colloidal gold method, immunochromatographic method, fluorescent PCR method, immunofluorescence technique or indirect immunofluorescence, enzyme-linked immunospot assay.Colloidal gold method is a kind of common labelling technique, it take collaurum as label, utilize specific antigen-antibody reaction, labelling technique that is qualitative and sxemiquantitative research is carried out to antigen-antibody, because it has the advantages such as operation fast and convenient, special sensitivity, stability are strong, cheap, obtained in clinical examination and other field and applied widely, but colloidal gold method can only carry out qualitative or half-quantitative detection, accuracy is poor, easy undetected weak positive sample.Immunochromatographic method is a kind of quick diagnosis technology of external rise in recent years, its principle is a certain zone special antibody being first fixed on nitrocellulose filter, after sample (urine or serum) is immersed in cellulose nitrate one end of this drying, due to capillarity, sample will move forward along this film, when moving to the region being fixed with antibody, in sample corresponding antigen namely with this antibody generation specific binding, if this region can be made to show certain color with immune colloid gold or Immunoperoxidase Staining, thus realize specific immunodiagnosis, although immunochromatographic method is simple to operate, do not need special instrument and equipment, but the selection of chromatographic material and process more difficult.Fluorescent PCR method is a kind of method that is more qualitative, specification, judge, but the method susceptibility is poor, comparatively easily occurs false-positive phenomenon than being easier to; Immunofluorescence technique or though its detection specificity of indirect immunofluorescence is strong, susceptibility is high, speed is fast it needs expensive immunofluorescence microscopy, staff need have certain experience, technical program yet more complicated; Enzyme-linked immunospot assay gets up based on ELISA developing test, antibody secreting cell can be detected from individual cell level, a kind of cellular immunology detection technique of secretory volume can be detected again, its principle is the cell factor with the secretion of antibody capture cultured cell, and showed in the mode of enzyme connection spot development, although enzyme-linked immunospot assay detection sensitivity is higher, during its operating cost, need tight Control experiment condition.
Summary of the invention
Technical matters to be solved by this invention is, a kind of method that can detect one or more pathogen antigen is fast provided, utilize the kit prepared by this method, method is easy, and the used time is shorter, detects efficiently and accurately, not easily there is undetected situation, can also eliminate disturbing factor to disturb the false positive of testing result, and the process that can make enzyme labelled antibody more rapidly and efficiently, extends the term of validity of kit simultaneously.
For solving the problems of the technologies described above, the technical scheme of the inventive method is: utilize antigen-antibody reaction, make antigen or two anti-immobilizations on nitrocellulose filter, when serum specimen passes through nitrocellulose filter due to transudation, pathogen IgM(IgG wherein) just corresponding on the film solid phase antigen of antibody or two anti-generation specific bindings form compound, other foreign matter are then filtered across, add enzyme labelled antibody or enzyme-labelled antigen, antigen antibody complex during filtration just on film is combined, then the purple trace that nitrite ion colour developing generation naked eyes can conveniently be observed is added.
Wherein, the compound method of described enzyme labelled antibody or enzyme-labelled antigen working fluid is divided into two parts, comprises the compound method of enzyme labelled antibody or enzyme-labelled antigen, and the compound method of the stable dilution buffer of enzyme labelled antibody or enzyme-labelled antigen;
The compound method of described enzyme labelled antibody or enzyme-labelled antigen, adopts following steps preparation:
(1) 1g horseradish peroxidase is dissolved in the 0.01M phosphate buffer of 25ml ~ 30ml, add 70 ~ 85mg ethylenediamine, with 0.1M hydrochloric acid, pH value is adjusted to 5.0, gained solution mixes with 115 ~ 125mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate again, after shaken at room temperature reaction, 0.01M phosphate buffer is adopted to dialyse to gained solution, obtained amination horseradish peroxidase solution;
(2) become by solution preparation obtained for step (1) 1ml, concentration to be the amination horseradish peroxidase solution of 5 ~ 5.2mg/ml, then in gained solution, add 60 ~ 85 μ l crosslinking chemicals, after mixing, under room temperature, react 25 ~ 35min;
(3) described reactant is added the super filter tube that molecular cut off is 10K, and centrifugal treating 8 ~ 12min is carried out to reactant, redissolve with the phosphate buffer of 0.01M again and be detained bond to 1ml, again with same centrifugal force 8 ~ 12min, to remove excessive crosslinking chemical, and again redissolve retentate to 1ml;
(4) add antibody or the antigenic solution of about 1mg, and react 25 ~ 35min under room temperature;
(5) solution making step (4) obtained, by SephadexG-200 chromatographic column, is collected the product of first peak, is enzyme labelled antibody or antigenic solution;
The stable dilution buffer of described enzyme labelled antibody or antigen adopts following methods preparation:
the preparation of brown alga extract: after fresh seaweed sample gathers, removing epibiont, frond top is got after repeatedly rinsing with distilled water, tissue mashing crusher machine is used after weighing, then add ethanol extract to extract, the method removing ethanol of extract decompression distillation, adds phosphate buffer, supernatant is got, i.e. obtained brown alga ethanol extract after high speed centrifugation;
the preparation of enzyme marker stability damping fluid: the methylisothiazolinone and the diagnostic reagent antiseptic PROCLIN300 that add 0.02-0.04wt% in the phosphate saline damping fluid of 0.01M, then 2 ~ 8wt% trehalose is added successively, 0.01 ~ 0.07wt% xanthans, 0.9wt% glycocoll and 0.7wt% serine, 0.3 ~ 1.2wt% brown alga extract is added again after mixing, the human-like collagen of 0.1 ~ 0.6wt% genetic recombination, finally add magnesium sulfate and the 0.9wt% zinc chloride of 1.8wt%, the glycerine of 0.1% vitamin B1 and 3% volume ratio, mix, obtain described enzyme marker stability damping fluid.
Preferably, the collocation method of described cleansing solution is, in the Tris-HCl damping fluid being configured to 0.01M, add 5.0g triton x-100,0.2gNaCl, 0.2gNaN
3with a certain amount of BSA, add water to 1000ml, shaken well is required cleansing solution.
Preferably, described nitrite ion comprises the sodium acetate buffer solution system of tetramethyl benzidine, urea peroxide, antioxidant chlorogenic acid, reductive agent ascorbic acid, ultraviolet light absorber 2-cyano group-3,3-diphenyl 2-Propenoic acid-2-ethylhexyl ester, pH5.0.
Preferably, described nitrite ion is adopted and is obtained with the following method:
8.3 ~ 8.5g sodium acetate trihydrate crystal is dissolved in 600ml ultrapure water, vibration mixing, and adjusts pH value of solution to 5.0;
again 0.8 ~ 0.83g tetramethyl benzidine is dissolved in 3ml absolute ethyl alcohol, obtained tetramethyl benzidine ethanolic solution;
by above-mentioned two kinds of solution mixing, vibration mixing;
respectively 70 ~ 90mg chlorogenic acid and 70 ~ 90mg ascorbic acid, 8 ~ 12ml2-cyano group-3,3-diphenyl 2-Propenoic acid-2-ethylhexyl ester, 2.5 ~ 2.9g phosphinylidyne glycocoll and 0.4 ~ 0.6g urea peroxide are joined step
in the mixed solution obtained, vibration mixing, obtains described nitrite ion.
Enzyme for detecting pathogen antigen fast exempts from the kit of percolation, comprise the check-out console be located in kit to go to the bottom, check-out console goes to the bottom top for adsorptive pads, is nitrocellulose filter above adsorptive pads, be check-out console upper cover above nitrocellulose filter, check-out console cover and is provided with well.
The described kit of exempting from percolation for the enzyme detecting pathogen antigen fast of preparation, adopts following steps:
method is as claimed in claim 3 adopted to prepare horseradish peroxidase marker solution;
prepare coated film: the coated film selecting applicable aperture, is cut into square piece for subsequent use;
assembling detects box: lid, coated film, adsorptive pads and box body are assembled in accordance with the order from top to bottom and fastening, make the window of upper cover be buckled in coated film center, and composition detects box;
(4) wrap quilt: the antigen after centrifugal with the 0.01M phosphate buffer dilution that with the addition of 3wt% trehalose or antibody wrap quilt respectively in the check point position of coated film, and Quality Control point position bag is by a kind of antibody that can react with enzyme marker;
(5) dry: to detect the atmosphere at room temperature dry 1 hour of box in humidity 10% ~ 40%;
packaging: dried detection box aluminium foil strip is packed, and places drying agent in bag, sealing.
After adopting technique scheme, the usefulness of this invention is: the advantage of enzyme labelled antibody working fluid that the inventive method uses: a, utilize horseradish peroxidase as label, it has greater activity, tolerable pH range is wider, and can save backup for a long time.The enzyme labelled antibody working fluid of b, use, eliminates the disturbing factor such as rheumatoid factor in sample to a great extent and disturbs the false positive of result, greatly reduce the antibody cost of kit simultaneously.
Further, the A in nitrite ion of the present invention, B component (i.e. superoxide with add lustre to thing TMB) in long-term co-existence same system, without the need to interim mixing during detection, directly can be added, simplify kit detecting step and saved cost.
Further, the enzyme labelled antibody dilution wherein used, utilize and screen through design and demonstrate the protectiveness dilution buffer fluid component that can make enzyme labelled antibody long-term stability, current existing disclosing of comparing is filled a prescription, this system component is less containing animal sources material, and can not biochemical reaction be there is between each composition, the protection to enzyme marker and stabilization can be played to greatest extent; Under c and enzyme labelled antibody protective agent can make enzyme marker normal temperature or 2-8 DEG C of stable long period (14 months), the test proof that accelerates the failure can in 37 DEG C stable at least 9 days, the long-time stability that also can be used for other enzyme marker are in theory preserved.
In sum, the inventive method has the following advantages: described in a, the inventive method, a certain item pathogen antigen detection kit is not subject to the interference of the disturbing factors such as rheumatoid factor, and false positive rate is lower; B, with in conjunction with high sensitivity enzyme marker for feature realizes quick and precisely detection to a certain item pathogen IgM/IgG antibody; Kit prepared by c, the inventive method can carry out one and even multinomially to detect fast simultaneously, detection chip (enzyme exempts from percolation) is exempted from the kit carrying out multinomial detection prepared as utilized the inventive method---TORCH family planning five (Tox-IgM/IgG, CMV-IgM/IgG, RV-IgG) fast enzyme, can detect Tox-IgM, Tox-IgG, CMV-IgM, CMV-IgG, RV-IgG five simultaneously, and the testing process used time is shorter, sensitivity is higher, and accuracy is stronger.D, the stable dilution buffer adopting the present invention to obtain and nitrite ion, can make kit storage life reach more than 12 months.The operating process of e, whole detection is simple and quick, without the need to specialized equipment equipment, sensitivity and accuracy higher, can be used for the medical diagnosis on disease research of hospital clinic, blood station, epidemic prevention station, health quarantine department, universities and colleges and scientific research institution.The method accuracy rate is high, high specificity, and testing result is reliable, and the testing process used time is shorter.
Accompanying drawing explanation
Fig. 1 is the structural representation that a kind of enzyme of quick detection pathogen antigen exempts from the kit of percolation.
In figure, 1 check-out console is gone to the bottom, 2 adsorptive pads, 3 nitrocellulose filters, 4 check-out console upper covers, 5 wells.
Embodiment
Below, by reference to the accompanying drawings and embodiment, the present invention is described further:
The technical scheme of the inventive method is: utilize antigen-antibody reaction, make antigen or two anti-immobilizations on nitrocellulose filter, when serum specimen passes through nitrocellulose filter due to transudation, pathogen IgM(IgG wherein) just corresponding on the film solid phase antigen of antibody or two anti-generation specific bindings form compound, other foreign matter are then filtered across, add enzyme labelled antibody or enzyme-labelled antigen, antigen antibody complex during filtration just on film is combined, and then adds the purple trace that nitrite ion colour developing generation naked eyes can conveniently be observed.
The involved relevant detection reagent of technique scheme is: enzyme labelled antibody or enzyme-labelled antigen working fluid, cleansing solution, nitrite ion, stop buffer.The compound method of liquid is as follows:
1.1 enzyme labelled antibodies or enzyme-labelled antigen working fluid.Compound method is divided into two parts:
(1) 1g horseradish peroxidase is dissolved in the 0.01M phosphate buffer of 25ml ~ 30ml, add 70 ~ 85mg ethylenediamine, with 0.1M hydrochloric acid, pH value is adjusted to 5.0, gained solution mixes with 115 ~ 125mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate again, after shaken at room temperature reaction, 0.01M phosphate buffer is adopted to dialyse to gained solution, obtained amination horseradish peroxidase solution;
(2) become by solution preparation obtained for step (1) 1ml, concentration to be the amination horseradish peroxidase solution of 5 ~ 5.2mg/ml, then in gained solution, add 60 ~ 85 μ l crosslinking chemicals, after mixing, under room temperature, react 25 ~ 35min;
(3) described reactant is added the super filter tube that molecular cut off is 10K, and centrifugal treating 8 ~ 12min is carried out to reactant, redissolve with the phosphate buffer of 0.01M again and be detained bond to 1ml, again with same centrifugal force 8 ~ 12min, to remove excessive crosslinking chemical, and again redissolve retentate to 1ml;
(4) add antibody or the antigenic solution of about 1mg, and react 25 ~ 35min under room temperature;
(5) solution making step (4) obtained, by SephadexG-200 chromatographic column, is collected the product of first peak, is enzyme labelled antibody or antigenic solution.
The stable dilution buffer of described enzyme labelled antibody or antigen adopts following methods preparation:
the preparation of brown alga extract: after fresh seaweed sample gathers, removing epibiont, frond top is got after repeatedly rinsing with distilled water, tissue mashing crusher machine is used after weighing, then add ethanol extract to extract, the method removing ethanol of extract decompression distillation, adds phosphate buffer, supernatant is got, i.e. obtained brown alga ethanol extract after high speed centrifugation.
the preparation of enzyme marker stability damping fluid: the methylisothiazolinone and the diagnostic reagent antiseptic PROCLIN300 that add 0.02-0.04wt% in the phosphate saline damping fluid of 0.01M, then 2 ~ 8wt% trehalose is added successively, 0.01 ~ 0.07wt% xanthans, 0.9wt% glycocoll and 0.7wt% serine, 0.3 ~ 1.2wt% brown alga extract is added again after mixing, the human-like collagen of 0.1 ~ 0.6wt% genetic recombination, finally add magnesium sulfate and the 0.9wt% zinc chloride of 1.8wt%, the glycerine of 0.1% vitamin B1 and 3% volume ratio, mix, obtain described enzyme marker stability damping fluid.
1.2 cleansing solution.
Its compound method is: in the Tris-HCl damping fluid being configured to 0.01M, add 5.0g triton x-100,0.2gNaCl, 0.2gNaN
3with a certain amount of BSA, add water to 1000ml, shaken well is required cleansing solution.
1.3 nitrite ion.
Configuration reagent: the sodium acetate buffer solution system of tetramethyl benzidine, urea peroxide, antioxidant chlorogenic acid, reductive agent ascorbic acid, ultraviolet light absorber 2-cyano group-3,3-diphenyl 2-Propenoic acid-2-ethylhexyl ester, pH5.0.
Collocation method:
8.3 ~ 8.5g sodium acetate trihydrate crystal is dissolved in 600ml ultrapure water, vibration mixing, and adjusts pH value of solution to 5.0;
again 0.8 ~ 0.83g tetramethyl benzidine is dissolved in 3ml absolute ethyl alcohol, obtained tetramethyl benzidine ethanolic solution;
by above-mentioned two kinds of solution mixing, vibration mixing;
respectively by 70 ~ 90mg chlorogenic acid and 70 ~ 90mg ascorbic acid, 8 ~ 12ml2-cyano group-3,3-diphenyl 2-Propenoic acid-2-ethylhexyl ester, 2.5 ~ 2.9g phosphinylidyne glycocoll and 0.4 ~ 0.6g urea peroxide join in the mixed solution that step (3) obtains, vibration mixing, obtains described nitrite ion.
1.4 stop buffer.
Described stop buffer compound method is 0.5M citric acid.
The present invention also provides the method for preparation and use of the kit of above-mentioned use; As shown in Figure 1, enzyme for detecting pathogen antigen fast exempts from the kit of percolation, comprise the check-out console be located in kit and go to the bottom 1, it is adsorptive pads 2 that check-out console is gone to the bottom above 1, it is nitrocellulose filter 3 above adsorptive pads 2, be check-out console upper cover 4 above nitrocellulose filter 3, check-out console upper cover 4 is provided with well 5.
The preparation method of kit comprises the following steps:
method as described in 1.1 is adopted to prepare horseradish peroxidase marker solution;
prepare coated film: the coated film selecting applicable aperture, is cut into square piece for subsequent use;
assembling detects box: lid, coated film, adsorptive pads and box body are assembled in accordance with the order from top to bottom and fastening, make the window of upper cover be buckled in coated film center, and composition detects box.
bag quilt: the antigen after centrifugal with the 0.01M phosphate buffer dilution that with the addition of 3wt% trehalose or antibody wrap quilt respectively in the check point position of coated film, and Quality Control point position bag is by a kind of antibody that can react with enzyme marker.
dry: to detect the atmosphere at room temperature dry 1 hour of box in humidity 10% ~ 40%;
packaging: dried detection box aluminium foil strip is packed, and places drying agent in bag, sealing.
2. a certain pathogen antigen detection kit (enzyme exempts from percolation) described in the present invention consists of: check-out console, enzyme mark liquid, cleansing solution, nitrite ion, stop buffer.Box and liquid component bottle and instructions will be detected above assemble with proper form, be a certain pathogen antigen detection kit (enzyme exempts from percolation).
The using method of mentioned reagent box:
(1) kit is taken out, equilibrium at room temperature 20 ~ 30 minutes; (2) test serum is prepared; (3) drip cleansing solution after equilibrium at room temperature in reaction zone, treat that liquid is completely moistening by film; (4) drip sample to be tested in reaction zone, treat that liquid fully sucks; (5) drip enzyme mark liquid in reaction zone, treat that liquid fully sucks; (6) drip cleansing solution in reaction zone, treat that liquid fully sucks; (7) drip nitrite ion in reaction zone, after liquid fully sucks, wait for 3 minutes; (8) drip stop buffer in reaction zone, after liquid fully sucks in 3 minutes observations.
Use above-mentioned kit, display result determination methods is as follows:
(1) negative---quality control region C position display purple trace, detection zone occurs without purple trace;
(2) positive---quality control region C position display purple trace, detection zone has purple trace to occur;
(3) invalid---do not develop the color and namely show that misoperation or reagent lost efficacy in quality control region C position.
3. the detection example of kit:
Multiple individual event detection kit can be prepared according to the inventive method, such as mycoplasma pneumoniae (MP) IgG antibody detection kit (enzyme exempts from percolation), mycoplasma pneumoniae (MP) IgM antibody detection kit (enzyme exempts from percolation), Chlamydia pneumoniae (Cpn) IgG antibody detection kit (enzyme exempts from percolation), Chlamydia pneumoniae (Cpn) IgM antibody detection kit (enzyme exempts from percolation), the quick enzyme of tubercle bacillus (TB) IgG antibody exempt from detection chip (enzyme exempts from percolation), and by this, several and domestic registration certificate kit that obtained carries out detection and contrasts:
(1) according to above detection mode, adopt mycoplasma pneumoniae IgG antibody detection kit (euzymelinked immunosorbent assay (ELISA)) kit in contrast obtaining registration certificate at home, prepare mycoplasma pneumoniae in kit (MP) IgG antibody detection kit (enzyme exempts from percolation) with the inventive method and detect yin and yang attribute serum simultaneously.Clinical serum obtains from relevant hospital, and testing result is as follows:
Table 1 the inventive method prepares kit and contrast agents box to Virus monitory result
。
Test shows, the sensitivity that this method prepares kit is higher, and with contrast agents box without marked difference.Mycoplasma pneumoniae IgG examines the false positive rate of reagent to be 0.7%, and the false positive rate of contrast agents box is 1.3%.Result shows, and the specificity that this method prepares kit mycoplasma pneumoniae (MP) IgG antibody detection kit (enzyme exempts from percolation) is better than contrast agents box.
(2) according to above detection mode, adopt mycoplasma pneumoniae IgM antibody detection kit (euzymelinked immunosorbent assay (ELISA)) kit in contrast obtaining registration certificate at home, prepare mycoplasma pneumoniae in kit (MP) IgM antibody detection kit (enzyme exempts from percolation) with the inventive method and detect yin and yang attribute serum simultaneously.Clinical serum obtains from relevant hospital, and testing result is as follows:
Table 2 the inventive method prepares kit and contrast agents box to Virus monitory result
。
Test shows, the sensitivity that this method prepares kit is higher, and with contrast agents box without marked difference.Mycoplasma pneumoniae IgM examines the false positive rate of reagent to be 0.3%, and the false positive rate of contrast agents box is 1.8%.Result shows, and the specificity that this method prepares kit mycoplasma pneumoniae (MP) IgM antibody detection kit (enzyme exempts from percolation) is better than contrast agents box.
(3) according to above detection mode, adopt CPn IgG antibody assay kit (euzymelinked immunosorbent assay (ELISA)) kit in contrast obtaining registration certificate at home, prepare Chlamydia pneumoniae in kit (Cpn) IgG antibody detection kit (enzyme exempts from percolation) with the inventive method and detect yin and yang attribute serum simultaneously.Clinical serum obtains from relevant hospital, and testing result is as follows:
Table 3 the inventive method prepares kit and contrast agents box to Virus monitory result
。
Test shows, the sensitivity that this method prepares kit is higher, and with contrast agents box without marked difference.The false positive rate of CPn IgG examination reagent is 0.7%, and the false positive rate of contrast agents box is 1.3%.Result shows, and the specificity that this method prepares kit Chlamydia pneumoniae (Cpn) IgG antibody detection kit (enzyme exempts from percolation) is better than contrast agents box.
(4) according to above detection mode, adopt anti-chlamydia pneumoniae (cp) IgM detection kit (enzyme linked immunosorbent assay) kit in contrast obtaining registration certificate at home, prepare Chlamydia pneumoniae in kit (Cpn) IgM antibody detection kit (enzyme exempts from percolation) with the inventive method and detect yin and yang attribute serum simultaneously.Clinical serum obtains from relevant hospital, and testing result is as follows:
Table 4 the inventive method prepares kit and contrast agents box to Virus monitory result
。
Test shows, the sensitivity that this method preparation method prepares kit is higher, and with contrast agents box without marked difference.Chlamydia pneumoniae IgM examines the false positive rate of reagent to be 0.4%, and the false positive rate of contrast agents box is 2.3%.Result shows, and the specificity that this method prepares kit Chlamydia pneumoniae (Cpn) IgM antibody detection kit (enzyme exempts from percolation) is better than contrast agents box.
(5) according to above detection mode, adopt and obtain tubercle bacillus IgG antibody detection kit (euzymelinked immunosorbent assay (ELISA)) kit in contrast of registration certificate at home, prepare the quick enzyme of tubercle bacillus in kit (TB) IgG antibody with the inventive method and exempt from detection chip (enzyme exempts from percolation) and detect yin and yang attribute serum simultaneously.Clinical serum obtains from relevant hospital, and testing result is as follows:
Table 5 the inventive method prepares kit and contrast agents box to Virus monitory result
。
Test shows, the sensitivity that this method prepares kit is higher, and with contrast agents box without marked difference.Tubercle bacillus IgG examines the false positive rate of reagent to be 0.3%, and the false positive rate of contrast agents box is 1.8%.Result shows, and this method is prepared the specificity that the quick enzyme of kit tubercle bacillus (TB) IgG antibody exempts from detection chip (enzyme exempts from percolation) and is better than contrast agents box.
Also multinomial detection kit can be prepared according to the inventive method, such as fast enzyme exempts from detection chip (enzyme exempts from percolation), seven quick enzymes of respiratory pathogen (MP, Cpn, RSV, Adv, PFV, FLU-A, FLU-B) IgM antibody exempt from detection chip (enzyme exempts from percolation) in TORCH family planning five (Tox-IgM/IgG, CMV-IgM/IgG, RV-IgG), by its with obtain registration certificate kit at home and carry out detection and contrast:
(1) according to above detection mode, adopt the toxoplasmosis IgM antigen testing reagent box (euzymelinked immunosorbent assay (ELISA)) obtaining registration certificate at home, toxoplasma antibody (IgG) detection kit (euzymelinked immunosorbent assay (ELISA)), human cytomegalovirus's IgM antibody detection kit (euzymelinked immunosorbent assay (ELISA)), human cytomegalovirus's antibody (IgG) detection kit (euzymelinked immunosorbent assay (ELISA)), Detecting Rubella Virus Antibodies In Human Sera (IgG) detection kit (euzymelinked immunosorbent assay (ELISA)) kit in contrast, TORCH family planning five (Tox-IgM/IgG in kit are prepared with the inventive method, CMV-IgM/IgG, RV-IgG) enzyme is exempted from detection chip (enzyme exempts from percolation) and is detected yin and yang attribute serum simultaneously fast.Clinical serum obtains from relevant hospital, and testing result is as follows:
Table 6 the inventive method prepares kit and contrast agents box to Virus monitory result--Tox-IgM
。
Table 7 the inventive method prepares kit and contrast agents box to Virus monitory result--Tox-IgG
。
Table 8 the inventive method prepares kit and contrast agents box to Virus monitory result--CMV-IgM
。
Table 9 the inventive method prepares kit and contrast agents box to Virus monitory result--CMV-IgG
。
Table 10 the inventive method prepares kit and contrast agents box to Virus monitory result--RV-IgG
。
Test shows, the sensitivity that this method prepares kit is higher, and with contrast agents box without marked difference.Tox-IgM examines the false positive rate of reagent to be 0.5%, and the false positive rate of contrast agents box is 1.8%; Tox-IgG examines the false positive rate of reagent to be 0.4%, and the false positive rate of contrast agents box is 1.6%; CMVIgM examines the false positive rate of reagent to be 0.1%, and the false positive rate of contrast agents box is 1.6%; CMVIgG examines the false positive rate of reagent to be 0.6%, and the false positive rate of contrast agents box is 1.4%; RVIgG examines the false positive rate of reagent to be 0.6%, and the false positive rate of contrast agents box is 1.9%.Result shows, and this method is prepared kit TORCH family planning five (Tox-IgM/IgG, CMV-IgM/IgG, RV-IgG) specificity that fast enzyme exempts from detection chip (enzyme exempts from percolation) and is better than contrast agents box.
(2) according to above detection mode, adopt respiratory pathogen spectrum antibody IgM detection kit (indirect immunofluorescence) kit in contrast obtaining registration certificate at home, prepare seven quick enzymes of respiratory pathogen (MP, Cpn, RSV, Adv, PFV, FLU-A, FLU-B) IgM antibody in kit with the inventive method and exempt from detection chip (enzyme exempts from percolation) and detect yin and yang attribute serum simultaneously.Clinical serum obtains from relevant hospital, and testing result is as follows:
Table 11 the inventive method prepares kit and contrast agents box to Virus monitory result-MP
。
Table 12 the inventive method prepares kit and contrast agents box to Virus monitory result-Cpn
。
Table 13 the inventive method prepares kit and contrast agents box to Virus monitory result-RSV
。
Table 14 the inventive method prepares kit and contrast agents box to Virus monitory result-Adv
。
Table 15 the inventive method prepares kit and contrast agents box to Virus monitory result-PFV
。
Table 16 the inventive method prepares kit and contrast agents box to Virus monitory result-FLU-A
。
Table 17 the inventive method prepares kit and contrast agents box to Virus monitory result-FLU-B
。
Test shows, the sensitivity that this method prepares kit is higher, and with contrast agents box without marked difference.MPIgM examines the false positive rate of reagent to be 0.5%, and the false positive rate of contrast agents box is 1.5%; CpnIgM examines the false positive rate of reagent to be 0.4%, and the false positive rate of contrast agents box is 0.2%; RSVIgM examines the false positive rate of reagent to be 0.7%, and the false positive rate of contrast agents box is 0.3%; AdvIgM examines the false positive rate of reagent to be 0.2%, and the false positive rate of contrast agents box is 0.8%; PFVIgM examines the false positive rate of reagent to be 0.3%, and the false positive rate of contrast agents box is 0.7%; FLU-AIgM examines the false positive rate of reagent to be 0.3%, and the false positive rate of contrast agents box is 0.6%; FLU-BIgM examines the false positive rate of reagent to be 0.3%, and the false positive rate of contrast agents box is 0.7%.So the specificity that this method prepares kit is better than contrast agents box.
Therefore can be found out by testing result, according to above detection technique scheme, the a certain pathogen antigen detection kit utilizing the inventive method to prepare detects the yin and yang attribute serum obtained from relevant hospital, testing result by with obtain the coherent detection kit testing result of registration certificate at home in contrast, the specificity of kit prepared by comparison the inventive method and contrast agents box, result shows, and the inventive method is prepared kit specificity and is better than contrast agents box.
The inventive method prepares stabilization of kit Journal of Sex Research: detection kit (choosing continuous three batches) is placed in 37 DEG C and places 9 days, and detect every day; Detection kit is placed in (2 ~ 8 DEG C) routinely to place 14 months, and regularly detects, examination criteria is as follows:
(1) physical detection: packing box is clean and tidy, lot number, term of validity content are correct, and internal composition is complete, and quantity is correct; The check-out console outward appearance loaded answers smooth cleaning, assembling tightly, and detect aperture interior diaphragm answers no marking, breakage and stain; The sealing of external packing aluminium foil bag is neat, tight, and content is correct; Various liquid bottle top tightens No leakage, label firm pasting.
(2) specificity: detect with 10 parts of negative reference product, result should be feminine gender (negative match-rate: 10/10).
(3) accuracy: detect by 10 parts of positive reference materials, result should be the positive (positive coincidence rate: 10/10).
Stabilization of kit testing result as shown in the table (+be positive;---be is negative)
9 days accelerated test testing results placed by table 1937 DEG C
。
Table 2014 month places (2 ~ 8 DEG C) laboratory test results routinely
。
Result shows: the kit of continuous three batches (100601,100701,100801), and place for 37 DEG C and place 14 months periods in 9 days and 2 ~ 8 DEG C, physical detection all meets the requirements; Negative quality-control product testing result is negative entirely, positive quality control product testing result is all positive, and property indices does not change substantially, can meet clinical examination requirement.
What the inventive method adopted is enzyme linked immunological percolation, and the method accuracy rate is high, high specificity, and testing result is reliable, and the testing process used time is shorter.Compared with prior art, this invention is simple to operate, convenient and swift, greatly saves the time, and testing result is apparent, the impact of the interference-free factor of testing result.
The advantage of enzyme labelled antibody working fluid that the inventive method uses: a, utilize horseradish peroxidase as label, it has greater activity, tolerable pH range is wider, and can save backup for a long time.The enzyme marker working fluid of b, use, eliminate the disturbing factor such as rheumatoid factor in sample to a great extent to disturb the false positive of result, greatly reduce the antibody cost of kit: the enzyme marker dilution wherein used simultaneously, utilize and screen through design and demonstrate the protectiveness dilution buffer fluid component that can make enzyme marker long-term stability, current existing disclosing of comparing is filled a prescription, this system component is less containing animal sources material, and can not biochemical reaction be there is between each composition, the protection to enzyme marker and stabilization can be played to greatest extent; Under c and enzyme marker protective agent can make enzyme marker normal temperature or 2-8 DEG C of stable long period (14 months), the test proof that accelerates the failure can in 37 DEG C stable at least 9 days, the long-time stability that also can be used for other enzyme marker are in theory preserved.
The nitrite ion advantage used in the inventive method: the A in this nitrite ion, B component (i.e. superoxide with add lustre to thing TMB) can in long-term co-existence same systems, without the need to interim mixing during detection, directly to add, simplify kit detecting step and saved cost.
The inventive method has the following advantages: described in a, the inventive method, a certain item pathogen antigen detection kit is not subject to the interference of the disturbing factors such as rheumatoid factor, and false positive rate is lower; B, with in conjunction with high sensitivity enzyme labeling mouse anti human IgM(IgG) monoclonal antibody is the quick and precisely detection that feature realizes to a certain item pathogen IgM/IgG antibody; Kit prepared by c, the inventive method can carry out one and even multinomially to detect fast simultaneously, detection chip (enzyme exempts from percolation) is exempted from the kit carrying out multinomial detection prepared as utilized the inventive method---TORCH family planning five (Tox-IgM/IgG, CMV-IgM/IgG, RV-IgG) fast enzyme, can detect Tox-IgM, Tox-IgG, CMV-IgM, CMV-IgG, RV-IgG five simultaneously, and the testing process used time is shorter, sensitivity is higher, and accuracy is stronger.D, the stable dilution buffer adopting the present invention to obtain and nitrite ion, can make kit storage life reach more than 12 months.The operating process of e, whole detection is simple and quick, without the need to specialized equipment equipment, sensitivity and accuracy higher, can be used for the medical diagnosis on disease research of hospital clinic, blood station, epidemic prevention station, health quarantine department, universities and colleges and scientific research institution.
Claims (9)
1. the enzyme for detecting pathogen antigen fast exempts from a percolation, it is characterized in that, detection method is as follows:
(1) add serum specimen to be checked, make serum specimen to be checked pass through film;
(2) pathogen IgM(IgG) the antibody solid phase antigen corresponding on film or two anti-generation specific bindings form compound, and add cleansing solution, other foreign matter are then filtered across;
(3) add enzyme labelled antibody or enzyme-labelled antigen working fluid, antigen antibody complex during filtration just on film is combined;
(4) nitrite ion colour developing is added;
(5) add stop buffer, carry out result interpretation.
2. the enzyme for detecting pathogen antigen fast according to claim 1 exempts from percolation, it is characterized in that: described film is nitrocellulose filter or PVDF membrane.
3. the enzyme for detecting pathogen antigen fast according to claim 1 exempts from percolation, it is characterized in that: the enzyme labelled antibody used in step (3) or the compound method of enzyme-labelled antigen working fluid are divided into two parts, comprise the compound method of enzyme labelled antibody or enzyme-labelled antigen, and the compound method of the stable dilution buffer of enzyme labelled antibody or enzyme-labelled antigen;
The compound method of described enzyme labelled antibody or enzyme-labelled antigen, adopts following steps preparation:
(1) 1g horseradish peroxidase is dissolved in the 0.01M phosphate buffer of 25ml ~ 30ml, add 70 ~ 85mg ethylenediamine, with 0.1M hydrochloric acid, pH value is adjusted to 5.0, gained solution mixes with 115 ~ 125mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate again, after shaken at room temperature reaction, 0.01M phosphate buffer is adopted to dialyse to gained solution, obtained amination horseradish peroxidase solution;
(2) become by the solution preparation that (1) step obtains 1ml, concentration to be the amination horseradish peroxidase solution of 5 ~ 5.2mg/ml, then in gained solution, add 60 ~ 85 μ l crosslinking chemicals, after mixing, under room temperature, react 25 ~ 35min;
(3) described reactant is added the super filter tube that molecular cut off is 10K, and centrifugal treating 8 ~ 12min is carried out to reactant, redissolve with the phosphate buffer of 0.01M again and be detained bond to 1ml, again with same centrifugal force 8 ~ 12min, to remove excessive crosslinking chemical, and again redissolve retentate to 1ml;
(4) add antibody or the antigenic solution of about 1mg, and react 25 ~ 35min under room temperature;
(5) the solution making step (4) obtain, by SephadexG-200 chromatographic column, is collected the product of first peak, is enzyme labelled antibody or antigenic solution;
The stable dilution buffer of described enzyme labelled antibody or antigen adopts following methods preparation:
(1) the preparation of brown alga extract: after fresh seaweed sample gathers, removing epibiont, frond top is got after repeatedly rinsing with distilled water, tissue mashing crusher machine is used after weighing, then add ethanol extract to extract, the method removing ethanol of extract decompression distillation, adds phosphate buffer, supernatant is got, i.e. obtained brown alga ethanol extract after high speed centrifugation;
(2) the preparation of enzyme marker stability damping fluid: the methylisothiazolinone and the diagnostic reagent antiseptic PROCLIN300 that add 0.02-0.04wt% in the phosphate saline damping fluid of 0.01M, then 2 ~ 8wt% trehalose is added successively, 0.01 ~ 0.07wt% xanthans, 0.9wt% glycocoll and 0.7wt% serine, 0.3 ~ 1.2wt% brown alga extract is added again after mixing, the human-like collagen of 0.1 ~ 0.6wt% genetic recombination, finally add magnesium sulfate and the 0.9wt% zinc chloride of 1.8wt%, the glycerine of 0.1% vitamin B1 and 3% volume ratio, mix, obtain described enzyme marker stability damping fluid.
4. the enzyme for detecting pathogen antigen fast according to claim 1 exempts from percolation, it is characterized in that: the collocation method of described cleansing solution is, in the Tris-HCl damping fluid being configured to 0.01M, add 5.0g triton x-100,0.2gNaCl, 0.2gNaN
3with a certain amount of BSA, add water to 1000ml, shaken well is required cleansing solution.
5. the enzyme for detecting pathogen antigen fast according to claim 1 exempts from percolation, it is characterized in that: described nitrite ion comprises the sodium acetate buffer solution system of tetramethyl benzidine, urea peroxide, antioxidant chlorogenic acid, reductive agent ascorbic acid, ultraviolet light absorber 2-cyano group-3,3-diphenyl 2-Propenoic acid-2-ethylhexyl ester, pH5.0.
6. the enzyme for detecting pathogen antigen fast according to claim 5 exempts from percolation, it is characterized in that: described nitrite ion is adopted and obtained with the following method:
8.3 ~ 8.5g sodium acetate trihydrate crystal is dissolved in 600ml ultrapure water, vibration mixing, and adjusts pH value of solution to 5.0;
again 0.8 ~ 0.83g tetramethyl benzidine is dissolved in 3ml absolute ethyl alcohol, obtained tetramethyl benzidine ethanolic solution;
by above-mentioned two kinds of solution mixing, vibration mixing;
respectively 70 ~ 90mg chlorogenic acid and 70 ~ 90mg ascorbic acid, 8 ~ 12ml2-cyano group-3,3-diphenyl 2-Propenoic acid-2-ethylhexyl ester, 2.5 ~ 2.9g phosphinylidyne glycocoll and 0.4 ~ 0.6g urea peroxide are joined step
in the mixed solution obtained, vibration mixing, obtains described nitrite ion.
7. the enzyme for detecting pathogen antigen fast exempts from the kit of percolation, it is characterized in that: comprise the check-out console be located in kit and go to the bottom (1), check-out console is gone to the bottom (1), and top is adsorptive pads (2), adsorptive pads (2) top is nitrocellulose filter (3), nitrocellulose filter (3) top is check-out console upper cover (4), and check-out console upper cover (4) is provided with well (5).
8. the preparation method exempting from the kit of percolation for the enzyme detecting pathogen antigen fast as claimed in claim 7, it is characterized in that, the preparation method of described kit comprises the following steps:
method is as claimed in claim 3 adopted to prepare horseradish peroxidase marker solution;
prepare coated film: the coated film selecting applicable aperture, is cut into square piece for subsequent use;
assembling detects box: lid, coated film, adsorptive pads and box body are assembled in accordance with the order from top to bottom and fastening, make the window of upper cover be buckled in coated film center, and composition detects box;
(4) wrap quilt: the antigen after centrifugal with the 0.01M phosphate buffer dilution that with the addition of 3wt% trehalose or antibody wrap quilt respectively in the check point position of coated film, and Quality Control point position bag is by a kind of antibody that can react with enzyme marker;
(5) dry: to detect the atmosphere at room temperature dry 1 hour of box in humidity 10% ~ 40%;
packaging: dried detection box aluminium foil strip is packed, and places drying agent in bag, sealing.
9. use the method exempting from the kit of percolation for the enzyme detecting pathogen antigen fast as claimed in claim 7, it is characterized in that: using method is as follows:
(1) kit is taken out, equilibrium at room temperature 20 ~ 30 minutes;
(2) test serum is prepared;
(3) drip cleansing solution after equilibrium at room temperature in reaction zone, treat that liquid is completely moistening by film;
(4) drip sample to be tested in reaction zone, treat that liquid fully sucks;
(5) drip enzyme mark liquid in reaction zone, treat that liquid fully sucks;
(6) drip cleansing solution in reaction zone, treat that liquid fully sucks;
(7) drip nitrite ion in reaction zone, after liquid fully sucks, wait for 3 minutes;
(8) drip stop buffer in reaction zone, after liquid fully sucks in 3 minutes observations.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107589250A (en) * | 2017-09-28 | 2018-01-16 | 国家食品安全风险评估中心 | TMB two-components nitrite ion and there is its kit |
| CN112946300A (en) * | 2021-03-19 | 2021-06-11 | 苏冬梅 | Alzheimer disease early detection kit based on Western Blot method and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110318747A1 (en) * | 2006-05-09 | 2011-12-29 | Beckman Coulter, Inc. | Nonseparation Assay Methods |
| CN103033615A (en) * | 2012-12-24 | 2013-04-10 | 青岛汉唐生物科技有限公司 | Method for detecting mycoplasma pneumoniae antibody, kit for detection and preparation method of kit |
| CN203011922U (en) * | 2012-12-24 | 2013-06-19 | 青岛汉唐生物科技有限公司 | Rapid double-antibody detection kit |
| CN103575904A (en) * | 2013-10-12 | 2014-02-12 | 北京健乃喜生物技术有限公司 | Mycobacterium-tuberculosis antibody detection kit capable of respectively detecting IgG and IgM antibodies simultaneously |
| CN104198704A (en) * | 2014-09-02 | 2014-12-10 | 张明 | Pertussis diagnostic kit and preparation method thereof |
-
2015
- 2015-07-27 CN CN201510445471.8A patent/CN105067809A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110318747A1 (en) * | 2006-05-09 | 2011-12-29 | Beckman Coulter, Inc. | Nonseparation Assay Methods |
| CN103033615A (en) * | 2012-12-24 | 2013-04-10 | 青岛汉唐生物科技有限公司 | Method for detecting mycoplasma pneumoniae antibody, kit for detection and preparation method of kit |
| CN203011922U (en) * | 2012-12-24 | 2013-06-19 | 青岛汉唐生物科技有限公司 | Rapid double-antibody detection kit |
| CN103575904A (en) * | 2013-10-12 | 2014-02-12 | 北京健乃喜生物技术有限公司 | Mycobacterium-tuberculosis antibody detection kit capable of respectively detecting IgG and IgM antibodies simultaneously |
| CN104198704A (en) * | 2014-09-02 | 2014-12-10 | 张明 | Pertussis diagnostic kit and preparation method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107589250A (en) * | 2017-09-28 | 2018-01-16 | 国家食品安全风险评估中心 | TMB two-components nitrite ion and there is its kit |
| CN112946300A (en) * | 2021-03-19 | 2021-06-11 | 苏冬梅 | Alzheimer disease early detection kit based on Western Blot method and preparation method thereof |
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