CN101949925B - Colloidal gold chromatographic test strip for rapidly testing prednisone acetate and derivative thereof and preparation method - Google Patents
Colloidal gold chromatographic test strip for rapidly testing prednisone acetate and derivative thereof and preparation method Download PDFInfo
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- CN101949925B CN101949925B CN 201010516358 CN201010516358A CN101949925B CN 101949925 B CN101949925 B CN 101949925B CN 201010516358 CN201010516358 CN 201010516358 CN 201010516358 A CN201010516358 A CN 201010516358A CN 101949925 B CN101949925 B CN 101949925B
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Abstract
The invention discloses a colloidal gold chromatographic test strip for rapidly testing prednisone acetate and a derivative thereof. The colloidal gold chromatographic test strip consists of a lining plate, a sample pad, a gold labeled conjugate pad, an encapsulating film and an absorbent pad which are connected on the lining plate sequentially. The gold labeled conjugate pad is a polyester fiber felt of a gold-labeled antibody of the prednisone acetate derivative; and the encapsulating film is provided with a straight line type detection line T which is encapsulated by solution of carrier protein coupled with the prednisone acetate derivative and a straight line type reference line C which is encapsulated by solution of goat-anti-mouse IgG, and the two lines are arranged parallelly and vertically. A preparation method comprises the specific steps of: preparing artificial conjugated antigen; preparing a monoclonal antibody of the prednisone acetate derivative; and preparing solution of colloidal gold to obtain a colloidal gold marker for resisting the monoclonal antibody of the prednisone acetate derivative and the gold labeled conjugate pad. The colloidal gold chromatographic test strip for rapidly testing the prednisone acetate and the derivative thereof and the preparation method have the advantages of high specificity, high sensitivity, simpleness, convenience, quickness, high timeliness, and visual and exact result display.
Description
Technical field
The present invention relates to the colloid gold chromatographic test paper strip and the preparation method of a kind of fast detecting prednisone acetate and derivant thereof, belong to the detection technique field of biology immunization method.
Background technology
Prednisone acetate is a kind of Aeroseb-Dex, has anti-inflammatory, antiallergy, antirheumatic, immunosuppressive action.In recent years, some illegal manufacturing enterprises are for seeking exorbitant profit, under without situation about getting the Green Light, in treatment immunity disease (lupus erythematosus for example, arthritis, ephritis or the like) illegally add hormones such as prednisone acetate in the Chinese medicine, the patient is under unwitting situation, use these problematic " pure Chinese medicinal preparations " in a short time, curative effect is good really, but these effects are exactly functions of hormones in fact, but use for a long time or drug withdrawal suddenly, problem has just been come out: the long-term use causes that adrenal gland suppresses, and brings out hypercortisolism (moon shaped face, female hirsutism, acne, hypertension, osteoporosis, sexual dysfunction etc.); Increase infection chance; Influence the wound healing; Increase the possibility that digestive tract ulcer is hemorrhage and bore a hole; Growth retardation, psychataxia, cataract, glaucoma, bone necrosis or the like.Suddenly drug withdrawal causes endogenous cortin low excessively (adrenal atrophy) to cause hormone response is lacked, and the state of an illness recurs rapidly, worsens even.The patient of some acute renal insufficiencies stops using hormone abnormally dangerous suddenly, causes death even.If use " pure Chinese medicinal preparation " added hormone, patient is also ignorant, and these patients are hormone contraindication and careful use patient just, for example active peptic ulcer, fracture, hypertension, diabetes, pregnant woman etc., in case use, danger is just big.So in order to guarantee the people's diet drug safety, we must go into overdrive to hit this phenomenon.Yet in recent years, the means of lawless person's illegal production are hidden day by day, and because the traditional Chinese medicine ingredients more complicated, detection difficulty is bigger, need accurate analytical instrument and professional and technical personnel, thereby cause supervision department's supervision examination at random cost very big, it is not satisfactory to produce effects.
Summary of the invention
The objective of the invention is in order to overcome above deficiency, provide a kind of energy fast, sensitive, detect the prednisone acetate colloidal gold chromatographic test strip and the preparation method of prednisone acetate derivant easily.
The present invention is achieved through the following technical solutions: the colloid gold chromatographic test paper strip of a kind of fast detecting prednisone acetate and derivant thereof, sample pad, gold-marking binding pad, coated film and the adsorptive pads of linking are formed successively by liner plate with on liner plate, gold-marking binding pad is the dacron felt of the golden labeling antibody of absorption prednisone acetate derivant, the carrier protein solution bag of useful prednisone acetate derivant coupling on coated film is by orthoscopic detection line T line, with with sheep anti-mouse igg solution bag by orthoscopic control line C line, two lines are vertically arranged in parallel.The PVC toughness material of liner plate for not absorbing water.Coated film is a nitrocellulose filter, or the cellulose mixture film.Adsorptive pads is the vegetable fibre cotton paper.The golden labeling antibody of prednisone acetate derivant is the monoclonal anti nanocrystal composition of the anti-prednisone acetate derivant of colloid gold label, and the carrier protein of coupling prednisone acetate derivant is bovine serum albumin(BSA) BSA.
The preparation method of the colloid gold chromatographic test paper strip of a kind of fast detecting prednisone acetate and derivant thereof, the carrier bovine serum albumin(BSA) BSA of preparation coupling prednisone acetate derivant is used to prepare relevant detection line T line; The pure albumen of carrier ovum gallinaceum of preparation coupling prednisone acetate derivant is used to prepare monoclonal antibody, and preparation prednisone acetate derivant gold labeling antibody is used to prepare the dacron felt that adsorbs prednisone acetate derivant gold labeling antibody; The preparation sheep anti-mouse igg antibody is used to prepare control line C line, comprises the steps:
(A) prednisone acetate derivant and the pure albumen OVA of carrier bovine serum albumin(BSA) BSA/ ovum gallinaceum are carried out coupling and prepare artificial antigen;
A, generation water-soluble carbodiimide (EDC): in reaction bulb, add dry dimethyl formamide (DMF) and that non-class raw material or draw non-class raw material, and logical N
2The protection, at room temperature stir, treat that dimethyl formamide (DMF) dissolves fully after; take by weighing concentration fast and be 60% sodium hydride (NaH), and it is added in the described reaction bulb, stirred 50-70 minute; not having bubble until solution produces; draw bromoacetate with syringe then and slowly inject described reaction bulb, after injection finishes, at room temperature stirred 11-13 hour; to disperse in the reactant liquor impouring water afterwards; extract with chloroform, merge extract, with anhydrous Na
2SO
4Carry out drying, filter afterwards, obtain solid through behind the concentrating under reduced pressure again, add the described solid of absolute ether making beating washing, filter afterwards, last vacuum drying obtains new solid, and this solid is an alkylates; The alkylates of absolute ethyl alcohol and above-mentioned steps gained is added in the new reaction bulb, carry out dispersed with stirring, add NaOH solution again, heating reflux reaction 2 hours, after stopping to heat cooling slightly, reactant liquor is changed in the single port bottle, concentrating under reduced pressure gets pastel, add entry again, be adjusted to the liquid that the pH value is 7-9 with hydrochloric acid, extract, merge extract with chloroform, again once, with anhydrous Na with the saturated common salt washing
2SO
4Drying is filtered, and concentrating under reduced pressure gets white solid, uses recrystallizing methanol, gets white powdery solid, and this solid is water-soluble carbodiimide (EDC);
B, generate artificial antigen: with the water-soluble carbodiimide (EDC) of gained in the above-mentioned steps, N-hydroxy-succinamide (NHS), dimethyl formamide (DMF), hapten compound balance to room temperature; Get water-soluble carbodiimide, N-hydroxy-succinamide and the hapten compound of gained in the above-mentioned steps, three's mol ratio is: hapten compound: water-soluble carbodiimide: N-hydroxy-succinamide=1:8-12:13-16, it is inserted round-bottomed flask, the dimethyl formamide dissolving that adds gained in the above-mentioned steps then, for guaranteeing to react completely, stir under the condition of room temperature lucifuge, the solution of Sheng Chenging is designated as reactant liquor No. two at last; After described water-soluble carbodiimide, N-hydroxy-succinamide and hapten compound react completely in dimethyl formamide, existing prestowage body protein solution (BSA/OVA), carrier protein and borate buffer are dissolved in borate buffer in another container by the amount of 1:0.15-0.2, change over to then in another round-bottomed flask, this solution is designated as reactant liquor No. three; No. two reactant liquors are splashed in No. three reactant liquors by per 10 seconds speed of 1, after No. two reactant liquor is added, under the condition of room temperature lucifuge, stir earlier, lucifuge stirs under 3-5 ℃ of condition afterwards, after question response is complete, generate solution, and the solution that generates is designated as reactant liquor No. four, carrier protein and hapten compound ratio are in No. four reactant liquors: 1:15-25; Deng institute respond finish after, place the 20000-30000 molecule to hold back bag filter No. four reactant liquors, the 0.01-0.02M phosphate buffer also is housed in the bag filter, the pH value is 7.0-7.5, bag filter is dialysed under 3-5 ℃ condition, changed one time phosphate buffer every 3-5 hour; Through the product of the final gained of above-mentioned dialysis step, be white cotton-shaped product through freeze drying, be artificial antigen.
(B) with the immune according to a conventional method cell strain of monoclonal antibody for preparing the prednisone acetate derivant of artificial antigen OVA, prepare antibody ascites with the Babc mouse immune, the ascites purifying is obtained highly purified monoclonal antibody protein, measure protein concentration, be equipped with colloid gold label and use;
(C) gold chloride reduction is made the colloidal gold solution of 20nm-40nm with the trisodium citrate reductive agent;
(D) use 0.1mol/LK
2CO
3Regulating colloidal gold solution to optimum PH is 8.5-9.0, behind the monoclonal antibody pure water dialysis 4hr with an amount of purifying, add in the colloid basin, room temperature electromagnetic agitation 15-30min, dropwise add 1% network protein solution, making network concentration is 0.1%, continue to stir 5min, 4 ℃ of centrifugal 10min of following 15000rpm, supernatant discarded is removed sediment, sediment is returned to the original volume mixing again with resuspended liquid, once centrifugal once more, it is standby with resuspended solution sediment to be suspended to 1/20,4 ℃ of preservation of original volume, promptly obtain the colloid gold label thing of anti-prednisone acetate derivant monoclonal antibody, resuspended liquid is the 0.02Tris-HCL damping fluid that contains 5% marine alga;
(E) the prednisone acetate derivant colloid of 1:20-1:10 dilution is advanced the labelled antibody spraying and be adsorbed in the dacron felt 27 ℃ of heat preservation and drynesses, preparation prednisone acetate derivant gold-marking binding pad;
(F) the carrier bovine serum albumin(BSA) BSA solution bag of using the coupling of prednisone acetate derivant on coated film is by orthoscopic detection line T line, and by orthoscopic control line C line, two lines are vertically arranged in parallel with sheep anti-mouse igg solution bag;
(G) assembling of test strips: stick to sample pad, gold mark combination mat, coated film and adsorptive pads on the liner plate successively, sample pad places an end of liner plate, adsorptive pads places the other end of liner plate, coated film places the centre of liner plate, gold-marking binding pad places between sample pad and the coated film, the prednisone acetate derivant colloidal gold chromatographic that promptly obtains being used for immune detection detects the big plate of ph test paper ph, and big plate is cut into the test strips of 2.5mm-7.0mm width specifications by customer requirement, and hermetically drying is preserved.
The present invention compared with prior art has the following advantages:
1, high specificity, the susceptibility height.This colloidal gold chromatographic test strip is that the basis is prepared from the monoclonal antibody of colloid gold label high specific high-affinity Electrostatic Absorption.
2, easy, quick, ageing strong, use colloidal gold chromatographic test strip and test card, need not any other reagent and instrument, but execute-in-place after sample mixed with extract, get supernatant and detect, be the decidable testing result at 5min.
3, the result shows image, directly perceived, accurate.Test strip is all to show that red line T line and C line are as the testing result positive and negative marker, when promptly on coated film, showing a red line C line, be illustrated in and contain checking matter in the test sample, when showing two red line T lines and C line, be illustrated in to be subtracted and do not contain checking matter in the sample.The result judges image, directly perceived, accurate, simple and clear, is not prone to artificial erroneous judgement such as false positive and false negative.
4, the preparation method adopts spraying coating process, and is fast simple, and spraying is even, fast drying.
Description of drawings:
Fig. 1 is a cross-sectional view of the present invention;
Fig. 2 is a plan structure synoptic diagram of the present invention;
Number in the figure: 1-liner plate, 2-sample pad, 3-gold-marking binding pad, 4-coated film, 5-adsorptive pads, 6-T line, 7-C line.
Embodiment:
In order to deepen the understanding of the present invention, the invention will be further described below in conjunction with embodiment and accompanying drawing, and this embodiment only is used to explain the present invention, does not constitute the qualification to protection domain of the present invention.
The colloid gold chromatographic test paper strip of a kind of as shown in Figure 1 and Figure 2 fast detecting prednisone acetate and derivant thereof, sample pad 2, gold-marking binding pad 3, coated film 4 and the adsorptive pads 5 of linking are formed successively by liner plate 1 with on liner plate 1, gold-marking binding pad 3 is the dacron felt of the golden labeling antibody of absorption prednisone acetate derivant, the carrier protein solution bag of useful prednisone acetate derivant coupling on coated film 4 is by orthoscopic detection line T line 6, be vertically arranged in parallel by 7, two lines of orthoscopic control line C line with usefulness sheep anti-mouse igg solution bag.The PVC toughness material of liner plate 1 for not absorbing water.Coated film 4 is a nitrocellulose filter, or the cellulose mixture film.Adsorptive pads 5 is the vegetable fibre cotton paper.The golden labeling antibody of prednisone acetate derivant is the monoclonal anti nanocrystal composition of the anti-prednisone acetate derivant of colloid gold label, and the carrier protein of coupling prednisone acetate derivant is bovine serum albumin(BSA) BSA.The carrier bovine serum albumin(BSA) BSA of preparation coupling prednisone acetate derivant is used to prepare relevant detection line T line 6; The pure albumen of carrier ovum gallinaceum of preparation coupling prednisone acetate derivant is used to prepare monoclonal antibody, and preparation prednisone acetate derivant gold labeling antibody is used to prepare the dacron felt that adsorbs prednisone acetate derivant gold labeling antibody; The preparation sheep anti-mouse igg antibody is used to prepare control line C line 7, comprises the steps:
(A) prednisone acetate derivant and the pure albumen OVA of carrier bovine serum albumin(BSA) BSA/ ovum gallinaceum are carried out coupling and prepare artificial antigen;
A, generation water-soluble carbodiimide (EDC): in reaction bulb, add dry dimethyl formamide (DMF) and that non-class raw material or draw non-class raw material, and logical N
2The protection, at room temperature stir, treat that dimethyl formamide (DMF) dissolves fully after; take by weighing concentration fast and be 60% sodium hydride (NaH), and it is added in the described reaction bulb, stirred 50-70 minute; not having bubble until solution produces; draw bromoacetate with syringe then and slowly inject described reaction bulb, after injection finishes, at room temperature stirred 11-13 hour; to disperse in the reactant liquor impouring water afterwards; extract with chloroform, merge extract, with anhydrous Na
2SO
4Carry out drying, filter afterwards, obtain solid through behind the concentrating under reduced pressure again, add the described solid of absolute ether making beating washing, filter afterwards, last vacuum drying obtains new solid, and this solid is an alkylates; The alkylates of absolute ethyl alcohol and above-mentioned steps gained is added in the new reaction bulb, carry out dispersed with stirring, add NaOH solution again, heating reflux reaction 2 hours, after stopping to heat cooling slightly, reactant liquor is changed in the single port bottle, concentrating under reduced pressure gets pastel, add entry again, be adjusted to the liquid that the pH value is 7-9 with hydrochloric acid, extract, merge extract with chloroform, again once, with anhydrous Na with the saturated common salt washing
2SO
4Drying is filtered, and concentrating under reduced pressure gets white solid, uses recrystallizing methanol, gets white powdery solid, and this solid is water-soluble carbodiimide (EDC);
B, generate artificial antigen: with the water-soluble carbodiimide (EDC) of gained in the above-mentioned steps, N-hydroxy-succinamide (NHS), dimethyl formamide (DMF), hapten compound balance to room temperature; Get water-soluble carbodiimide, N-hydroxy-succinamide and the hapten compound of gained in the above-mentioned steps, three's mol ratio is: hapten compound: water-soluble carbodiimide: N-hydroxy-succinamide=1:8-12:13-16, it is inserted round-bottomed flask, the dimethyl formamide dissolving that adds gained in the above-mentioned steps then, for guaranteeing to react completely, stir under the condition of room temperature lucifuge, the solution of Sheng Chenging is designated as reactant liquor No. two at last; After described water-soluble carbodiimide, N-hydroxy-succinamide and hapten compound react completely in dimethyl formamide, existing prestowage body protein solution (BSA/OVA), carrier protein and borate buffer are dissolved in borate buffer in another container by the amount of 1:0.15-0.2, change over to then in another round-bottomed flask, this solution is designated as reactant liquor No. three; No. two reactant liquors are splashed in No. three reactant liquors by per 10 seconds speed of 1, after No. two reactant liquor is added, under the condition of room temperature lucifuge, stir earlier, lucifuge stirs under 3-5 ℃ of condition afterwards, after question response is complete, generate solution, and the solution that generates is designated as reactant liquor No. four, carrier protein and hapten compound ratio are in No. four reactant liquors: 1:15-25; Deng institute respond finish after, place the 20000-30000 molecule to hold back bag filter No. four reactant liquors, the 0.01-0.02M phosphate buffer also is housed in the bag filter, the pH value is 7.0-7.5, bag filter is dialysed under 3-5 ℃ condition, changed one time phosphate buffer every 3-5 hour; Through the product of the final gained of above-mentioned dialysis step, be white cotton-shaped product through freeze drying, be artificial antigen.
(B) with the immune according to a conventional method cell strain of monoclonal antibody for preparing the prednisone acetate derivant of artificial antigen OVA, prepare antibody ascites with the Babc mouse immune, the ascites purifying is obtained highly purified monoclonal antibody protein, measure protein concentration, be equipped with colloid gold label and use;
(C) gold chloride reduction is made the colloidal gold solution of 20nm-40nm with the trisodium citrate reductive agent;
(D) use 0.1mol/LK
2CO
3Regulating colloidal gold solution to optimum PH is 8.5-9.0, behind the monoclonal antibody pure water dialysis 4hr with an amount of purifying, add in the colloid basin, room temperature electromagnetic agitation 15-30min, dropwise add 1% network protein solution, making network concentration is 0.1%, continue to stir 5min, 4 ℃ of centrifugal 10min of following 15000rpm, supernatant discarded is removed sediment, sediment is returned to the original volume mixing again with resuspended liquid, once centrifugal once more, it is standby with resuspended solution sediment to be suspended to 1/20,4 ℃ of preservation of original volume, promptly obtain the colloid gold label thing of anti-prednisone acetate derivant monoclonal antibody, resuspended liquid is the 0.02Tris-HCL damping fluid that contains 5% marine alga;
(E) the prednisone acetate derivant colloid of 1:20-1:10 dilution is advanced the labelled antibody spraying and be adsorbed in the dacron felt 27 ℃ of heat preservation and drynesses, preparation prednisone acetate derivant gold-marking binding pad 3;
(F) the carrier bovine serum albumin(BSA) BSA solution bag of using the coupling of prednisone acetate derivant on coated film 4 is vertically arranged in parallel by 7, two lines of orthoscopic control line C line with sheep anti-mouse igg solution bag by orthoscopic detection line T line 6;
(G) assembling of test strips: sample pad 2, gold mark combination mat 3, coated film 4 and adsorptive pads 5 are sticked on the liner plate 1 successively, sample pad 2 places an end of liner plate 1, adsorptive pads 5 places the other end of liner plate 1, coated film 4 places the centre of liner plate 1, gold-marking binding pad 3 places between sample pad 2 and the coated film 4, promptly obtain being used for the big plate of prednisone acetate derivant colloidal gold chromatographic detection ph test paper ph of immune detection, big plate is cut into the test strips of 2.5mm-7.0mm width specifications by customer requirement, and hermetically drying is preserved.
The detection reaction principle of prednisone acetate colloid gold chromatographic test paper strip:
Prednisone acetate belongs to small-molecule substance.The present invention adopts competition law, and promptly the prednisone acetate in the sample competes the anti-prednisone acetate monoclonal antibody that colloid advances mark with the envelope antigen M2-BSA that is fixed on the coated film 4.After test strips is with sample pad 2 terminal immersion samples, sample solution passes through capillary action swimming from the bottom up along test strips, dissolve gold mark pad is gone up dry golden labeling antibody, if do not have medicine to be measured in the testing sample, immune response can direct swimming take place to the M2-BSA on detection line and the nitrocellulose membrane in then golden labeling antibody, thereby colloid gold particle is assembled, form red lines, other unconjugated golden labeling antibodies continue by capillarity swimming forward then, with the sheep anti mouse two anti-generation immune responses for the second time on the control line, the red lines of same formation just have two red lines like this on the coated film 4, the expression sample is negative.If have medicine to be measured in the testing sample, then golden labeling antibody at first can with the detection thing generation immune response in the sample, when the golden labeling antibody that does not react has residue, can with M2-BSA immune response take place just on detection line, form red lines, the lines intensity when its color intensity is weaker than feminine gender; And when golden labeling antibody all with sample in prednisone acetate take place immunity in conjunction with the time, just do not have again antibody to combine, thereby detection line does not just have red lines appearance with the control line coating antigen.Control line is that for whether check gold-marking immunity chromatography method itself exists medicine to be measured control line all should manifest.If control line does not develop the color, then illustrate what test paper was not effectively set, so be that bar lost efficacy in the sample no matter.
High specificity of the present invention, the susceptibility height detects minimum limiting the quantity of and can reach 5ppb; Easy, quick, ageing strong, need not any other reagent and instrument, but execute-in-place, test strips was the decidable testing result in 15 minutes after adding test sample liquid; The result shows image, directly perceived, accurate; Cost saving, applied widely, be convenient to promote.
Claims (1)
1. the preparation method of the colloid gold chromatographic test paper strip of fast detecting prednisone acetate and derivant thereof, it is characterized in that: colloid gold chromatographic test paper strip is by liner plate (1) and the sample pad (2) that is connected successively on liner plate (1), gold-marking binding pad (3), coated film (4) and adsorptive pads (5) are formed, gold-marking binding pad (3) is the dacron felt of the golden labeling antibody of absorption prednisone acetate derivant, go up the carrier protein solution bag of useful prednisone acetate derivant coupling by orthoscopic detection line T line (6) at coated film (4), with with sheep anti-mouse igg solution bag by orthoscopic control line C line (7), article two, line is vertically arranged in parallel, the PVC toughness material of described liner plate (1) for not absorbing water, described coated film (4) is a nitrocellulose filter, or cellulose mixture film, described adsorptive pads (5) is the vegetable fibre cotton paper, the golden labeling antibody of described prednisone acetate derivant is the monoclonal anti nanocrystal composition of the anti-prednisone acetate derivant of colloid gold label, and the carrier protein of described coupling prednisone acetate derivant is bovine serum albumin(BSA) BSA;
The carrier bovine serum albumin(BSA) BSA of preparation coupling prednisone acetate derivant is used to prepare relevant detection line T line (6); The pure albumen of carrier ovum gallinaceum of preparation coupling prednisone acetate derivant is used to prepare monoclonal antibody, and preparation prednisone acetate derivant gold labeling antibody is used to prepare the dacron felt that adsorbs prednisone acetate derivant gold labeling antibody; The preparation sheep anti-mouse igg antibody is used to prepare control line C line (7), comprises the steps:
(A) prednisone acetate derivant and the pure albumen OVA of carrier bovine serum albumin(BSA) BSA/ ovum gallinaceum are carried out coupling and prepare artificial antigen;
A, generation water-soluble carbodiimide (EDC): in reaction bulb, add dry dimethyl formamide (DMF) and that non-class raw material or draw non-class raw material, and logical N
2The protection, at room temperature stir, treat that dimethyl formamide (DMF) dissolves fully after; take by weighing concentration fast and be 60% sodium hydride (NaH), and it is added in the described reaction bulb, stirred 50-70 minute; not having bubble until solution produces; draw bromoacetate with syringe then and slowly inject described reaction bulb, after injection finishes, at room temperature stirred 11-13 hour; to disperse in the reactant liquor impouring water afterwards; extract with chloroform, merge extract, with anhydrous Na
2SO
4Carry out drying, filter afterwards, obtain solid through behind the concentrating under reduced pressure again, add the described solid of absolute ether making beating washing, filter afterwards, last vacuum drying obtains new solid, and this solid is an alkylates; The alkylates of absolute ethyl alcohol and above-mentioned steps gained is added in the new reaction bulb, carry out dispersed with stirring, add NaOH solution again, heating reflux reaction 2 hours, after stopping to heat cooling slightly, reactant liquor is changed in the single port bottle, concentrating under reduced pressure gets pastel, add entry again, be adjusted to the liquid that the pH value is 7-9 with hydrochloric acid, extract, merge extract with chloroform, again once, with anhydrous Na with the saturated common salt washing
2SO
4Drying is filtered, and concentrating under reduced pressure gets white solid, uses recrystallizing methanol, gets white powdery solid, and this solid is water-soluble carbodiimide (EDC);
B, generate artificial antigen: with the water-soluble carbodiimide (EDC) of gained in the above-mentioned steps, N-hydroxy-succinamide (NHS), dimethyl formamide (DMF), hapten compound balance to room temperature; Get water-soluble carbodiimide, N-hydroxy-succinamide and the hapten compound of gained in the above-mentioned steps, three's mol ratio is: hapten compound: water-soluble carbodiimide: N-hydroxy-succinamide=1:8-12:13-16, it is inserted round-bottomed flask, the dimethyl formamide dissolving that adds gained in the above-mentioned steps then, for guaranteeing to react completely, stir under the condition of room temperature lucifuge, the solution of Sheng Chenging is designated as reactant liquor No. two at last; After described water-soluble carbodiimide, N-hydroxy-succinamide and hapten compound react completely in dimethyl formamide, existing prestowage body protein solution B SA/OVA, carrier protein and borate buffer are dissolved in borate buffer in another container by the amount of 1:0.15-0.2, change over to then in another round-bottomed flask, this solution is designated as reactant liquor No. three; No. two reactant liquors are splashed in No. three reactant liquors by per 10 seconds speed of 1, after No. two reactant liquor is added, under the condition of room temperature lucifuge, stir earlier, lucifuge stirs under 3-5 ℃ of condition afterwards, after question response is complete, generate solution, and the solution that generates is designated as reactant liquor No. four, carrier protein and hapten compound ratio are in No. four reactant liquors: 1:15-25; Deng institute respond finish after, place the 20000-30000 molecule to hold back bag filter No. four reactant liquors, the 0.01-0.02M phosphate buffer also is housed in the bag filter, the pH value is 7.0-7.5, bag filter is dialysed under 3-5 ℃ condition, changed one time phosphate buffer every 3-5 hour; Through the product of the final gained of above-mentioned dialysis step, be white cotton-shaped product through freeze drying, be artificial antigen;
(B) with the immune according to a conventional method cell strain of monoclonal antibody for preparing the prednisone acetate derivant of artificial antigen OVA, prepare antibody ascites with the Babc mouse immune, the ascites purifying is obtained highly purified monoclonal antibody protein, measure protein concentration, be equipped with colloid gold label and use;
(C) gold chloride reduction is made the colloidal gold solution of 20nm-40nm with the trisodium citrate reductive agent;
(D) regulating colloidal gold solution to optimum PH with 0.1mol/LK2CO3 is 8.5-9.0, behind the monoclonal antibody pure water dialysis 4hr with an amount of purifying, add in the colloid basin, room temperature electromagnetic agitation 15-30min, dropwise add 1% network protein solution, making network concentration is 0.1%, continue to stir 5min, 4 ℃ of centrifugal 10min of following 15000rpm, supernatant discarded is removed sediment, sediment is returned to the original volume mixing again with resuspended liquid, once centrifugal once more, it is standby with resuspended solution sediment to be suspended to 1/20,4 ℃ of preservation of original volume, promptly obtain the colloid gold label thing of anti-prednisone acetate derivant monoclonal antibody, described resuspended liquid is the 0.02 mol Tris-HCl damping fluid that contains 5% marine alga;
(E) the prednisone acetate derivant colloid of 1:20-1:10 dilution is advanced the labelled antibody spraying and be adsorbed in the dacron felt 27 ℃ of heat preservation and drynesses, preparation prednisone acetate derivant gold-marking binding pad (3);
(F) the carrier bovine serum albumin(BSA) BSA solution bag of using the coupling of prednisone acetate derivant on coated film (4) is by orthoscopic detection line T line (6), and by orthoscopic control line C line (7), two lines are vertically arranged in parallel with sheep anti-mouse igg solution bag;
(G) assembling of test strips: with sample pad (2), gold mark combination mat (3), coated film (4) and adsorptive pads (5) stick on the liner plate (1) successively, sample pad (2) places an end of liner plate (1), adsorptive pads (5) places the other end of liner plate (1), coated film (4) places the centre of liner plate (1), gold-marking binding pad (3) places between sample pad (2) and the coated film (4), promptly obtain being used for the big plate of prednisone acetate derivant colloidal gold chromatographic detection ph test paper ph of immune detection, big plate is cut into the test strips of 2.5mm-7.0mm width specifications by customer requirement, and hermetically drying is preserved.
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| CN1807601A (en) * | 2006-01-09 | 2006-07-26 | 华中农业大学 | Immune colloid gold test paper strip for detecting sulfadiazine residue and its preparation method |
| CN1907953A (en) * | 2005-08-04 | 2007-02-07 | 曾立波 | Monoclonal antibody for ketamine detection and immune detection board |
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| CN1907953A (en) * | 2005-08-04 | 2007-02-07 | 曾立波 | Monoclonal antibody for ketamine detection and immune detection board |
| CN1807601A (en) * | 2006-01-09 | 2006-07-26 | 华中农业大学 | Immune colloid gold test paper strip for detecting sulfadiazine residue and its preparation method |
| CN101666799A (en) * | 2009-07-23 | 2010-03-10 | 江苏省农业科学院 | Test strip for detecting colibacillus O157:H7 mouse serum antibody by colloidal gold immunochromatography |
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Address after: 226000 Jiangsu Province, Nantong City Lake Economic and Technological Development Zone, Road No. 1692 No. 15 building block A Patentee after: NANTONG EGENS BIOTECHNOLOGY CO., LTD. Address before: 226000 Jiangsu Province, Nantong City Lake Economic and Technological Development Zone, Road No. 1692 No. 15 building block A Patentee before: Nantong Egens Biotechnology Co., Ltd. |
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