CN100445746C - Method for detecting 19-nortestosterone and special enzyme-linked immune reagent kit thereof - Google Patents
Method for detecting 19-nortestosterone and special enzyme-linked immune reagent kit thereof Download PDFInfo
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- CN100445746C CN100445746C CNB2006100072876A CN200610007287A CN100445746C CN 100445746 C CN100445746 C CN 100445746C CN B2006100072876 A CNB2006100072876 A CN B2006100072876A CN 200610007287 A CN200610007287 A CN 200610007287A CN 100445746 C CN100445746 C CN 100445746C
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Abstract
The present invention discloses a method for detecting 19-nortestosterone and a special enzyme-linked immunity reagent box thereof. The enzyme-linked immunity reagent box of the detected 19-nortestosterone comprises a 19-nortestosterone specificity antibody, a coating source and an enzyme-labeled article, wherein the coating source is a coupling article or an anti-antibody of a 19-nortestosterone half anti-source and carrier protein, and the enzyme-labeled article is an enzyme-labeled anti-antibody or an enzyme-labeled 19-nortestosterone half anti-source; when the coating source is the coupling article of the 19-nortestosterone half anti-source and the carrier protein, the enzyme-labeled article is the enzyme-labeled anti-antibody; when the coating source is the anti-antibody, the enzyme-labeled article is the enzyme-labeled 19-nortestosterone half anti-source. The method of the present invention has the advantages of easy operation, low price, high sensitivity and on-site monitoring; furthermore, the present invention is suitable for detecting the residual quantity of 19-nortestosterone medicines in animal tissues and urine specimens in a large-amount sample filtration way.
Description
Technical field
The present invention relates to a kind of method and special ELISA reagent kit thereof of the 19-of detection nortestosterone.
Background technology
(19-nortestosterone is a kind of synthetic hormone NT) to the 19-nortestosterone, is to have androgenic effect, belongs to anabolism class steroids.The clinical treatment anaemia senile osteoporosis that is mainly used in can also promote growing of muscle, increases training endurance and training load.But there is also very serious adverse, the parent absorption can make female child manlike, causes the genitality deformity.The women takes for a long time and can produce manlike feature, the male sex take for a long time can cause bald too early.The short-term large dose oral administration can cause hepatosis, also may cause cancer, diabetes, amentia etc.Therefore, the detection of strengthening this medicine is very necessary.
The chemical method that detects 19-nortestosterone residual quantity mainly contains thin-layered chromatography (TLC), vapor-phase chromatography (GC), high performance liquid chromatography (HPLC), gas-matter online (GC/MS), liquid-matter online (HPLC/MS), Capillary Electrophoresis (CE) etc., because required instrument and equipment is complicated and process is loaded down with trivial details, is not suitable for on-site supervision and great amount of samples examination.
Summary of the invention
The method and the special ELISA reagent kit thereof that the purpose of this invention is to provide a kind of 19-of detection nortestosterone.
The enzyme linked immunological kit of detection 19-nortestosterone provided by the present invention comprises 19-nortestosterone specific antibody and coating antigen and enzyme labeling thing; Described coating antigen is the conjugate or the antiantibody of 19-nortestosterone haptens and carrier protein; Described enzyme labeling thing is enzyme mark antiantibody or enzyme mark 19-nortestosterone haptens; When described coating antigen was the conjugate of 19-nortestosterone haptens and carrier protein, described enzyme labeling thing was an enzyme mark antiantibody; When described coating antigen was antiantibody, described enzyme labeling thing was an enzyme mark 19-nortestosterone haptens.
The conjugate of described 19-nortestosterone haptens and carrier protein can obtain by 19-nortestosterone haptens and carrier protein are carried out coupling with mixed anhydride method or active ester method or water-soluble carbodiimide method (EDC); Described 19-nortestosterone haptens obtains 19-nortestosterone and succinic anhydride by acylation reaction; Described carrier protein can be ovalbumin (OVA), albumin rabbit serum (RSA) or mouse serum albumin (MSA) etc.
The marker enzyme of described enzyme labeling thing is horseradish peroxidase or alkaline phosphatase, wherein preferred alkaline phosphatase; Alkaline phosphate ester enzyme labeling antiantibody can adopt several different methods of the prior art such as glutaraldehyde method or sodium periodate method that enzyme is crosslinked on antiantibody; The 19-nortestosterone haptens of alkaline phosphate ester enzyme labeling can adopt mixed anhydride method that alkaline phosphatase and 19-nortestosterone hapten conjugation are obtained.Described 19-nortestosterone haptens obtains 19-nortestosterone and succinic anhydride by acylation reaction.
Described 19-nortestosterone specific antibody can be 19-nortestosterone monoclonal antibody or 19-nortestosterone polyclonal antibody; They all are that conjugate with 19-nortestosterone haptens and carrier protein obtains as immunogene; Polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, and described 19-nortestosterone monoclonal antibody is a 19-nortestosterone mouse monoclonal antibody, and described 19-nortestosterone polyclonal antibody is preferably 19-nortestosterone rabbit polyclonal antibody.Described antiantibody is sheep anti mouse or goat-anti rabbit antiantibody, is preferably goat-anti rabbit antiantibody.
Described 19-nortestosterone mouse monoclonal antibody is preferably the antibody of the monoclonal hybridoma strain A-3-3 CGMCC No.1613 secretion of 19-nortestosterone.
The monoclonal hybridoma strain A-3-3 CGMCC No.1613 of 19-nortestosterone has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on February 9th, 2006.
Above antibody all can prepare as immunogene according to a conventional method with the conjugate of 19-nortestosterone haptens and carrier protein.Described carrier protein can be common carrier albumen such as mouse haemocyanin, bovine serum albumin(BSA), rabbit anteserum albumen, human serum albumins, ovalbumin or hemocyanin; The conjugate of described 19-nortestosterone haptens and carrier protein can obtain by 19-nortestosterone haptens and carrier protein are carried out coupling with active ester method or mixed anhydride method; Described 19-nortestosterone haptens obtains 19-nortestosterone and succinic anhydride by acylation reaction.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises 19-nortestosterone standard solution, developer, stop buffer, concentrated cleaning solution, concentrates redissolution liquid.
Described concentrated cleaning solution is 0.01M～0.05M, contain 0.01% sodium azide (Na
3N) and the phosphate buffer of 1% Tween 80; Described percentage composition is the quality percentage composition.
The Sodium azide that contains in the concentrated cleaning solution of kit of the present invention suppresses the growth of bacterium in solution, to the stability of solution its to a protective effect.
When marker enzyme was horseradish peroxidase, described developer was made up of colour developing liquid A liquid and colour developing liquid B liquid, and described colour developing liquid A liquid is hydrogen peroxide or urea peroxide, and described colour developing liquid B liquid is o-phenylenediamine or tetramethyl benzidine; When marker enzyme was alkaline phosphatase, described developer was a 4-nitrophenols phosphate buffer.
Described concentrated redissolution liquid can be the phosphate buffer that contains 0.5% bovine serum albumin(BSA) and 0.1% polysorbas20,0.01～0.05M; Described percentage composition is the quality percentage composition.。
It is pH9.6 that described bag is cushioned liquid, the carbonate buffer solution of 0.05mol/L;
Described confining liquid is the 0.01～0.05M phosphate buffer that contains 5% skimmed milk power; Described percentage composition is the quality percentage composition.；
Described stop buffer is sulfuric acid, hydrochloric acid or the sodium hydrate buffer solution of 1～2mol/L.
The method of detection 19-nortestosterone provided by the present invention may further comprise the steps:
1) sample pre-treatments
When sample is animal tissue or feed, with homogenizer homogeneous animal tissue's sample or feed, taking by weighing 2g sample and 0.5g ascorbic acid joins in the mixed liquor of 8ml acetonitrile and 2ml 1mol/L NaOH, the vibration mixing, more than the room temperature 3000g, 15 ℃, centrifugal 10-15 minute, get in the supernatant 1ml dislocation centrifuge tube, add 1ml 1mol/L NaOH and 2ml acetonitrile, mixing, mixed liquor (volume ratio of normal hexane and methylene chloride is 8: 2) the vibration mixing that adds 3ml normal hexane and methylene chloride again, more than the 3000g, 15 ℃, centrifugal 10-15 minute, remove upper strata normal hexane phase, get the middle layer organic phase and flow down bone dry,, can analyze with the above-mentioned dry residue water intaking phase of the concentrated redissolution liquid dissolving with 2 times of deionized water dilutions of 1ml at nitrogen;
When sample is urine, get the 2ml urine in centrifuge tube, more than the 3000g, 15 ℃, centrifugal 10-15 minute until limpid, pipette the 1ml urine in centrifuge tube, the acetonitrile that adds 2ml adds mixed liquor (volume ratio of normal hexane and methylene chloride is 8: the 2) mixing of 3ml normal hexane and methylene chloride, more than the 3000g again, 15 ℃, centrifugal 10-15 minute, remove upper strata normal hexane phase, get the middle layer organic phase and flow down bone dry at nitrogen, with the above-mentioned dry residue of concentrated redissolution liquid dissolving with 2 times of deionized water dilutions of 1ml, water intaking is analyzed mutually.
2) utilize the enzyme linked immunological kit test sample of above-mentioned detection 19-nortestosterone.
The carrier mass that wherein can be used as the conjugate of fixedly 19-nortestosterone haptens and carrier protein or 19-nortestosterone antiantibody is a lot, as polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber, Ago-Gel etc.The form of this carrier can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc.Wherein be preferably the micro-reaction plate shrinkage pool that polystyrene is made, its advantage is: polystyrene has the performance of stronger adsorbed proteins, still keeps original immunocompetence after antigen adsorbs on it, and blank value is low, transparency height at the bottom of the hole is between each plate, performance is close between each hole of same plate.
The enzyme linked immunological kit of detection of the present invention 19-nortestosterone mainly adopts the residual quantity of 19-nortestosterone in the qualitative or samples such as detection by quantitative animal tissue, feed and urine of indirect competitive ELISA method; Pre-treatment requirement to sample is low, and sample pretreatment process is simple, simultaneously the fast detecting gross sample.
19-nortestosterone and succinic anhydride are synthesized haptens by acylation reaction; its advantage is to pick out a spacerarm that contains 4 carbochains to the 19-nortestosterone; given prominence to characteristic group---the testosterone in the 19-nortestosterone molecular structure, made the 19-nortestosterone antibody of preparation very high recognition capability all be arranged 19-nortestosterone immunogene or coating antigen.Adopt mixed anhydride method and carrier protein couplet to obtain immunogene 19-nortestosterone haptens again.It is low or too high all unfavorable to immunity that haptens and the ratio that combines of carrier protein are crossed, and haptens was respectively 12: 1,17: 1 and 17: 1 with the mol ratio that combines of KLH, RSA and HSA.
But the residual quantity of 19-nortestosterone in the samples such as kit qualitative and quantitative analysis of the present invention animal tissue, urine sample.Detection principle of the present invention is when wrapping by the conjugate of 19-nortestosterone haptens and carrier protein on the capillary strip in advance, after adding series standard product or sample solution and 19-nortestosterone antibody working fluid, in the sample on residual 19-nortestosterone and the capillary strip coupled antigen of pre-bag quilt compete the antibody of anti-19-nortestosterone, add the enzyme labeling thing and carry out the enzymatic activity amplification, the colour developing back stops; When bag is by antiantibody on the capillary strip, behind the adding 19-nortestosterone antibody working fluid, add series standard product or sample solution and enzyme mark haptens again, 19-nortestosterone that sample is residual and enzyme mark haptens are competed anti-19-nortestosterone antibody, colour developing; Colour developing stops the back and measures every hole absorbance (OD value) with microplate reader, and the content of sample absorbance and its residue 19-nortestosterone is negative correlation, relatively can draw the content of corresponding residue 19-nortestosterone with typical curve.Also can be according to the depth of the sample solution color on the ELISA Plate, with the 19-nortestosterone titer color of the series concentration concentration range of 19-nortestosterone in the judgement sample relatively.
The enzyme linked immunological kit of detection of the present invention 19-nortestosterone mainly adopts the residual quantity of 19-nortestosterone in the qualitative or samples such as detection by quantitative animal tissue and urine of indirect competitive ELISA method; Pre-treatment requirement to sample is low, and sample pretreatment process is simple, simultaneously the fast detecting gross sample; Adopt the 19-nortestosterone monoclonal antibody of high specific, main agents provides with the form of working fluid, and the method for inspection is convenient and easy, has specificity height, highly sensitive, characteristics such as degree of accuracy is high, accuracy height.Enzyme linked immunological kit of the present invention, simple in structure, easy to use, low price, carrying convenience, detection method is efficient, accurate, easy, be suitable for the qualitative, quantitative of batch samples screening.
Description of drawings
Fig. 1 is the enzyme linked immunological kit 19-nortestosterone canonical plotting of coating antigen for the conjugate with 19-nortestosterone antigen and carrier protein
Fig. 2 is for being the enzyme linked immunological kit 19-nortestosterone canonical plotting of coating antigen with the antiantibody
Embodiment
The method of following embodiment is conventional method if no special instructions.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
With the conjugate of 19-nortestosterone haptens and carrier protein is that the enzyme linked immunological kit of coating antigen comprises:
(1) is coated with the ELISA Plate of 19-nortestosterone and carrier protein couplet thing;
(2) the sheep anti mouse antiantibody working fluid of alkaline phosphate ester enzyme labeling: the sheep anti mouse antiantibody of alkaline phosphate ester enzyme labeling is diluted to the enzyme labeling antiantibody working fluid that protein concentration is 0.1～1 μ g/L, 12ml/ bottle, 1 bottle with dilution.Dilution is for containing 0.1 ‰ glycerine (can prevent that the enzyme labeling thing of putting into-20 ℃ of environment from freezing, also can keep the biologically active of enzyme labeling thing for a long time), 1% calf serum, the solution of 1% thimerosal antiseptic (being convenient to preserve).
(3) 19-nortestosterone standard solution: 6 bottles of 19-nortestosterone series standard solution, 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 3ml/ bottle.For the dilution of standard solution be the dilution of 19-nortestosterone: pH 8.3, the phosphate buffer of 0.03mol/L
(4) colour developing liquid: colour developing liquid is 4-nitrophenols phosphate buffer, the 7ml/ bottle.
(5) 19-nortestosterone mouse monoclonal antibody working fluid: with the pH 7.9 that contains 1%BSA, 0.5mol/L it is that 2.0 μ g/L use that phosphate buffer becomes protein concentration with the antibody dilution of the monoclonal hybridoma strain A-3-3 CGMCC No.1613 of 19-nortestosterone secretion, the 12ml/ bottle, 1 bottle.
(6) concentrated cleaning solution: 0.01M～0.05M, contain 0.1 ‰ (mass concentration) sodium azide (Na
3N) and the phosphate buffer of 1% (mass concentration) Tween 80.The 40ml/ bottle, 1 bottle.Be 20 times of normal working concentration.
(7) stop buffer: 2mol/L NaOH, 8ml/ bottle, 1 bottle.
(8) concentrate redissolution liquid: contain the phosphate buffer of 0.5% bovine serum albumin(BSA) and 1 ‰ polysorbas20s, 0.01～0.05M, be divided into the 50ml/ bottle, 1 bottle.Be 3 times of normal working concentration.
(9) bag is cushioned liquid: pH9.6, the carbonate buffer solution of 0.05mol/L.
(10) confining liquid: 0.01 phosphate buffer that contains 5% skimmed milk power.
Wherein, the preparation method of the sheep anti mouse antiantibody of the antibody of the monoclonal hybridoma strain A-3-3 CGMCC No.1613 of 19-nortestosterone haptens and carrier protein couplet thing, 19-nortestosterone secretion, alkaline phosphate ester enzyme labeling is as follows:
One, the preparation of ELISA Plate
1, the haptenic synthetic method of 19-nortestosterone:
Haptenic synthetic: that 19-nortestosterone and succinic anhydride are synthesized haptens by acylation reaction.Its advantage is to pick out a spacerarm that contains 4 carbochains to the 19-nortestosterone, given prominence to characteristic group--the testosterone in the 19-nortestosterone molecular structure, made the 19-nortestosterone antibody of preparation very high recognition capability all be arranged 19-nortestosterone immunogene or coating antigen.
2, coating antigen: adopt water-soluble carbodiimide method (EDC) to carry out coupling 19-nortestosterone haptens and albumin rabbit serum (RSA) and obtain coating antigen.
The concrete preparation method of coating antigen is as follows:
(1) gets 19-nortestosterone haptens 2g and be dissolved in 20ml, in the sodium hydroxide solution of 0.5M;
(2) getting the 1.5g carbodiimides again is dissolved in the 5ml pure water and was added in the solution of step (1) the stirring at room reaction 2 hours;
(3) get albumin rabbit serum 20g and be dissolved in the carbonate buffer solution of 75ml pH9.6 and be added drop-wise in the solution of step (2) 4 ℃ of stirrings and spend the night, the phosphate buffer dialysis of 0.1M 7 days is changed liquid every day 3～4 times, at last antigen is concentrated or freeze-drying is preserved.
3, the preparation of ELISA Plate:
Be cushioned liquid with bag 19-nortestosterone and albumin rabbit serum conjugate are diluted to 1 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h or 4 ℃ spend the night, and the coating buffer that inclines is with cleansing solution (concentrated cleaning solution with 19 times of deionized water dilutions) washing 3 times, each 30 seconds, pat dry, in every hole, add 200 μ l confining liquids, 37 ℃ of incubation 1-2h then, the liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
Two, the preparation of 19-nortestosterone mouse monoclonal antibody
1, immunogene: adopt mixed anhydride method to carry out coupling 19-nortestosterone haptens and hemocyanin (KLH) and obtain immunogene.
Immunogenic preparation method: 1) get 19-nortestosterone haptens 2g and be dissolved in 30ml, 50% N is in the dinethylformamide solution; 2) getting the 0.5ml isobutyl chlorocarbonate again is dissolved in the no Shui diox of 5ml and is added in the step 1) solution stirring at room reaction 4 hours; 3) get in the carbonate buffer solution that hemocyanin 32g is dissolved in 70ml pH9.6 and be added to step 2) in the solution 4 ℃ of stirrings spend the night, dialysed 7 days at the phosphate buffer of 0.2M then, change liquid every day 3～4 times, at last enzyme labeling haptens antigen is concentrated or the freeze-drying preservation.
2,19-nortestosterone mouse monoclonal antibody preparation
It is immune animal that the animal immune program adopts the Balb/c mouse, 19-nortestosterone haptens and hemocyanin conjugate are immunogene, immunizing dose is 80-100 μ g/, Freund's complete adjuvant with immunogenic and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, At intervals of two to three weeks are got the same dose immunogene and are added equivalent incomplete Freund mixing and emulsifying, and booster immunization once, four exempt from the pneumoretroperitoneum booster immunization once, extracting spleen cell after 3 days.
The splenocyte of immune Balb/c mouse is got in Fusion of Cells and cloning, merges in 10: 1 ratios and SP2/0 myeloma cell, adopts indirect competitive ELISA to measure cell conditioned medium liquid, screens positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, until the monoclonal antibody of the monoclonal hybridoma strain A-3-3 of the hybridoma cell strain that obtains the stably excreting monoclonal antibody-19-nortestosterone CGMCC No.1613 secretion.
Cell cryopreservation and recovery are got the hybridoma that is in exponential phase and are made 1 * 10 with cryopreserving liquid
6～5 * 10
6The cell suspension of individual/mL is sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying adopt in the body and induce method, and Balb/c mouse (8 age in week) abdominal cavity is only injected sterilization paraffin oil 0.5ml/, the monoclonal hybridoma strain A-3-3 CGMCC No.1613 5 * 10 of 14 days pneumoretroperitoneum injection 19-nortestosterones
6Individual/as only, to gather ascites after 10 days.Carry out the ascites purifying through sad saturated ammonium sulfate method, bottle packing ,-20 ℃ of preservations.
Three, the preparation of enzyme mark antiantibody
With mouse source antibody is that immunogene is carried out immunity to the pathogen-free domestic goat, obtains the sheep anti mouse antiantibody.
Sheep anti mouse antiantibody and alkaline phosphatase adopt glutaraldehyde method to carry out coupling, obtain the antiantibody of alkaline phosphate ester enzyme labeling.
Sheep anti mouse antiantibody and alkaline phosphatase are carried out coupling, the preferred glutaraldehyde method of method that adopts, with ratio and the goat-anti rabbit antiantibody coupling of alkaline phosphatase with 2: 1,60%～70% enzyme and 8% antiantibody coupling are arranged, the rate ratio of enzyme labeling thing uses the horseradish peroxidase height.
Enzyme mark sheep anti mouse antiantibody concrete steps are as follows:
1) takes by weighing alkaline phosphatase 25mg and be dissolved in 1.25% glutaraldehyde solution, in the room temperature standing over night.
2) reacted enzyme solutions is used the physiological saline wash-out through Sephadex G-25 chromatographic column.Flow speed control is collected brown effluent at 1ml/1min.Greater than 5ml, then be concentrated into 5ml as volume with poly-hexanediol.Place in the 25ml small beaker, slowly stir.
3) get sheep anti mouse antiantibody 12.5mg and be diluted to 5ml, dropwise add in the enzyme solutions under stirring with physiological saline.
4) with 1M pH9.5 carbonic acid buffer 0.25ml, continue to stir 3h.
5) add 0.2M lysine 0.25ml, behind the mixing, put room temperature 2h.
6) under agitation dropwise add the equal-volume saturated ammonium sulfate, put 4 ℃ of 1h.
7) 3000rpm centrifugal half an hour, abandon supernatant.Sediment is washed secondary with semi-saturation ammonium sulfate, and last sediment is dissolved in the phosphate buffer of a small amount of 0.15M pH7.4.
8) above-mentioned solution is packed in the bag filter,, remove (detecting) behind the ammonium ion with Nai Shi reagent with the phosphate buffer dialysis of 0.15M pH7.4,10, the centrifugal 30min of 000rpm removes precipitation, and supernatant is enzyme conjugates, after the packing, stored frozen.
Utilize the method for 19-nortestosterone residual in this kit test sample as follows:
One, sample pre-treatments
A, animal tissue and feed:
With homogenizer homogeneous animal tissue's sample or feed, taking by weighing 2g sample and 0.5g ascorbic acid joins in the mixed liquor of 8ml acetonitrile and 2ml NaOH (1mol/L), vibration 10min, more than the 3000g, 15 ℃, centrifugal 10min gets in the supernatant 1ml dislocation centrifuge tube, add 1ml NaOH (1mol/L) and 2ml acetonitrile, vibration 1min adds mixed liquor (volume ratio of normal hexane and methylene chloride is 8: 2) the vibration 10min of 3ml normal hexane and methylene chloride, more than the 3000g, 15 ℃, centrifugal 10min removes upper strata normal hexane phase, get the middle layer organic phase and flow down bone dry with the residue water intaking phase of 1ml, get 50 μ l and analyze with the concentrated redissolution liquid dissolving drying of 2 times of deionized water dilutions at nitrogen;
B, urine:
Get the 2ml urine in centrifuge tube, more than the 3000g, 15 ℃, centrifugal 10min, until limpid, pipette the 1ml urine in centrifuge tube, add the 2ml acetonitrile, vibration 1min, mixed liquor (volume ratio of normal hexane and methylene chloride is 8: 2) the vibration 10min of adding 3ml normal hexane and methylene chloride, more than the 3000g, 15 ℃, centrifugal 10min removes upper strata normal hexane phase, gets the middle layer organic phase and flows down bone dry at nitrogen, with the concentrated redissolution liquid dissolving dry residue of 1ml, get 50 μ l waters and analyze with 2 times of deionized water dilutions.
Two, detection method
A, in the ELISA Plate micropore of 19-nortestosterone and albumin rabbit serum conjugate bag quilt, add series standard product solution or sample solution (each 2 hole) 50 μ l, add 19-nortestosterone mouse monoclonal antibody working fluid 50 μ l then, with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens.
B, pour out liquid in the hole, every hole adds 250 μ l cleansing solutions, pours out liquid in the hole after 30 seconds, and so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.The sheep anti mouse antiantibody working fluid 100 μ l that add the alkaline phosphate ester enzyme labeling with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens.
C, taking-up ELISA Plate are washed plate 5 times as described above.Every hole adds substrate colour developing liquid 100 μ l, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 15-30min.
D, every hole add stop buffer 50 μ l, and the mixing that vibrates gently is determined at 400nm wavelength place with microplate reader with wavelength coverage, measures every hole absorbance (OD value).
Three, interpretation of result
Each the concentration standard solution that is obtained or the mean value (B) of sample absorbance are divided by the absorbance (B of first standard (0 standard)
0) multiply by 100% again, i.e. percentage absorbance.
B is the mean light absorbency value of standard solution or sample solution in the formula, B
0It is the mean light absorbency value of 0 μ g/L standard solution.Natural logarithm value with 19-nortestosterone concentration is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard curve map, as shown in Figure 1.The concentration of 19-nortestosterone can be read from typical curve in corresponding each sample.Also can use regression equation method, calculate the concentration of 19-nortestosterone in the sample solution.Utilize computer professional software, the express-analysis of a large amount of samples of being more convenient for.Whole testing process only needed just can finish in 1.5 hours, and lowest detection is limited to 0.1 μ g/L.
Embodiment 2, with goat-anti rabbit antiantibody as enzyme linked immunological kit of coating antigen and preparation method thereof
Comprise with the enzyme linked immunological kit of goat-anti rabbit antiantibody as coating antigen:
(1) is coated with the ELISA Plate of goat-anti rabbit antiantibody;
(2) the 19-nortestosterone haptens working fluid of alkaline phosphate ester enzyme labeling: with the 19-nortestosterone haptens dilution of alkaline phosphate ester enzyme labeling is the working fluid of 0.1～1 μ g/L, 12ml/ bottle, 1 bottle.Used dilution is for containing the solution of 0.1 ‰ glycerine (can prevent that the enzyme labeling thing of putting into-20 ℃ of environment from freezing, also can keep the biologically active of enzyme labeling thing for a long time), 1% thimerosal antiseptic (being convenient to preserve).
(3) 19-nortestosterone standard solution: 6 bottles of 19-nortestosterone series standard product solution, 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 3ml/ bottle.For the dilution of standard solution be the dilution of 19-nortestosterone: the pH value is 8.3, the phosphate buffer of 0.03mol/L.
(4) colour developing liquid: colour developing liquid is 4-nitrophenols phosphate buffer, the 7ml/ bottle.
(5) 19-nortestosterone rabbit polyclonal antibody working fluid: with the pH 7.9 that contains 1%BSA, it is that 2.0 μ g/L use 12ml/ bottle, 1 bottle that the 0.5mol/L phosphate buffer becomes protein concentration with antibody dilution.
(6) concentrated cleaning solution: 0.01M～0.05M, contain 0.1 ‰ (mass concentration) sodium azide (Na
3N) and the phosphate buffer of 1% (mass concentration) Tween 80.The 40ml/ bottle, 1 bottle.Be 20 times of normal working concentration.
(7) stop buffer: 2mol/L NaOH, 8ml/ bottle, 1 bottle.
(8) concentrate redissolution liquid: contain the phosphate buffer of 0.5% bovine serum albumin(BSA) and 1 ‰ polysorbas20s, 0.01～0.05M, be divided into the 50ml/ bottle, 1 bottle.Be 3 times of normal working concentration.
(9) bag is cushioned liquid: pH9.6, the carbonate buffer solution of 0.05mol/L.
(10) confining liquid: the potpourri with ratio mixing in 1: 5 of phosphate buffer and 5% skimmed milk power.
Wherein, the haptenic preparation method of 19-nortestosterone of goat-anti rabbit antiantibody coating antigen, 19-nortestosterone rabbit polyclonal antibody, alkaline phosphate ester enzyme labeling is as follows:
One, the preparation of ELISA Plate
1, the preparation of coating antigen: with rabbit source antibody is that immunogene is carried out immunity to the pathogen-free domestic goat, obtains goat-anti rabbit antiantibody.
2, be coated with the ELISA Plate preparation method of goat-anti rabbit antiantibody: be cushioned liquid with bag goat-anti rabbit antiantibody is diluted to 1 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h or 4 ℃ spend the night, and the coating buffer that inclines is with cleansing solution (concentrated cleaning solution with 19 times of deionized water dilutions) washing 3 times, each 30 seconds, pat dry, in every hole, add 200 μ l confining liquids, 37 ℃ of incubation 1-2h then, the liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
Two, the preparation of 19-nortestosterone rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, with immunogene (19-nortestosterone haptens with and the conjugate of hemocyanin) immunizing dose is 1mg/kg, Fu Shi Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, getting the same dose immunogene 3～4 weeks adds equivalent freund 's incomplete adjuvant mixing and emulsifying at interval, booster immunization once immune 5 times altogether, does not add adjuvant for the last time.Take a blood sample behind last immunity 7～10d, measure serum antibody titer, heart is taken a blood sample, and obtains the polyclonal antibody of purifying through ammonium sulfate precipitation.
Three, enzyme is marked haptenic preparation
The haptenic preparation of alkaline phosphatase mark: the haptenic preparation of 19-nortestosterone is with the 19-nortestosterone haptens preparation method among the embodiment 1.
Enzyme mark haptens preparation: adopt mixed anhydride method that alkaline phosphatase and 19-nortestosterone hapten conjugation are obtained enzyme labeling 19-nortestosterone haptens.
Concrete grammar is as follows: 1) get the N that 19-nortestosterone haptens 2g is dissolved in 30ml 50% (quality percentage composition), in the dinethylformamide solution; 2) get the 0.5ml isobutyl chlorocarbonate again and be dissolved in the no Shui diox of 5ml, be added to then in the solution that step 1) obtains, stirring at room reaction 4 hours; 3) get in the carbonate buffer solution that alkaline phosphatase 32g is dissolved in 70ml pH9.6, be added to step 2 then) in the solution that obtains 4 ℃ of stirrings spend the night, dialysed 7 days at the phosphate buffer of 0.2mol/L then, change liquid every day and obtain enzyme labeling 19-nortestosterone haptens for 3～4 times, at last the enzyme labeling haptens is concentrated or the freeze-drying preservation.
Utilize the method for 19-nortestosterone residual in this kit test sample as follows:
The concrete steps of sample pre-treatments are with the sample pre-treatments step among the embodiment 1
Detection method:
A, in the ELISA Plate micropore of goat-anti rabbit antiantibody bag quilt, add 19-nortestosterone rabbit polyclonal antibody working fluid 100 μ l, 37 ℃ of reaction 30min.
B, pour out liquid in the hole, every hole adds the cleansing solution after the dilution, pours out liquid in the hole after 30 seconds, and so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.Every hole adds the 19-nortestosterone haptens working fluid 50 μ l of series standard product solution or sample solution 50 μ l and alkaline phosphate ester enzyme labeling, 37 ℃ of reaction 30min.
C, pour out liquid in the hole, repeat to wash the plate step, add substrate colour developing liquid 100 μ l, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuges colour developing 15～30min.
D, every hole add stop buffer 50 μ l, and the mixing that vibrates gently is set in 400nm wavelength place with microplate reader with wavelength coverage, measures every hole absorbance (OD value).
The method of interpretation of result is with the interpretation of result method among the embodiment 1, the canonical plotting of this kit, as shown in Figure 2.Interpretation of result shows that the whole testing process of the kit of preparation only needed just can finish in 1.5 hours, and lowest detection is limited to 0.1 μ g/L.
Embodiment 3, kit precision, accuracy and storage life test
1, kit precision test
(1) standard items precision test
The kit of preparation among embodiment 1 and the embodiment 2 is got three batches respectively carry out the precision experiment, every batch of kit extracts 10 kits, from the elisa plate of each kit, respectively extract 20 micropores again out, measure the absorbance (OD value) of 0.9 μ g/L standard solution, calculate the coefficient of variation.The measurement result of three batches of kits among the embodiment 1 is as shown in table 1, and the result shows that coefficient of variation scope is between 5.4%～10.9%.Meet precision less than 15% requirement.
The repeatable test of table 1 standard
The measurement result of three batches of kits among the embodiment 2 is as shown in table 2, and the result shows that coefficient of variation scope is between 5.9%～10.6%.Meet precision less than 15% requirement.
The repeatable test of table 2 standard
(2) the repeatable test of sample
Each chicken meat sample, feed sample, urine sample sample add 19-nortestosterone standard items by 25 μ g/kg concentration, get each three of the kits of three different batches of preparation among embodiment 1 and the embodiment 2 respectively, and each concentration repeats 5 times, calculates the coefficient of variation respectively.The measurement result of three batches of kits among the embodiment 1 is shown in table 3-table 5, and the result shows that the chicken sample coefficient of variation all is lower than 22%, and the Variation Lines number average of feed sample is lower than 20%, and the Variation Lines number average of urine sample is lower than 18%.Meet the coefficient of variation less than 25% requirement.
The repeatable test of table 3 chicken meat sample
The repeatable test of table 4 feed sample
The repeatable test of table 5 urine sample sample
The measurement result of three batches of kits among the embodiment 2 is shown in table 6-table 8, and the result shows that the chicken sample coefficient of variation all is lower than 20%, and the Variation Lines number average of feed sample is lower than 20%, and the Variation Lines number average of urine sample is lower than 20%.
The repeatable test of table 6 chicken meat sample
The repeatable test of table 7 feed sample
The repeatable test of table 8 urine sample sample
2, the accuracy determination of kit
Each chicken meat sample, feed sample, urine sample sample are pressed 25 μ g/kg concentration respectively, are added 19-nortestosterone standard items by 50 μ g/kg concentration, utilize the method detection halofuginone hydrobromide of the kit of embodiment 1 or embodiment 2 respectively according to embodiment 1 or embodiment 2, each concentration do 4 parallel, accuracy in computation respectively.The kit measurement result of embodiment 1 is as shown in table 9, and the result shows the accuracy of chicken interpolation between 71.9%-103.2%, and feed adds accuracy between 82.4%-112.4%, and urine is added accuracy between 67.5%-108.7%.
The accuracy determination test μ g/kg (L) of the kit of table 9 embodiment 1
The kit measurement result of embodiment 2 is as shown in table 10, and the result shows the accuracy of chicken interpolation between 68.5-96.4, and feed adds accuracy between 65.2-102.3, and urine is added accuracy between 73.5-101.3.
The accuracy determination test μ g/kg (L) of the kit of table 10 embodiment 2
3, kit storage life test
The kit of embodiment 1 and embodiment 2 preparations is kept at 2-8 ℃ respectively, after 6 months, maximum absorbance value (zero standard), 50% inhibition concentration, the 19-nortestosterone of measuring kit add the practical measurement value, the result shows maximum absorbance value (zero standard), 50% inhibition concentration of the kit of embodiment 1, and the maximum absorbance value (zero standard) of the kit of embodiment 2,50% inhibition concentration are all within normal range.Consideration has improper preservation condition and occurs in transportation and use, and the mentioned reagent box was placed 6 days under the condition of 37 ℃ of preservations, carries out the accelerated deterioration experiment, and the result shows that the every index of kit of embodiment 1 and embodiment 2 preparations meets the requirements fully.Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 5 days, measurement result shows that also the every index of kit of embodiment 1 and embodiment 2 preparations is normal fully.The kit that can draw embodiment 1 and embodiment 2 preparations from above result can be preserved more than 6 months at least at 2-8 ℃.
Claims (9)
1, a kind of enzyme linked immunological kit that detects the 19-nortestosterone comprises 19-nortestosterone specific antibody and coating antigen and enzyme labeling thing; The monoclonal antibody of the monoclonal hybridoma strain A-3-3 CGMCC No.1613 secretion that described 19-nortestosterone specific antibody is the 19-nortestosterone; Described coating antigen is the conjugate or the antiantibody of 19-nortestosterone haptens and carrier protein; Described enzyme labeling thing is enzyme mark antiantibody or enzyme mark 19-nortestosterone haptens; When described coating antigen was the conjugate of 19-nortestosterone haptens and carrier protein, described enzyme labeling thing was an enzyme mark antiantibody; When described coating antigen was antiantibody, described enzyme labeling thing was an enzyme mark 19-nortestosterone haptens; Described 19-nortestosterone haptens obtains 19-nortestosterone and succinic anhydride by acylation reaction.
2, enzyme linked immunological kit according to claim 1 is characterized in that: described kit also comprises 19-nortestosterone standard solution, developer, concentrated cleaning solution, stop buffer, concentrates redissolution liquid; Described concentrated redissolution liquid is the phosphate buffer that contains 0.5% bovine serum albumin(BSA) and 0.1% polysorbas20,0.01～0.05M, and described percentage composition is the quality percentage composition; Described carrier protein is ovalbumin, albumin rabbit serum or mouse serum albumin.
3, enzyme linked immunological kit according to claim 1 and 2 is characterized in that: the marker enzyme of described enzyme labeling thing is horseradish peroxidase or alkaline phosphatase.
4, enzyme linked immunological kit according to claim 1 and 2 is characterized in that: described antiantibody is sheep anti mouse or goat-anti rabbit antiantibody.
5, enzyme linked immunological kit according to claim 2 is characterized in that: described concentrated cleaning solution is 0.01M～0.05M, contain the phosphate buffer of 0.01% sodium azide and 1% Tween 80; Described percentage composition is the quality percentage composition.
6, enzyme linked immunological kit according to claim 2, it is characterized in that: developer is made up of colour developing liquid A liquid and colour developing liquid B liquid when marker enzyme is horseradish peroxidase, described colour developing liquid A liquid is hydrogen peroxide or urea peroxide, and described colour developing liquid B liquid is o-phenylenediamine or tetramethyl benzidine; When marker enzyme was alkaline phosphatase, developer was a 4-nitrophenols phosphate buffer.
7, enzyme linked immunological kit according to claim 2 is characterized in that: described stop buffer is sulfuric acid, hydrochloric acid or the sodium hydrate buffer solution of 1～2mol/L; Described percentage composition is the quality percentage composition.
8, enzyme linked immunological kit according to claim 1, it is characterized in that: to be cushioned liquid be pH 9.6 to used bag in the process of bag quilt, 0.05mol/L carbonate buffer solution, confining liquid is the 0.01～0.05M phosphate buffer that contains 5% skimmed milk power, and described percentage composition is the quality percentage composition.
9, a kind of method that detects the 19-nortestosterone may further comprise the steps:
1) sample pre-treatments:
When sample is animal tissue or feed, taking by weighing 2g animal tissue or feed homogenate and 0.5g ascorbic acid joins in the mixed liquor of 8ml acetonitrile and 2ml 1mol/L NaOH, the vibration mixing, more than the 3000g, 15 ℃, centrifugal 10-15 minute, get in the supernatant 1ml dislocation centrifuge tube, add 1ml 1mol/L NaOH and 2ml acetonitrile, mixing, adding the 3ml volume ratio again is 8: 2 the normal hexane and the mixed liquor vibration mixing of methylene chloride, 3000g, 15 ℃, centrifugal 10-15 minute, remove upper strata normal hexane phase, get the middle layer organic phase and flow down bone dry at nitrogen, with the dry residue water intaking phase of the described concentrated redissolution liquid dissolving of the claim 2 of 2 times of 1ml deionized water dilutions, can analyze;
When sample is urine, get the 2ml urine in centrifuge tube, more than the 3000g, 15 ℃, centrifugal 10-15 minute until limpid, pipette the 1ml urine in centrifuge tube, add the acetonitrile of 2ml, adding the 3ml volume ratio again is 8: 2 the normal hexane and the mixed liquor mixing of methylene chloride, more than the 3000g, 15 ℃, centrifugal 10-15 minute, remove upper strata normal hexane phase, get the middle layer organic phase and flow down bone dry at nitrogen, with the dry residue of the described concentrated redissolution liquid dissolving of the claim 2 of 2 times of 1ml deionized water dilutions, water intaking is analyzed mutually;
2) utilize the enzyme linked immunological kit test sample of arbitrary described detection 19-nortestosterone among the claim 1-8.
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