CN101407580B - Method for synthesizing melamine complete antigen and ELISA reagent kit for detecting melamine - Google Patents
Method for synthesizing melamine complete antigen and ELISA reagent kit for detecting melamine Download PDFInfo
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- CN101407580B CN101407580B CN2008100514885A CN200810051488A CN101407580B CN 101407580 B CN101407580 B CN 101407580B CN 2008100514885 A CN2008100514885 A CN 2008100514885A CN 200810051488 A CN200810051488 A CN 200810051488A CN 101407580 B CN101407580 B CN 101407580B
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- trimeric cyanamide
- monoclonal antibody
- enzyme
- complete antigen
- tripolycyanamide
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Abstract
The invention provides a method for synthesizing tripolycyanamide complete antigen and a directly competitive ELISA kit which uses monoclonal antibody for detecting tripolycyanamide. The kit comprises a tripolycyanamide enzyme standard, a tripolycyanamide standard liquid, an ELISA plate used for coating tripolycyanamide monoclonal antibody, substrate colorimetric solution, cleaning solution, stopping solution, enzyme standard diluent, sample diluent, and the like. The invention provides a method for artificially synthesizing the tripolycyanamide complete antigen, which uses polylysine as carriers for preparing the monoclonal antibody; and the ELISA kit prepared by the monoclonal antibody is characterized by being simple and fast, and having large sample processing amount, high sensitivity, strong specificity, low cost, and the like, is suitable for factory production, and can be used for fast detection of tripolycyanamide in liquid milk, powdered milk and solid samples.
Description
Technical field
The present invention relates to a kind of method of synthetic melamine complete antigen, the ELISA test kit of the detection trimeric cyanamide of using monoclonal antibody art development especially is provided, also disclose its preparation method simultaneously, belong to the Enzyme-multiplied immune technique field.
Background technology
Trimeric cyanamide (melamine) is called for short triamine, formal name used at school three ammonia triazines, and another name melamine, melon, three polymeric amide are a kind of important nitrogen heterocyclic Organic Chemicals.This chemical often is used to produce plastics, glue and fire retardant.The trimeric cyanamide nitrogen content is 66.6%, amounting to into crude protein content is 416.27%, mix on a small quantity and just can increase percent protein rapidly, product is seemed in routine inspection contain higher protein, often being masquerading as the high protein material by illegal retailer adds, protein detection numerical value in the food can also reduce cost significantly when detecting to improve.Because trimeric cyanamide is not dissolved in the water, anti-ammonia nitrogen determination adds that trimeric cyanamide is a white powder, easily is colored, and is easy to sneak in the raw material and is not found.So general analytical test department is difficult to analyze.
" the international chemical security manual " that IPCS and European Commission EC compile in collaboration with (the 3rd volume) shows: taking in trimeric cyanamide for a long time or repeatedly in a large number may exert an influence to kidney and bladder, cause producing calculus, so trimeric cyanamide is a kind of chemical substance of forbidding being used for food and animal-feed.
At present, the trimeric cyanamide detection method mainly is vapor-phase chromatography and high performance liquid chromatography.Though high performance liquid chromatography is simple to operate, detection limit is higher, can't satisfy the requirement that the trimeric cyanamide low residue is limited the quantity of; Vapor-phase chromatography sensitivity is higher, but sample pretreatment process complexity, analysis speed are slow, are not suitable for promoting the use of in basic unit; And vapor-phase chromatography and high performance liquid chromatography need expensive Special Equipment, are not suitable for on-the-spot the detection, and be higher to testing staff's technical requirements.
Based on the ELISA detection method that antigen and antibody specific reaction is set up, have simple, fast, handle that sample size is big, sensitivity is higher, high specificity and plurality of advantages such as cheap, can be directly used in production practice.
Summary of the invention:
The present invention discloses a kind of method of synthetic melamine complete antigen, is used to prepare monoclonal antibody or polyclonal antibody.
The present invention also provides using monoclonal antibody to detect the preparation method of the direct competitive ELISA detection kit of trimeric cyanamide, is fit to batch production production.
Synthetic melamine complete antigen disclosed by the invention obtains by the following method:
With the trimeric cyanamide is haptens, prepares complete antigen by glutaraldehyde method and poly-lysine coupling.
The composition of ELISA detection kit provided by the invention comprises:
Trimeric cyanamide enzyme mark thing, trimeric cyanamide reference liquid, bag are by the enzyme plate of trimeric cyanamide monoclonal antibody, substrate colour developing liquid, washings, stop buffer, enzyme mark thing diluent and sample diluting liquid etc.
Described monoclonal antibody is for being haptens with the trimeric cyanamide, by preparing complete antigen with the poly-lysine coupling, and cultivates by mouse immune, cytogamy, screening and cloning and to make; Described trimeric cyanamide enzyme mark thing adopts the chemical process coupling to obtain, and marker enzyme is a horseradish peroxidase, and described method is the sodium periodate method; Described sample diluting liquid is the PBST damping fluid that contains 0.1% tween 20, and the pH value is 7.4; Described washings is the PBST damping fluid that contains 0.05% tween 20, and the pH value is 7.4; Described enzyme mark thing diluent is the PBST damping fluid that contains 0.01% tween 20, and the pH value is 7.4; Substrate colour developing liquid: A liquid is that 0.1mol/LpH value is that sodium-acetate-citrate buffer solution of 5.0 is mixed with 0.3% hydrogen peroxide storing solution, and every 1mL damping fluid adding 14uL storing solution is made use liquid; B liquid is tetramethyl benzidine, earlier is made into 0.2mg/mL solution with acetone, is that sodium-acetate-citrate buffer solution of 5.0 is mixed with 0.2mg/L solution with the 0.1mol/LpH value.
On the other hand, the present invention also provides the method for content of melamine in the test sample, and step is as follows:
(1) sample pre-treatments
(2) detect with the described ELISA detection kit of claim 2, in wrapping, add standard substance or sample solution by the enzyme plate hole of trimeric cyanamide monoclonal antibody, add trimeric cyanamide enzyme mark thing, hatched 30 minutes, the washings washing, add colour developing liquid and hatched 20 minutes, the stop buffer termination reaction is measured absorbance with microplate reader.
(3) analyzing and testing result
The principle of the artificial synthesizing melamine complete antigen of the present invention is:
Glutaraldehyde (GA) is a kind of homotype bi-functional cross-linking agent, and its two functional groups can combine with the amino on poly-lysine and the trimeric cyanamide, forms the binding substances of poly-lysine-GA-trimeric cyanamide.First with two-step approach with poly-lysine and excessive GA reaction, form the binding substances of the two, and then amino crosslinked with this binding substances and antibody protein molecule, form the binding substances of poly-lysine-GA-trimeric cyanamide, be prepared into the complete antigen of trimeric cyanamide, can be used for immune animal and prepare monoclonal antibody or polyclonal antibody.
The detection principle of test kit of the present invention is:
With the monoclonal antibody bag of anti-melamine by on solid phase carrier, add sample or trimeric cyanamide standard solution and add trimeric cyanamide enzyme mark thing, trimeric cyanamide in the sample to be checked and trimeric cyanamide enzyme mark thing competition bag are by the monoclonal antibody on solid phase carrier, remove unconjugated trimeric cyanamide enzyme mark thing by washing, add colour developing liquid colour developing back termination reaction, the working sample absorbance, the amount of trimeric cyanamide is negative correlation in revaluate and the sample, with typical curve relatively can working sample in the concentration of trimeric cyanamide.
Positively effect of the present invention is:
The present invention discloses a kind of method of new synthesizing melamine complete antigen first, for preparation monoclonal antibody or polyclonal antibody are laid a good foundation; And the first Application monoclonal antibody set up a kind of direct competitive ELISA test kit that detects trimeric cyanamide, can be qualitative or the quantitative analysis sample in trimeric cyanamide, accommodate batch production production.Test kit of the present invention has shortened detection time, and low to the pre-treatment requirement of sample, treating processes is simple, and has been equipped with relevant sample preparation reagent, can detect gross sample simultaneously rapidly.
All reagent of test kit of the present invention are the working fluid that has prepared, can need not to handle by direct control, reduced operation steps, can be quick, trimeric cyanamide in Sensitive Detection liquid milk, milk powder, the solid sample has been avoided complicated operations, and is low for equipment requirements, can adapt to multiple testing environment, and multiple detection needs; And test kit sample consumption of the present invention is little, and the reagent shelf time is long, and the level of automation height is to operator's nontoxicity, environmentally safe etc.; The ELISA detection kit that using monoclonal antibody therefore of the present invention is set up has important social meaning and vast market prospect.
Description of drawings
Fig. 1. trimeric cyanamide suppresses curve.
Embodiment
By following examples the present invention is described for example further, and do not limit the present invention in any way, under the prerequisite that does not deviate from technical solution of the present invention, any change or change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope of the present invention.
The preparation melamine complete antigen
Make trimeric cyanamide and poly-lysine coupling prepare complete antigen by glutaraldehyde method, concrete steps are as follows:
1. get the 100mg poly-lysine and be dissolved among the 20mL pH7.2PBS, add the 2mL50% glutaraldehyde, 40 ℃ of effect 18h.
2. the Sephadex G-50 gel column wash-out of crossing with the 0.15mol/LNaCl balance is removed the free glutaraldehyde, or is used 0.01mol/L, pH7.2PBS, 4 ℃ of dialysed overnight, during change dialyzate three times.
3. regulate elutriant or dialyzate pH value to 9.0～9.6 with 1mol/L pH9.6 carbonate buffer solution, the 100mg trimeric cyanamide directly is dissolved in elutriant or the dialyzate, 37 ℃ of effect 18h.
4. the dialysis tubing of packing into, with 0.01mol/L, pH7.2PBS, 4 ℃ of dialysed overnight.
5. use the PEG8000 concentrate dialysate, add equivalent 50% glycerine, packing in a small amount, 4 ℃ or 20 ℃ of preservations.But with the complete antigen direct immunization animal of this method preparation, preparation monoclonal antibody or polyclonal antibody.
The preparation of trimeric cyanamide monoclonal antibody specific
As immunogen, select for use healthy 8 ages in week female BALB/C mice as immune animal with the complete antigen of embodiment 1 method preparation, immunizing dose 1mg/ only takes abdominal injection, and each immunity was spaced apart for 2 weeks, immunity 5 times.Initial immunity immunogen and Freund's complete adjuvant (volume ratio=1:1) injection, two, three, four immunizing antigens mix with the equivalent Freund's incomplete adjuvant, it is immune with 2 times of amounts not add adjuvant for the last time, get mouse boosting cell after 3 days and Sp2/0 myeloma cell is hybridized fusion, the preparation hybridoma.Take limiting dilution assay screening hybridoma, the hybridoma cell strain that screening can stably excreting trimeric cyanamide monoclonal antibody.The production of monoclonal antibody and purifying, mouse peritoneal injection hybridoma is gathered ascites, and through ammonium sulfate precipitation, SPA-Sepharose CL-4B affinity column purified monoclonal antibody adds 0.01% sodium azide, through vacuum lyophilization postposition-20 ℃ preservation.
Embodiment 3
The preparation of trimeric cyanamide enzyme mark thing
Enzyme in the enzyme-labelled antigen can be horseradish peroxidase (HRP) or alkaline phosphatase (AP), is preferably HRP, and HRP-trimeric cyanamide marker adopts the preparation of sodium periodate method, and is specific as follows:
1. taking by weighing 10mgHRP is dissolved in the 1ml distilled water.
2. in last liquid, add the 0.1M NaIO that 0.2mL newly joins
4Solution, lucifuge stirred 20 minutes under the room temperature.
3. above-mentioned solution is packed in the dialysis tubing into 4 ℃ of dialysed overnight of the sodium-acetate buffer of 1mM PH4.4.
4. add 20 μ L0.2M PH9.5 carbonate buffer solutions, make the PH of above hydroformylation HRP be elevated to 9.0～9.5.
5.0.5mg haptens is dissolved in the 1mL0.01M carbonate buffer solution, is divided into two parts, joins among the HRP of hydroformylation, the room temperature lucifuge stirred 2 hours gently.
6. add the 4mg/mL NaBH that 0.1ml newly joins
4Liquid, mixing, put again 4 ℃ 2 hours.
7. above-mentioned liquid is packed in the dialysis tubing, to 0.15M PH7.4PBS dialysis, 4 ℃ are spent the night.
8. under agitation dropwise add the equal-volume saturated ammonium sulphate, put 4 ℃ 1 hour.
9.3000rpm centrifugal half an hour, abandon supernatant.Throw out is washed secondary with semi-saturation ammonium sulfate, and last throw out is dissolved among the PBS of a small amount of 0.15M PH7.4.
10. above-mentioned solution is packed in the dialysis tubing, the PBS damping fluid dialysis to 0.15M PH7.4 (detects with Nai Shi reagent) behind the removal ammonium ion, 10,000rpm removed precipitation in centrifugal 30 minutes, and supernatant liquor is enzyme conjugates, add equivalent 50% glycerine, packing in a small amount, 4 ℃ or-20 ℃ of preservations.
Embodiment 4
The foundation of direct competitive ELISA method
1. monoclonal antibody the best is wrapped determining by concentration and trimeric cyanamide enzyme conjugates optimum dilution degree
Monoclonal antibody coated elisa plate with doubling dilution, after bag is spent the night, 37 ℃ of sealings of 1% BSA 1h, after the washing, by detecting the positive and negative sample respectively, the OD value of positive and negative sample relatively, select higher, the negative OD value of positive OD value is lower and the P/N value is higher monoclonal antibody working concentration as the best bag by concentration; Best after measured bag is 1.41ug/mL by concentration.Monoclonal antibody by the concentration coated elisa plate, detects the enzyme conjugates optimum dilution degree with best bag, after measured trimeric cyanamide enzyme conjugates optimum dilution degree be 8000 *.
2.ELISA detect monoclonal antibody IC50 value:
With the best bag by concentration monoclonal antibody coated elisa plate, prepare 0ug/L, 5ug/L, 10ug/L, 50ug/L, 100ug/L and 200ug/L trimeric cyanamide standard substance, add 50uL standard substance and 50uL optimum dilution degree enzyme mark binding substances, room temperature reaction 30 minutes, 20 minutes termination reactions of washing back colour developing, microplate reader is measured the OD450nm value.Detected result is seen Fig. 1.Trimeric cyanamide monoclonal antibody IC50 shows that the monoclonal antibody that the present invention prepares has specificity preferably about 10ug/L as seen from Figure 1, can satisfy the requirement that detects trimeric cyanamide ELISA test kit.
Detect the establishment of trimeric cyanamide direct competitive ELISA test kit
The ELISA detection kit comprises following component:
1. trimeric cyanamide standardized solution: concentration is respectively 0ug/L, 5ug/L, 10ug/L, 50ug/L, 100ug/L and 200ug/L, totally six bottles.
2. bag is by 96 hole polystyrene enzyme plates of trimeric cyanamide monoclonal antibody.
3. the trimeric cyanamide enzyme is marked thing: HRP-trimeric cyanamide enzyme mark substrate concentration is 1mg/L.
4. substrate colour developing liquid: A liquid is that 0.1mol/LpH value is that sodium-acetate-citrate buffer solution of 5.0 is mixed with 0.3% hydrogen peroxide storing solution, and every 1mL damping fluid adding 14uL storing solution is made use liquid; B liquid is tetramethyl benzidine, earlier is made into 0.2mg/mL solution with acetone, is that sodium-acetate-citrate buffer solution of 5.0 is mixed with 0.2mg/L solution with the 0.1mol/LpH value.
5. washings: contain the PBST damping fluid of 0.05% tween 20, the pH value is 7.4.
6. enzyme is marked the thing diluent: contain the PBST damping fluid of 0.01% tween 20, the pH value is 7.4.
7. sample diluting liquid: contain the PBST damping fluid of 0.1% tween 20, the pH value is 7.4.
8. stop buffer: 2mol/L sulfuric acid.
The storage temperature of test kit is 4 ℃ of preservations.
Embodiment 6
The detection of melamine residual in the sample
1. sample pre-treatments:
Liquid state milk: with sample diluting liquid 1:10 dilution;
Milk powder: press the sample packaging explanation and replace the water dilution, again with sample diluting liquid 1:10 dilution with sample diluting liquid;
Solid sample: take by weighing sample and add 5 times of volume DMSO vibrations 1 hour, the centrifuging and taking supernatant, with the sample diluting liquid dilution, the preparation sample solution.
2. test kit detects:
(1) will in wrapping, add 50uL sample or trimeric cyanamide standard solution, and add 50uL trimeric cyanamide enzyme mark thing by the enzyme plate of trimeric cyanamide monoclonal antibody;
(2) vibrated 30 seconds room temperature reaction 30 minutes gently;
(3) washing: the liquid in the hole of inclining, fill with each hole (about 300 μ l) with washings, outwell washings then, 3 times so repeatedly, on thieving paper, pat dry for the last time;
(4) add 50uL trimeric cyanamide enzyme mark thing, 37 ℃ were reacted 20 minutes;
(5) add 50uL stop buffer termination reaction;
(6) read the OD450nm value with microplate reader.
3. interpretation of result:
The drawing standard curve, the content of trimeric cyanamide can be read from typical curve in corresponding each sample, also can go out the content of trimeric cyanamide in the sample with regression equation calculation.Absorbancy that records and the content of melamine in the sample are inversely proportional to, and can calculate the content of trimeric cyanamide by typical curve.
Embodiment 7
Toward no trimeric cyanamide fresh milk in add 50ug/L trimeric cyanamide as actual sample, detect with test kit.
1. sample pre-treatments:
With sample diluting liquid 1:10 dilution, the preparation sample solution.
2. test kit detects:
(1) will in wrapping, add 50uL sample or trimeric cyanamide standard solution, and add 50uL trimeric cyanamide enzyme mark thing by the enzyme plate of trimeric cyanamide monoclonal antibody;
(2) vibrated 30 seconds room temperature reaction 30 minutes gently;
(3) washing: the liquid in the hole of inclining, fill with each hole (about 300 μ 1) with washings, outwell washings then, 3 times so repeatedly, on thieving paper, pat dry for the last time;
(4) add 50uL trimeric cyanamide enzyme mark thing, 37 ℃ were reacted 20 minutes;
(5) add 50uL stop buffer termination reaction;
(6) read the OD450nm value with microplate reader, numerical value is 1.012 after measured.
3. interpretation of result:
By typical curve relatively, the content of trying to achieve the sample solution trimeric cyanamide is 5ug/L, because sample solution has diluted 10 times through pre-treatment, so the content of trimeric cyanamide is 50ug/L in the working sample milk.
Claims (4)
1. the method for a synthetic melamine complete antigen is characterized in that: be haptens with the trimeric cyanamide, prepare complete antigen by glutaraldehyde method and poly-lysine coupling.
2. the monoclonal antibody of complete antigen preparation according to claim 1 detects the direct competitive ELISA test kit of trimeric cyanamide, and composition comprises: trimeric cyanamide enzyme mark thing, trimeric cyanamide reference liquid, bag are by the enzyme plate of trimeric cyanamide monoclonal antibody, substrate colour developing liquid, washings, stop buffer, enzyme mark thing diluent and sample diluting liquid.
3. the preparation method of the described test kit of claim 2 is characterized in that: be haptens with the trimeric cyanamide, by preparing complete antigen with the poly-lysine coupling; Prepare monoclonal antibody by complete antigen immune mouse, cytogamy, screening and cloning culturing process; The monoclonal antibody bag is wrapped by the enzyme plate of trimeric cyanamide monoclonal antibody in the enzyme plate preparation, add trimeric cyanamide enzyme mark thing, trimeric cyanamide reference liquid, substrate colour developing liquid, washings, stop buffer, enzyme mark thing diluent and sample diluting liquid again, form the ELISA test kit.
4. the preparation method of the described ELISA test kit of claim 3 is characterized in that: the used enzyme of described trimeric cyanamide enzyme mark thing is a horseradish peroxidase, prepares the trimeric cyanamide enzyme by sodium periodate method and trimeric cyanamide coupling and marks thing.
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Cited By (2)
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| CN102435729A (en) * | 2011-12-02 | 2012-05-02 | 杭州迪恩科技有限公司 | One-step enzyme-linked immunoassay method for quickly detecting melamine and kit thereof |
| US8829150B2 (en) | 2012-09-21 | 2014-09-09 | National Taiwan Normal University | Methods for polymering haptens into immunogens |
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| CN101717444B (en) * | 2009-11-27 | 2012-01-25 | 北京生命科学研究所 | Kit for detecting melamine and application thereof |
| CN102086230B (en) * | 2009-12-04 | 2012-11-14 | 北京维德维康生物技术有限公司 | Enzyme-linked immunosorbent assay kit for detecting melamine and application thereof |
| CN102590495A (en) * | 2012-02-10 | 2012-07-18 | 北京沙屏研科技有限公司 | Chemiluminescence assay kit for detecting melamine and preparation method thereof |
| CN102661950A (en) * | 2012-05-31 | 2012-09-12 | 福建省产品质量检验研究院 | Test card for quickly detecting melamine |
| CN110133268A (en) * | 2019-05-20 | 2019-08-16 | 北京组学生物科技有限公司 | For the ELISA kit and its application of AP25 |
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| US8829150B2 (en) | 2012-09-21 | 2014-09-09 | National Taiwan Normal University | Methods for polymering haptens into immunogens |
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