CN102128923A - One-step ELISA (Enzyme Linked Immunosorbent Assay) method for neomycin (NEO) residues in milk - Google Patents

Abstract

The invention relates to a one-step ELISA (Enzyme Linked Immunosorbent Assay) method for neomycin (NEO) residues in milk, which belongs to the technical field of immunoassay chemicals. The method comprises the following steps of: coupling NEO with bovine serum albumin (BSA) or ovalbumin (OVA) to synthetize a NEO antigen (immunogen NEO-BSA or coating antigen NEO-OVA) by a glutaraldehyde method; authenticating the NEO-BSA by utilizing an infrared spectroscopy and an SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis) method; obtaining NEO antiserum by immunization and detecting the valence of the NEO antiserum by an indirect ELISA method; and detecting the specificity of the NEO antiserum by the one-step ELISA method. Compared with the indirect competition ELISA method, the invention greatly shortens the detecting time of the specificity of the NEO antibody, has higher sensitivity, achieves the aim of simply, rapidly and sensitively detecting NEO residues in milk and can be widely used for rapid field detection.

Description

The residual step ELISA detection method of neomycin in a kind of milk

Technical field

A kind of at the residual step ELISA detection method of neomycin in the milk, belong to the immunoassay technical field of chemistry.

Background technology

(neomycin is the polycomponent microbiotic that is produced by streptomysin Pseudomonas streptomyces fradiae NEO) to neomycin, contains two kinds of isomerss of actiline and C (structural formula is as follows), but has only actiline to have antibacterial activity.Neomycin is water miscible alkaline antibiotic, and common dosage forms is the neomycinsulphate of white or off-white powder shape on the market, has stronger hydroscopicity, and long term exposure is understood because absorb moisture deliquescence in air.And neomycin itself has extremely strong stability, to acid, alkali, the tolerance of environmental factors such as heat obviously is better than other microbiotic.Neomycin is very easily water-soluble, but is dissolved in hardly in the organic solvents such as ethanol, ether, acetone or chloroform, has optically active.

Neomycin belongs to the aminoglycoside antibiotics medicine, because its broad spectrum antibacterial, the growth of inhibition Gram-negative bacteria that can be fabulous has been widely used in medicine and field of veterinary at present.On veterinary clinic, neomycin becomes one of choice drug of prevention and treatment fowl bacterial enteritis, and its effect spy is good.At present, China and some other country also add neomycin in the animal feed to, improve on the one hand efficiency of feed utilization, promote the growth of animal, prevent the disease that is caused by bacterial infection simultaneously and avoid loss.It is big that but neomycin absorbs toxicity, mainly shows as ototoxicity and renal toxicity, and neomycin is residual bigger to human health damage.In order to ensure the common people's food security, strengthen the international competitiveness of animal-derived food, must the detection that neomycin in the animal-derived food is residual include in the conventional sense and go.

The method of relevant neomycin assay is a lot, and both at home and abroad main report has microbiology method, thin-layered chromatography, high performance liquid chromatography, vapor-phase chromatography, LC-MS method, immunoassay, capillary electrophoresis and an ion exchange process etc.In these residue analysis methods, the TLC method is mainly as a kind of purification means, and electrophoresis and micro-biological process are not high because of complex operation, sensitivity, detection speed is slow and specificity is lower and be restricted, so high performance liquid chromatography and immunological method become the main analytical approach of neomycin.High performance liquid chromatography has higher sensitivity and specificity than microbial method, but apparatus expensive, experimenter's quality is also had higher requirements, analyze time-consuming and difficult popularization, it also needs complicated loaded down with trivial details sample pre-treatments and can not screen a large amount of samples synchronously simultaneously, the existence of these influence factors is not suitable for timely monitoring beyond doubt, on-the-spot detection.Immunoassay is to utilize the analytical technology of the specificity association reaction of antigen and antibody for the basis at present, is core reagent with antibody, has characteristics such as high selectivity, high sensitivity.It is at present external that what adopt all is that (enzyme-linked immunosorbent assay ELISA), in particular for the residue detection in milk, blood plasma and the honey sample, can reach good detection effect to enzyme linked immunosorbent assay basically.Jin etc. successfully prepare the monoclonal antibody of anti-neomycin, and adopt direct competitive ELISA method to measure the residual half-inhibition concentration IC of neomycin in blood plasma and the milk ₅₀=50 ng/mL. are at phosphate buffered solution, blood plasma, and the detectability in the milk (LOD) is respectively 6.85 ng/mL, 3.61 ng/mL, 2.73 ng/mL.

But present neomycin immune analysis method is relatively still longer detection time, and detectability is not high enough.Therefore exploitation is set up residual method for quick of neomycin and corresponding technical standard and is very important at residual more convenient, quick, the sensitive determination techniques of neomycin in the animal-derived food.

Summary of the invention

The purpose of this invention is to provide a kind of quicker, simple, a sensitive step ELISA method, the neomycin that is used for detecting milk is residual.

Technical scheme of the present invention: the residual step ELISA detection method of neomycin in a kind of milk comprises the synthetic of neomycin antigen and identifies the mensuration of neomycin antiserum titre and the specific detection of neomycin antiserum.

Processing step is:

(1) utilize glutaraldehyde method to synthesize neomycin antigen (immunogene and coating antigen);

(2) utilize infra-red sepectrometry (IR method) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that antigen is identified;

(3) obtain anti-neomycin antiserum by immunity;

(4) utilize indirect elisa method to measure tiring of neomycin antibody;

(5) utilize a step ELISA method to detect the specificity of neomycin antibody;

Described neomycin antigen: immunogene and coating antigen synthetic:

1. take by weighing 80 mg neomycin NEO, 40mg bovine serum albumin(BSA) BSA or ovalbumin OVA are dissolved in the phosphate buffer of 10 mL, 0.01 mol/L;

2. fully dropwise add 1.5mL mass concentration 1% glutaraldehyde solution after the dissolving, stirring at room is reacted 30 min;

3. add 112 mg NaBH ₄, leave standstill 1h in 4 ℃ of refrigerators;

4. reactant liquor was dialysed 3 days with the 0.01mol/L phosphate buffer, and 6h changes liquid at interval;

Gained NEO-BSA conjugate is an immunogene, and gained NEO-OVA conjugate is a coating antigen.

The described infra-red sepectrometry that utilizes carries out the immunogenic evaluation of neomycin:

1. take by weighing the NEO standard items respectively, each 2 mg of BSA and NEO-BSA sample;

2. add dry potassium bromide KBr 200 mg respectively, potpourri places agate mortar, mixing, fully grinding under the infrared lamp irradiation;

3. the compression mold of packing at 10 tons of pressure lower sheeting 10 min, makes the transparent KBr medicine compressing tablet of thickness 1 mm;

4. produce corresponding KBr-NEO respectively, KBr-BSA, and 3 sample compressing tablets of KBr-NEO-BSA go up the infrared spectrometer Test Identification subsequently.

The described SDS-PAGE of utilization gel electrophoresis carries out the immunogenic evaluation of neomycin:

1. separation gel 10%, concentrates glue 5%, voltage 100V, electric current 20mA, electrophoresis time 1.5h;

2. immobile liquid is 12.5% trichloroacetic acid solution, fixedly 30min;

3. the immobile liquid dyeing 1h that contains 0.1% Coomassie brilliant blue G250;

4. ultra-low molecular amount standard protein molecular weight is respectively: rabbit myosin 200000, calmodulin 130000, rabbit phosphorylase B 97400, bovine serum albumin(BSA) 66200, rabbit actin 43000.With the logarithm mapping of the mobility of the standard protein of known molecular amount, obtain a typical curve to molecular weight;

5. when molecular weight when 15KD is between 200KD, the logarithm of the molecular weight of the mobility of protein in the SDS-PAGE electrophoresis and it is linear, meets following formula: log (Mw)=k-b * R _f, in the formula: Mw is a protein molecular weight, R _fBe mobility, k, b are constant, and the testing protein of unknown molecular amount is carried out the SDS-PAGE electrophoresis under the same conditions, try to achieve its molecular weight according to its electrophoretic mobility on typical curve.

Described preparation neomycin antiserum, and carry out the mensuration of neomycin antibody titer:

1. wrap quilt: neomycin coating antigen NEO-OVA is cushioned liquid with bag does to add in the 96 hole ELISA Plate behind the serial gradient dilution and wrap quilt, 100 μ L/ holes are in 4 ℃ of refrigerator overnight.Take out ELISA Plate and be back to room temperature next day, dries, and 200 μ L 0.01mol/L PBST solution are injected as cleansing solution in every hole, and the 3min that vibrates on the horizontal shaking table firmly gets rid of cleansing solution, on thieving paper, pat dry, and repeated washing 3 times, following washing methods is identical;

2. sealing: fully after the washing, add the PBST confining liquid sealase target of 0.01M, 200 μ L/ holes, behind the incubation 2h, repeated washing 3 times is put into baking oven 10min oven dry back and is taken out stand-by in 37 ℃ of baking ovens;

3. competition: will resist the positive antiserum dilution series of neomycin gradient, correspondence joins ELISA Plate 7 row, and eighth row adds negative serum, and 100 μ L/ holes are hatched washing behind the 30min, patted dry for 37 ℃;

4. add two anti-: every hole adds 100 μ L, and the ELIAS secondary antibody HRP-IgG of 1:3000 dilution is hatched washing behind 30 min, patted dry in 37 ℃ of baking ovens;

5. colour developing: every hole adds 100 μ L colour developing liquid, and TMB and substrate buffer solution volume proportion are 1:5, and 15 min are reacted in 37 ℃ of baking oven dark places;

6. stop: take out every hole, back and add 100 μ L stop buffers, i.e. the sulfuric acid of 2 mol/L is measured the light absorption value A at 450nm place with microplate reader ₄₅₀

Select the A of positive serum ₄₅₀Value is at 1.5-1.9, simultaneously with the A of negative serum ₄₅₀Value difference is a best effort concentration apart from coating antigen dilute concentration, the antibody dilution concentration of maximum.

The A of positive serum ₄₅₀2.1 times of 〉=negative control hole, and A ₄₅₀Greater than 0.1, this moment, the extension rate of serum was tiring of serum.

The described mensuration of utilizing a step ELISA method to carry out the neomycin antibody specificity:

1. wrap quilt: get the ELISA Plate in two 8 holes, detect the selected the best bag that comes out with the working point and by concentration coating antigen is carried out serial gradient dilution and wraps quilt, 100 μ L/ holes, two contrasts are set, place 4 ℃ of refrigerators to preserve and spend the night, PBST washing lotion washing three times, each 3 min pat dry;

2. sealing: with the PBST confining liquid sealing of 0.01M, 200 μ L/ holes place 4 ℃ of refrigerators to preserve and spend the night, PBST washing lotion washing three times, and each 3 min pat dry;

3. competition: first row adds antibody diluent, second queues the neomycin standard solution that adds 7 concentration respectively, and selected best dilute concentration dilution antibody is detected with the working point in 50 μ L/ holes, add in 50 μ L/ holes, each hole all adds the ELIAS secondary antibody of 1:3000 dilution simultaneously, 100 μ L/ holes, fully vibration on oscillator, place 30 min for 37 ℃, PBST washing lotion washing five times, each 3 min pat dry;

4. colour developing: add colour developing liquid, substrate buffer solution and TMB volume ratio 5:1,100 μ L/ holes, 37 ℃ of reaction 15 min;

5. stop: add stop buffer, the 2 mol/L concentrated sulphuric acids, each hole absorbance is detected in 100 μ L/ holes on the microplate reader;

6. obtain the B/B of each concentration ₀: B/B ₀=B _{Corrected value}/ B _{0 corrected value}

B _{Corrected value}=Ab _{The actual measurement average}-Ab _{Blank average}

B _{0 corrected value}Light absorption value when being 0 ng/mL, the i.e. corrected value of negative control hole for neomycin standard solution interpolation concentration.

IC ₅₀For reaching light absorption value is maximum light absorption value A _MaxOne half added neomycin standard items concentration, with B/B ₀Be ordinate, the logarithm value of the serial gradient concentration that adds with the neomycin standard solution is that horizontal ordinate is mapped on EXCEL, promptly gets the inhibition typical curve of neomycin.

Beneficial effect of the present invention: the present invention has prepared a kind of at the residual step ELISA detection method of neomycin in the milk, and with respect to the indirect competitive ELISA method, owing to greatly shortened the detection time of neomycin antibody specificity, and has very high sensitivity, the neomycin that has reached in simple, quick, the sensitive detection milk is residual, can be widely used in quick on-the-spot the detection.

Description of drawings

Fig. 1 infra-red sepectrometry identifies that neomycin is immunogenic synthetic.

Fig. 2 SDS-PAGE gel electrophoresis is identified the neomycin immunogene.A、BSA，B、NEO-BSA，C，Marker。

One step ELISA of Fig. 3 neomycin antibody suppresses typical curve.

Embodiment

Embodiment 1

(1) the synthetic neomycin antigen of glutaraldehyde method

1. take by weighing 80 mg neomycins, 40mg bovine serum albumin(BSA) (BSA) or ovalbumin (OVA) are dissolved among 10 mL, the 0.01 mol/L PBS;

2. fully dropwise add 1.5mL 1% glutaraldehyde solution after the dissolving, stirring at room is reacted 30 min;

3. add 112 mg NaBH ₄, leave standstill 1h in 4 ℃ of refrigerators;

4. reactant liquor was dialysed 3 days with the 0.01mol/L phosphate buffer, and 6h changes liquid at interval;

Gained NEO-BSA conjugate is an immunogene, and gained NEO-OVA conjugate is a coating antigen.

(2) infra-red sepectrometry (IR method) is identified neomycin immunogene (NEO-BSA)

1. take by weighing the NEO standard items respectively, each 2 mg of BSA and NEO-BSA sample;

2. add dry potassium bromide (KBr) 200 mg respectively, potpourri places agate mortar, mixing, fully grinding under the infrared lamp irradiation;

3. the compression mold of packing at 10 tons of pressure lower sheeting 10 min, makes the transparent KBr medicine compressing tablet of thickness 1 mm

4. produce corresponding KBr-NEO respectively, KBr-BSA, and 3 sample compressing tablets of KBr-NEO-BSA go up the infrared spectrometer Test Identification subsequently.

(3) the SDS-PAGE gel electrophoresis is identified NEO-BSA

1. separation gel 10%, concentrates glue 5%, voltage 100V, electric current 20mA, electrophoresis time 1.5h;

2. immobile liquid is 12.5% trichloroacetic acid solution, fixedly 30min;

3. the immobile liquid dyeing 1h that contains 0.1% Coomassie brilliant blue G250;

4. ultra-low molecular amount standard protein molecular weight is respectively: rabbit myosin 200000, calmodulin 130000, rabbit phosphorylase B 97400, bovine serum albumin(BSA) 66200, rabbit actin 43000.With the logarithm mapping of the mobility of the standard protein of known molecular amount, obtain a typical curve to molecular weight;

5. when molecular weight when 15KD is between 200KD, the logarithm of the molecular weight of the mobility of protein in the SDS-PAGE electrophoresis and it is linear, meets following formula: log (Mw)=k-b * R _f(in the formula: Mw is a protein molecular weight, R _fBe mobility, k, b are constant), the testing protein of unknown molecular amount is carried out the SDS-PAGE electrophoresis under the same conditions, on typical curve, try to achieve its molecular weight according to its electrophoretic mobility.

(4) indirect elisa method is measured tiring of neomycin antibody

Adopt the indirect competitive ELISA method, the square formation titrimetry is determined the suitableeest working concentration of coating antigen and immune serum, measures under best effort concentration that NEO is sero-fast to tire, and the goat anti-rabbit igg of HRP mark (HRP-IgG) is selected recommended density 1:3000 for use.

1. wrap quilt: neomycin coating antigen (NEO-OVA) is cushioned liquid with bag does to add in the 96 hole ELISA Plate behind the serial gradient dilution and wrap quilt, 100 μ L/ holes are in 4 ℃ of refrigerator overnight.Take out ELISA Plate and be back to room temperature next day, dries, and 200 μ L 0.01mol/L PBST solution are injected as cleansing solution in every hole, and the 3min that vibrates on the horizontal shaking table firmly gets rid of cleansing solution, pats dry repeated washing 3 times on thieving paper.Following washing methods is identical.

2. sealing: fully after the washing, add the PBST confining liquid sealase target of 0.01M, 200 μ L/ holes, behind the incubation 2h, repeated washing 3 times is put into baking oven 10min oven dry back and is taken out stand-by in 37 ℃ of baking ovens.

3. competition: will resist the positive antiserum dilution series of neomycin gradient, correspondence joins ELISA Plate 7 row, and eighth row adds negative serum, and 100 μ L/ holes are hatched washing behind the 30min, patted dry for 37 ℃.

4. add two anti-: every hole adds 100 μ L, and the ELIAS secondary antibody (HRP-IgG) of 1:3000 dilution is hatched washing behind 30 min, patted dry in 37 ℃ of baking ovens.

5. colour developing: every hole adds 100 μ L colour developing liquid (TMB and substrate buffer solution volume proportion are 1:5), and 15 min are reacted in 37 ℃ of baking oven dark places.

6. stop: take out every hole, back and add 100 μ L stop buffers (i.e. the sulfuric acid of 2 mol/L), measure the light absorption value A at 450nm place with microplate reader ₄₅₀

Select the A of positive serum ₄₅₀Value is at 1.5-1.9, simultaneously with the A of negative serum ₄₅₀Value difference is a best effort concentration apart from coating antigen dilute concentration, the antibody dilution concentration of maximum.

The A of positive serum ₄₅₀2.1 times and A of 〉=negative control hole ₄₅₀Greater than 0.1, this moment, the extension rate of serum was tiring of serum.

(5) one step ELISA methods detect the specificity of neomycin antibody

1. wrap quilt: get the ELISA Plate in two 8 holes, detect the selected the best bag that comes out with the working point and by concentration coating antigen is carried out serial gradient dilution and wraps quilt, 100 μ L/ holes are provided with two contrasts.Placing 4 ℃ of refrigerators to preserve spends the night.PBST washing lotion washing three times, each 3 min pat dry.

2. sealing: with the PBST confining liquid sealing of 0.01M, 200 μ L/ holes place 4 ℃ of refrigerators to preserve and spend the night.PBST washing lotion washing three times, each 3 min pat dry.

3. competition: first row adds antibody diluent, and second queues the neomycin standard solution that adds 7 concentration respectively, 50 μ L/ holes.Detect selected best dilute concentration dilution antibody with the working point, add in 50 μ L/ holes, and each hole all adds the ELIAS secondary antibody of 1:3000 dilution simultaneously, 100 μ L/ holes, and fully vibration on oscillator is placed 30 min for 37 ℃.PBST washing lotion washing five times, each 3 min pat dry.

4. colour developing: add colour developing liquid (substrate buffer solution and TMB volume ratio 5:1), 100 μ L/ holes, 37 ℃ of reaction 15 min.

5. stop: add stop buffer (2 mol/L concentrated sulphuric acid sulfuric acid), each hole absorbance is detected in 100 μ L/ holes on the microplate reader.

6. obtain the B/B of each concentration ₀: B/B ₀=B _{Corrected value}/ B _{0 corrected value}

B _{Corrected value}=Ab _{Measured value (average)}-Ab _{Blank value (average)}

B _{0 corrected value}Light absorption value when being 0 ng/mL, the i.e. corrected value of negative control hole for neomycin standard solution interpolation concentration.

IC ₅₀For reaching light absorption value is maximum light absorption value (A _Max) half added neomycin standard items concentration.With B/B ₀Be ordinate, the logarithm value of the serial gradient concentration that adds with the neomycin standard solution is that horizontal ordinate is mapped on EXCEL, promptly gets the inhibition typical curve of neomycin.

Claims (6)

1. the residual step ELISA detection method of neomycin in the milk is characterized in that comprising the synthetic of neomycin antigen and identifying the mensuration of neomycin antiserum titre and the specific detection of neomycin antiserum;

(1) neomycin antigen is synthetic

Utilize synthetic neomycin immunogene of glutaraldehyde method and coating antigen;

(2) the immunogenic evaluation of neomycin

The neomycin immunogene of utilizing step (1) to obtain is identified antigen by infra-red sepectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE;

(3) preparation neomycin antiserum

The neomycin immunogene of utilizing step (1) to obtain prepares anti-neomycin antiserum, i.e. neomycin antibody by immunity;

(4) mensuration of neomycin antibody titer

The neomycin antibody that utilizes step (3) to obtain utilizes neomycin coating antigen coated elisa plate, measures tiring of neomycin antibody by indirect elisa method;

(5) mensuration of neomycin antibody specificity

The neomycin antibody that utilizes step (3) to obtain according to tiring and the suitableeest working concentration of step (4) gained neomycin antibody, utilizes a step ELISA method to detect the specificity of neomycin antibody.

2. the residual step ELISA detection method of neomycin in the milk according to claim 1 is characterized in that neomycin antigen: immunogene and coating antigen synthetic:

1. take by weighing 80 mg neomycin NEO, 40mg bovine serum albumin(BSA) BSA or ovalbumin OVA are dissolved in the phosphate buffer of 10 mL, 0.01 mol/L;

2. fully dropwise add 1.5mL mass concentration 1% glutaraldehyde solution after the dissolving, stirring at room is reacted 30 min;

3. add 112 mg NaBH ₄, leave standstill 1h in 4 ℃ of refrigerators;

4. reactant liquor was dialysed 3 days with the 0.01mol/L phosphate buffer, and 6h changes liquid at interval;

Gained NEO-BSA conjugate is an immunogene, and gained NEO-OVA conjugate is a coating antigen.

3. the residual step ELISA detection method of neomycin in the milk according to claim 1 is characterized in that utilizing infra-red sepectrometry to carry out the immunogenic evaluation of neomycin:

1. take by weighing the NEO standard items respectively, each 2 mg of BSA and NEO-BSA sample;

2. add dry potassium bromide KBr 200 mg respectively, potpourri places agate mortar, mixing, fully grinding under the infrared lamp irradiation;

3. the compression mold of packing at 10 tons of pressure lower sheeting 10 min, makes the transparent KBr medicine compressing tablet of thickness 1 mm;

4. produce corresponding KBr-NEO respectively, KBr-BSA, and 3 sample compressing tablets of KBr-NEO-BSA go up the infrared spectrometer Test Identification subsequently.

4. the residual step ELISA detection method of neomycin in the milk according to claim 1 is characterized in that utilizing the SDS-PAGE gel electrophoresis to carry out the immunogenic evaluation of neomycin:

1. separation gel 10%, concentrates glue 5%, voltage 100V, electric current 20mA, electrophoresis time 1.5h;

2. immobile liquid is 12.5% trichloroacetic acid solution, fixedly 30min;

3. the immobile liquid dyeing 1h that contains 0.1% Coomassie brilliant blue G250;

4. ultra-low molecular amount standard protein molecular weight is respectively: rabbit myosin 200000, calmodulin 130000, rabbit phosphorylase B 97400, bovine serum albumin(BSA) 66200, rabbit actin 43000;

With the logarithm mapping of the mobility of the standard protein of known molecular amount, obtain a typical curve to molecular weight;

5. when molecular weight when 15KD is between 200KD, the logarithm of the molecular weight of the mobility of protein in the SDS-PAGE electrophoresis and it is linear, meets following formula: log (Mw)=k-b * R _f, in the formula: Mw is a protein molecular weight, R _fBe mobility, k, b are constant, and the testing protein of unknown molecular amount is carried out the SDS-PAGE electrophoresis under the same conditions, try to achieve its molecular weight according to its electrophoretic mobility on typical curve.

5. the residual step ELISA detection method of neomycin in the milk according to claim 1 is characterized in that preparing the neomycin antiserum, and carries out the mensuration of neomycin antibody titer:

1. wrap quilt: neomycin coating antigen NEO-OVA is cushioned liquid with bag does to add in the 96 hole ELISA Plate behind the serial gradient dilution and wrap quilt, 100 μ L/ holes, in 4 ℃ of refrigerator overnight, take out ELISA Plate and be back to room temperature next day, dries, 200 μ L 0.01mol/L PBST solution are injected as cleansing solution in every hole, the 3min that vibrates on the horizontal shaking table firmly gets rid of cleansing solution, pats dry on thieving paper, repeated washing 3 times, following washing methods is identical;

2. sealing: fully after the washing, add the PBST confining liquid sealase target of 0.01M, 200 μ L/ holes, behind the incubation 2h, repeated washing 3 times is put into baking oven 10min oven dry back and is taken out stand-by in 37 ℃ of baking ovens;

3. competition: will resist the positive antiserum dilution series of neomycin gradient, correspondence joins ELISA Plate 7 row, and eighth row adds negative serum, and 100 μ L/ holes are hatched washing behind the 30min, patted dry for 37 ℃;

4. add two anti-: every hole adds 100 μ L, and the ELIAS secondary antibody HRP-IgG of 1:3000 dilution is hatched washing behind 30 min, patted dry in 37 ℃ of baking ovens;

5. colour developing: every hole adds 100 μ L colour developing liquid, and TMB and substrate buffer solution volume proportion are 1:5, and 15 min are reacted in 37 ℃ of baking oven dark places;

6. stop: take out every hole, back and add 100 μ L stop buffers, i.e. the sulfuric acid of 2 mol/L is measured the light absorption value A at 450nm place with microplate reader ₄₅₀

Select the A of positive serum ₄₅₀Value is at 1.5-1.9, simultaneously with the A of negative serum ₄₅₀Value difference is a best effort concentration apart from coating antigen dilute concentration, the antibody dilution concentration of maximum;

The A of positive serum ₄₅₀2.1 times of 〉=negative control hole, and A ₄₅₀Greater than 0.1, this moment, the extension rate of serum was tiring of serum.

6. the residual step ELISA detection method of neomycin in the milk according to claim 1 is characterized in that utilizing a step ELISA method to carry out the mensuration of neomycin antibody specificity:

1. wrap quilt: get the ELISA Plate in two 8 holes, detect the selected the best bag that comes out with the working point and by concentration coating antigen is carried out serial gradient dilution and wraps quilt, 100 μ L/ holes, two contrasts are set, place 4 ℃ of refrigerators to preserve and spend the night, PBST washing lotion washing three times, each 3 min pat dry;

2. sealing: with the PBST confining liquid sealing of 0.01M, 200 μ L/ holes place 4 ℃ of refrigerators to preserve and spend the night, PBST washing lotion washing three times, and each 3 min pat dry;

3. competition: first row adds antibody diluent, second queues the neomycin standard solution that adds 7 concentration respectively, and selected best dilute concentration dilution antibody is detected with the working point in 50 μ L/ holes, add in 50 μ L/ holes, each hole all adds the ELIAS secondary antibody of 1:3000 dilution simultaneously, 100 μ L/ holes, fully vibration on oscillator, place 30 min for 37 ℃, PBST washing lotion washing five times, each 3 min pat dry;

4. colour developing: add colour developing liquid, substrate buffer solution and TMB volume ratio 5:1,100 μ L/ holes, 37 ℃ of reaction 15 min;

5. stop: add stop buffer, the 2 mol/L concentrated sulphuric acids, each hole absorbance is detected in 100 μ L/ holes on the microplate reader;

6. obtain the B/B of each concentration ₀: B/B ₀=B _{Corrected value}/ B _{0 corrected value}

B _{Corrected value}=Ab _{The actual measurement average}-Ab _{Blank average}

B _{0 corrected value}Light absorption value when being 0 ng/mL, the i.e. corrected value of negative control hole for neomycin standard solution interpolation concentration;

IC ₅₀For reaching light absorption value is maximum light absorption value A _MaxOne half added neomycin standard items concentration, with B/B ₀Be ordinate, the logarithm value of the serial gradient concentration that adds with the neomycin standard solution is that horizontal ordinate is mapped on EXCEL, promptly gets the inhibition typical curve of neomycin.

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