CN104569398B - The enzyme linked immunological kit of detection ethoxy quinoline and application thereof - Google Patents

The enzyme linked immunological kit of detection ethoxy quinoline and application thereof Download PDF

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CN104569398B
CN104569398B CN201310499100.9A CN201310499100A CN104569398B CN 104569398 B CN104569398 B CN 104569398B CN 201310499100 A CN201310499100 A CN 201310499100A CN 104569398 B CN104569398 B CN 104569398B
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ethoxy quinoline
quinoline
ethoxy
antibody
test kit
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CN104569398A (en
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罗晓琴
朱亮亮
万宇平
何方洋
冯才伟
崔海峰
余厚美
扶胜
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Beijing Kwinbon Biotechnology Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes

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Abstract

The invention provides a kind of enzyme linked immunological kit detecting ethoxy quinoline, it includes: be coated with the ELISA Plate of coating antigen, ethoxy quinoline standard solution, ELIAS secondary antibody, ethoxy quinoline specific antibody, substrate nitrite ion, stop buffer, cleaning mixture, redissolution liquid, sample extraction agent, described coating antigen is ethoxy quinoline coupled antigen, and described ELIAS secondary antibody is the sheep anti mouse anti antibody of enzyme labelling。The invention also discloses a kind of method applying above-mentioned enzyme linked immunological kit detection ethoxy quinoline, it includes: first carries out sample pre-treatments, then detects with test kit, ultimate analysis testing result。Enzyme linked immunological kit provided by the invention can be used for detecting the content of ethoxy quinoline in tilapia, shrimp sample, it is easy and simple to handle, low cost, highly sensitive, can the examination of on-site supervision and applicable great amount of samples。

Description

The enzyme linked immunological kit of detection ethoxy quinoline and application thereof
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, be specifically related to a kind of enzyme linked immunological kit for detecting ethoxy quinoline, can the residual quantity of ethoxy quinoline medicine in qualitative and quantitative analysis tilapia, shrimp sample。
Background technology
Ethoxy quinoline (Ethoxyquin) has another name called 6-ethyoxyl-2,2,4-trimethyl-1,2-dihyaroquinoline, it is one of the feed antioxidant of function admirable, is most economical antioxidant, it is adaptable to premix material, fish flour and add the product of fat, can prevent vitamin A. D. E therein etc. and fat oxygen from dividing rotten natural pigment oxidation stain, and have certain mildew-resistant and preservation；Separately can as food antioxidant, fruit antistaling agent, rubber antioxidant。Owing to ethoxy quinoline is to the toxic effect of human body, therefore, the developed country such as American-European in succession forbids or strictly prohibits the use of。
At present, the detection method of ethoxy quinoline residual is less, mainly has high-efficient liquid phase technique to coordinate UV-detector, and gas chromatography coordinates nitrogen phosphorous detector, Liquid Chromatography-Mass Spectrometry。These methods have the advantages such as highly sensitive, result is accurate, but the input cost such as fund and personnel is higher。Present invention application euzymelinked immunosorbent assay (ELISA), measures the residual quantity of ethoxy quinoline medicine in tilapia and shrimp, has that detection limit is low, high specificity, easy and simple to handle, speed is fast, testing cost is low in detection, is very easy to the advantages such as popularization。
Summary of the invention
It is an object of the invention to provide a kind of enzyme linked immunological kit of ethoxy quinoline drug residue in tilapia, shrimp sample that can detect, and provide a kind of efficiently, accurately, easy, be suitable to the qualitative and quantitative analysis method of batch samples screening。
Test kit of the present invention, it includes: be coated with the ELISA Plate of coating antigen, ethoxy quinoline standard solution, ELIAS secondary antibody, ethoxy quinoline specific antibody, substrate nitrite ion, stop buffer, cleaning mixture, redissolution liquid, sample extraction agent, described coating antigen is ethoxy quinoline coupled antigen, and described ELIAS secondary antibody is the sheep anti mouse anti antibody of enzyme labelling。
Described ethoxy quinoline coupled antigen is to be obtained by ethoxy quinoline hapten and carrier protein couplet, described ethoxy quinoline hapten is to be obtained by ethoxy quinoline and phthalic anhydride, and described carrier protein can be Mus serum albumin, thyroprotein, bovine serum albumin, rabbit serum proteins, human albumin, ovalbumin, hemocyanin or Fibrinogen。
Described ethoxy quinoline specific antibody is to prepare using ethoxy quinoline coupled antigen as immunogen, described ethoxy quinoline specific antibody can be ethoxy quinoline monoclonal antibody or ethoxy quinoline polyclonal antibody, wherein preferred ethoxy quinoline monoclonal antibody。
The marker enzyme of described enzyme marker is horseradish peroxidase or antibacterial extraction alkaline phosphatase, wherein preferred horseradish peroxidase；ELIAS secondary antibody is to adopt the Over-voltage protection after improveing to carry out coupling to obtain。
For more convenient on-site supervision and great amount of samples examination, described test kit also includes ethoxy quinoline standard solution, substrate nitrite ion, stop buffer, cleaning mixture, redissolution liquid, sample extraction agent。
Described ethoxy quinoline standard solution 5 bottles, concentration respectively 0 μ g/L, 0.75 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L。
When marker enzyme is horseradish peroxidase, described substrate nitrite ion is made up of substrate solution A liquid and substrate solution B liquid, and A liquid is hydrogen peroxide or urea peroxide, and B liquid is o-phenylenediamine or tetramethyl benzidine, and described stop buffer is sulphuric acid or the hydrochloride buffer of 1～2mol/L；When marker enzyme is antibacterial extraction alkaline phosphatase, described substrate nitrite ion is to nitro phosphate buffer, and described stop buffer is 1～2mol/L sodium hydroxide solution。
It is 7.4 that described cleaning mixture is preferably pH value, containing 0.5%～1.0% tween 20,0.01 ‰～0.03 ‰ sodium azide preservatives, 0.1～0.3mol/L phosphate buffer, described percentage ratio is percent weight in volume。
Described redissolution liquid be preferably pH value be 7.0, the phosphate buffer of 0.02mol/L, described percentage ratio is percent weight in volume。
Described sample extraction agent is the hydrochloric acid of 0.1mol/L。
Wherein in ELISA Plate preparation process, the used buffer that is coated be pH value is 9.6, the carbonate buffer solution of 0.05mol/L, confining liquid is pH value is 7.1～7.5, and containing the phosphate buffer of 1%～3% casein, 0.1～0.3mol/L, described percentage ratio is percent weight in volume。
In the present invention, the preparation process of ELISA Plate is: with being coated buffer, coating antigen is diluted to 20 μ g/ml, every hole adds 100 μ l, 25 DEG C of lucifuges hatch 2h or 4 DEG C overnight, and liquid in hole of inclining washs 2 times with cleaning mixture, each 30s, patting dry, then add 150～200 μ l confining liquids in every hole, 25 DEG C of lucifuges hatch 1～2h, the liquid in hole that inclines pats dry, and dried sealing by aluminum film vacuum preserves。
The Cleaning Principle of the present invention is:
This test kit adopts competitive ELISA method, pre-coated coupled antigen on ELISA Plate capillary strip, coupled antigen competition ethoxy quinoline antibody pre-coated on the ethoxy quinoline remained in sample and capillary strip, after adding ELIAS secondary antibody, develop the color with substrate, sample absorbance with its contained by the content of residue ethoxy quinoline become negative correlation, be multiplied by its corresponding extension rate with standard curve more again, the residual quantity of ethoxy quinoline in sample can be drawn。
Present invention also offers a kind of method applying above-mentioned enzyme linked immunological kit detection ethoxy quinoline, it includes step:
(1) sample pre-treatments；
(2) detect with test kit；
(3) testing result is analyzed。
The present invention detects the enzyme linked immunological kit of ethoxy quinoline and mainly adopts ELISA method qualitative or the content of ethoxy quinoline in detection by quantitative sample；Requiring low to the pre-treatment of sample, sample pretreatment process is simple, can quickly detect batch samples simultaneously；Main agents provides with the form of working solution, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, degree of accuracy is high, accuracy high。The enzyme linked immunological kit of the present invention, simple in construction, easy to use, low price, carrying convenience, detection method efficiently, accurately, easy, be suitable to the qualitative, quantitative of batch samples screening。
Accompanying drawing explanation
Fig. 1: ethoxy quinoline hapten synthesis route map
Fig. 2: ethoxy quinoline hapten hydrogen nuclear magnetic resonance spectrogram
Fig. 3: kit standard curve chart
Detailed description of the invention
The present invention is expanded on further below in conjunction with specific embodiment。Should be understood that these embodiments are merely to illustrate the present invention, without limiting the scope of the present invention。
The preparation of embodiment 1 reagent constituents
1, the haptenic preparation of ethoxy quinoline
440mg ethoxy quinoline, 360mg phthalic anhydride and 2mL pyridine mixed liquor in 25mlDMSO, stirring reaction 15h at 80 DEG C, solvent is evaporated off, column chromatography purification obtains phthalic acid monosubstituted ethoxy quinoline amides。
Take above-mentioned product through nuclear magnetic resonance hydrogen spectruming determining, as in figure 2 it is shown, 7.8 and 8.3ppm near two groups of aromatic ring signal peaks increasing, and the carboxyl signal peak that about 13.3ppm increases, illustrates that hapten synthesis is successfully。
2, the preparation of antigen
Immunogen is prepared ethoxy quinoline hapten and is obtained immunogen with bovine serum albumin (BSA) coupling。
Take the 15mg ethoxy quinoline hapten prepared as stated above, be dissolved in 1mLDMF, obtain solution (1)；Take after 30mgEDC and NHS 0.2ml water fully dissolves in adding in (1), stir 24h under room temperature, reactant liquor A can be obtained；Weigh BSA100mg, so as to be substantially dissolved in 3.8mLPBS(PH7.2) in, reactant liquor A is dropwise slowly added dropwise in protein solution, and under room temperature, stirs 24h；With 0.01mol/lPBS, 4 DEG C of dialysis 3d, change 3 dialysis solution every day, obtain immunogen。
Coating antigen is prepared ethoxy quinoline hapten and is obtained immunogen with ovalbumin (OVA) coupling。
Take the 15mg ethoxy quinoline hapten prepared as stated above, be dissolved in 1mLDMF, obtain solution (1)；Take after 30mgEDC and NHS 0.2ml water fully dissolves in adding in (1), stir 24h under room temperature, reactant liquor A can be obtained；Weigh OVA100mg, so as to be substantially dissolved in 3.8mLPBS(PH7.2) in, reactant liquor A is dropwise slowly added dropwise in protein solution, and under room temperature, stirs 24h；With 0.01mol/lPBS, 4 DEG C of dialysis 3d, change 3 dialysis solution every day, obtain immunogen。
3, the preparation of ethoxy quinoline monoclonal antibody
Animal immune: immunogen above-mentioned steps obtained is injected in Balb/c Mice Body, immunizing dose is 150 μ g/ so that it is produce antiserum。
Cell fusion and cloning: after mice serum measurement result is higher, take its splenocyte, in 8:1(quantitative proportion) ratio and SP2/0 myeloma cell fusion, adopt indirect competitive ELISA to measure cell conditioned medium liquid, the positive hole of screening。Utilize limiting dilution assay that positive hole is carried out cloning, until obtaining the hybridoma cell strain of secretion ethoxy quinoline monoclonal antibody。
Cell cryopreservation and recovery: monoclonal hybridoma strain frozen stock solution is made 1 × 106The cell suspension of individual/ml, preserves for a long time in liquid nitrogen。Take out cryopreservation tube during recovery, be immediately placed in 37 DEG C of water-bath middling speeds and melt, after centrifugal segregation frozen stock solution, move into and cultivate culture in glassware。
The production of monoclonal antibody and purification: Balb/c mouse peritoneal is injected sterilizing paraffin oil 0.5ml/ only, the monoclonal hybridoma strain 5 × 10 that pneumoretroperitoneum injection in 7 days is stable5Individual/only, gather ascites after 7 days。Carry out ascites by sad-saturated ammonium sulfate method to purify ,-20 DEG C of preservations。
4, the preparation of enzyme mark disome
Over-voltage protection after sheep anti mouse anti antibody is adopted improvement with horseradish peroxidase (HRP) carries out coupling。
5, the preparation of ELISA Plate
With being coated buffer, coating antigen is diluted to 20 μ g/ml, every hole adds 100 μ l, 25 DEG C of lucifuges hatch 2h, liquid in hole of inclining, and wash 2 times with cleaning mixture, each 30s, patting dry, then add 200 μ l confining liquids in every hole, 25 DEG C of lucifuges hatch 2h, the liquid in hole that inclines pats dry, and dried sealing by aluminum film vacuum preserves。
Embodiment 2 detects the establishment of the enzyme linked immunological kit of ethoxy quinoline
Set up the enzyme linked immunological kit of detection ethoxy quinoline so that it is comprise following component:
(1) ELISA Plate of ethoxy quinoline coupled antigen it is coated；
(2) ethoxy quinoline standard solution 5 bottles, concentration respectively 0 μ g/L, 0.75 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L；
(3) with the sheep anti mouse anti antibody of horseradish peroxidase-labeled；
(4) ethoxy quinoline specific antibody；
(5) substrate nitrite ion is made up of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine；
(6) stop buffer is 2mol/L sulphuric acid；
(7) cleaning mixture is pH value is 7.4, containing 0.5%～1.0% tween 20,0.01 ‰～0.03 ‰ sodium azide preservatives, 0.1～0.3mol/L phosphate buffer, described percentage ratio is percent weight in volume；
(8) redissolve liquid to be pH value be 7.0, the phosphate buffer of 0.02mol/L, described percentage ratio is percent weight in volume；
(9) sample extraction agent is the hydrochloric acid of 0.1mol/L。
The detection of ethoxy quinoline in embodiment 3 tilapia, shrimp sample
1, sample pre-treatments
With homogenizer homogenizing sample；Weighing the tissue samples after 3.0 ± 0.05g homogenizing to 50ml polystyrene centrifuge tube, add 100 μ l sample extraction agent and 900 μ l deionized waters, whirling motion mixes, add 5ml acetonitrile, with vortex instrument whirling motion 2min, mixing, 3000g room temperature (20-25 DEG C) is centrifuged 5min；Pipette 3ml supernatant to 50ml polystyrene centrifuge tube, add the mixing of 1ml2M sodium hydroxide solution, add 6ml normal hexane, with the centrifugal 5min of vortex instrument whirling motion 3min, 3000g room temperature (20-25 DEG C)；Pipette 2ml supernatant to 10ml clean dried teat glass, flow down in 50-60 DEG C of nitrogen or air and dry up, add 1ml redissolution working solution vortex instrument whirling motion 2min；Take 50 μ l for analyzing。
2, detect with test kit
Adding standard substance working solution/sample 50 μ l in corresponding micropore, add antibody working solution 50 μ l/ hole, mixing of vibrating gently, with reacting 30min in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate。Carefully opening cover plate film, dried by liquid in hole, add wash operating solution 250 μ l/ hole, fully washing 4-5 time, every minor tick 10s, pat dry with absorbent paper。Add ELIAS secondary antibody 100 μ l/ hole, mixing of vibrating gently, with the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate react 30min, take out and wash plate。Adding substrate solution A liquid 50 μ l/ hole, add substrate solution B liquid 50 μ l/ hole, mixing of vibrating gently, with the 15min that develops the color in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate。Add stop buffer 50 μ l/ hole, mixing of vibrating gently, set microplate reader in 450nm place, measure every hole absorbance (OD)。
3, Analysis of test results
The percentage absorptance of standard substance or sample is equal to the meansigma methods (diplopore) meansigma methods divided by the absorbance of first standard substance (0 standard) of the absorbance of standard substance or sample, then is multiplied by 100%, obtains the percentage absorbance of standard substance or sample。With standard substance percentage absorptance for vertical coordinate, with the logarithm of ethoxy quinoline standard concentration (μ g/L) for abscissa, drawing standard curve chart。The percentage absorptance of sample is substituted in standard curve, from standard curve, read the concentration corresponding to sample, be multiplied by the actual concentrations that the extension rate of its correspondence is in sample ethoxy quinoline。
The determination test of embodiment 4 ethoxy quinoline technical parameter
1, test kit sensitivity and detection limit
Conventionally measure test kit sensitivity, standard curve range for 0.75～27 μ g/L, IC50(50% inhibition concentration) domain of walker is 1.8～2.5 μ g/L；20 parts of samples are detected, the concentration corresponding to each percentage absorbance is found from standard curve, representing detection limit with the meansigma methods of 20 parts of concentration of specimens plus 3 times of standard deviations, result shows, the detection of tilapia, shrimp sample is limit and is 2.25 μ g/kg by the method。
2, sample preci-sion and accuracy test
Using the response rate as accuracy estimating index, the testing result relative standard deviation (RSD%) of a certain concentration samples of replication is as precision evaluation index。Computing formula is: the response rate (%)=practical measurement value/theoretical value × 100%, and wherein theoretical value is the interpolation concentration of sample；Relative standard deviation RSD%=SD/X × 100%, wherein SD is standard deviation, and X is the meansigma methods of determination data。
Respectively by 2.25 μ g/kg, 4.50 μ g/kg, tri-concentration ethoxy quinoline of 9.00 μ g/kg tilapia, shrimp sample are added reclaiming and measure, each sample do 4 parallel, being measured with three batches of different reagent, the average recovery rate and the precision result that calculate sample are shown in following table。
Table 1 tilapia sample precision and accuracy test
Table 2 shrimp tissue samples precision and accuracy test
With 2.25 μ g/kg, 4.50 μ g/kg, 9.00 tri-concentration of μ g/kg ethoxy quinoline respectively tilapia, shrimp sample are added, average recovery rate is between 80.5%～99.4%；Batch in, batch between relative standard deviation be respectively less than 10%。
3, stabilization of kit test
Test kit preservation condition is 2～8 DEG C, and through the mensuration of 12 months, the maximum absorbance value (zero standard) of test kit, 50% inhibition concentration, ethoxy quinoline added practical measurement value all within normal range。Considering in transport and use procedure, have improper preservation condition and occur, being placed 7 days under 37 DEG C of preservation conditions by test kit, be accelerated senile experiment, result shows that this test kit indices complies fully with requirement。Considering that test kit freezing situation occurs, test kit is put into-20 DEG C of refrigerator freezings 7 days, measurement result also indicates that test kit indices is completely normal。Can show that test kit at least can preserve more than 12 months at 2～8 DEG C from the result above。

Claims (8)

1. the enzyme linked immunological kit detecting ethoxy quinoline, it is characterized in that including: be coated with the ELISA Plate of coating antigen, ethoxy quinoline standard solution, ELIAS secondary antibody, ethoxy quinoline specific antibody, substrate nitrite ion, stop buffer, cleaning mixture, redissolution liquid, sample extraction agent, described coating antigen is ethoxy quinoline coupled antigen, described ELIAS secondary antibody is the sheep anti mouse anti antibody of enzyme labelling, described ethoxy quinoline coupled antigen is to be obtained by ethoxy quinoline hapten and carrier protein couplet, described ethoxy quinoline hapten is to be obtained by ethoxy quinoline and phthalic anhydride, molecular structural formula is:
2. test kit as claimed in claim 1, it is characterised in that the haptenic preparation method of described ethoxy quinoline is as follows:
440mg ethoxy quinoline, 360mg phthalic anhydride and 2mL pyridine mixed liquor in 25mlDMSO, stirring reaction 15h at 80 DEG C, solvent is evaporated off, column chromatography purification obtains phthalic acid monosubstituted ethoxy quinoline amides。
3. test kit as claimed in claim 1, it is characterised in that described carrier protein is Mus serum albumin, thyroprotein, bovine serum albumin, rabbit serum proteins, human albumin, oralbumin, hemocyanin or Fibrinogen。
4. test kit as claimed in claim 1, it is characterized in that described ethoxy quinoline specific antibody is to prepare using ethoxy quinoline coupled antigen as immunogen, described ethoxy quinoline specific antibody is ethoxy quinoline monoclonal antibody or ethoxy quinoline polyclonal antibody。
5. test kit as claimed in claim 1, it is characterized in that the marker enzyme of described ELIAS secondary antibody is horseradish peroxidase or antibacterial extraction alkaline phosphatase, when marker enzyme is horseradish peroxidase, substrate nitrite ion A liquid is hydrogen peroxide or urea peroxide, substrate nitrite ion B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is the sulphuric acid of 1～2mol/L；When marker enzyme is antibacterial extraction alkaline phosphatase, stop buffer is 1～2mol/L sodium hydroxide。
6. test kit as claimed in claim 1, it is characterized in that described cleaning mixture be pH value is 7.4, containing the tween 20 that percent weight in volume is 0.5%～1.0%, 0.01 ‰～0.03 ‰ sodium azide preservatives, 0.1～0.3mol/L phosphate buffer；Described redissolution liquid is pH value is 7.0, the phosphate buffer of 0.02mol/L, and described sample extraction agent is the hydrochloric acid of 0.1mol/L。
7. test kit as claimed in claim 1, it is characterised in that the concentration of described ethoxy quinoline standard solution respectively 0 μ g/L, 0.75 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L。
8. detect a method for ethoxy quinoline content in sample, including step:
(1) sample pre-treatments；
(2) detect with the test kit described in any one of claim 1～7；
(3) testing result is analyzed。
CN201310499100.9A 2013-10-22 2013-10-22 The enzyme linked immunological kit of detection ethoxy quinoline and application thereof Active CN104569398B (en)

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CN103099061A (en) * 2012-12-13 2013-05-15 济南和美华饲料有限公司 Feed for improving the production performance of sows

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CN1963507A (en) * 2006-12-06 2007-05-16 华中农业大学 Immune testing method for enzyme linked retained 3-methylquinoxaline-2-carboxylic acid and reagent set
CN103099061A (en) * 2012-12-13 2013-05-15 济南和美华饲料有限公司 Feed for improving the production performance of sows

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