WO2009127094A1 - 检测林可霉素的酶联免疫试剂盒 - Google Patents

检测林可霉素的酶联免疫试剂盒 Download PDF

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WO2009127094A1
WO2009127094A1 PCT/CN2008/001668 CN2008001668W WO2009127094A1 WO 2009127094 A1 WO2009127094 A1 WO 2009127094A1 CN 2008001668 W CN2008001668 W CN 2008001668W WO 2009127094 A1 WO2009127094 A1 WO 2009127094A1
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Prior art keywords
lincomycin
solution
enzyme
antibody
liquid
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PCT/CN2008/001668
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English (en)
French (fr)
Inventor
沈建忠
何方洋
万宇平
冯才伟
赵正苗
冯才茂
汪善良
罗晓琴
马孝斌
Original Assignee
北京望尔康泰生物技术有限公司
北京望尔生物技术有限公司
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Priority to US12/988,111 priority Critical patent/US8507214B2/en
Publication of WO2009127094A1 publication Critical patent/WO2009127094A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1292—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Actinomyces; from Streptomyces (G)
    • 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
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9446—Antibacterials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates

Definitions

  • the invention relates to an enzyme-linked immunoassay technology, in particular to an enzyme-linked immunoassay kit for detecting lincomycin and an application thereof.
  • Lincomycin also known as lincomycin or lincomycin, is a lincosamide antibiotic produced by Streptomyces (structure shown in Figure 1) and has strong antibacterial activity. The accumulation of residues through the food chain can lead to bacterial resistance. Therefore, all countries have limited requirements.
  • the Ministry of Agriculture's Document No. 235 stipulates that the residue limit is 0.05ppm (eggs).
  • the Japanese affirmative list stipulates that the residue limit is 0.02ppm (chicken edible internal organs).
  • Another object of the invention is to provide a lincomycin antibody and a method of producing the same.
  • the lincomycin antibody has binding specificity to lincomycin which is free or bound to animal tissues or proteins and the like.
  • the lincomycin antibody may be a monoclonal antibody or a polyclonal antibody.
  • a further object of the present invention is to provide a lincomycin hapten and a method for synthesizing the same, and coupling the hapten with a macrocarrier protein to obtain a lincomycin immunogen.
  • the present invention provides an enzyme-linked immunosorbent kit for detecting lincomycin, which comprises a coating original and an enzyme label selected from the group consisting of lincomycin hapten and a carrier. a protein conjugate, a lincomycin antibody, and an anti-antibody; when the coating is originally a conjugate of a lincomycin hapten and a carrier protein, the enzyme label is an enzyme-labeled anti-antibody; When the coating is originally a lincomycin antibody, the enzyme label is an enzyme-labeled lincomycin-conjugated antigen; when the coating is originally an anti-antibody, the enzyme label is an enzyme The conjugated antigen; the lincomycin hapten is obtained by a condensation reaction of lincomycin and succinic anhydride.
  • the above kit may further comprise a lincomycin standard solution, a color developing solution, a concentrated washing liquid, a stopping liquid and a concentrated complex solution;
  • the concentrated washing liquid has a pH of 7.3-7.9 and contains 0.8-1.3 wt% Tween- 20, 0.1-0.6 wt% 3 ⁇ 4 lanthanum mercury 0.1-0.3 mol / L phosphate buffer;
  • the concentrated complex solution is a pH value of 7.6-8.0 0.3-0.7 mol / L phosphate buffer;
  • the coating buffer The solution is a 0.1-0.3 mol/L boric acid buffer having a pH of 8.6-9.0;
  • the blocking solution contains 8-12 wt% calf serum and 0.03-0.07 wt%.
  • the substrate color developing solution is composed of a color developing liquid A liquid and a color developing liquid liquid B, and the color developing liquid A liquid is hydrogen peroxide.
  • the color solution B solution is o-phenylenediamine or tetramethylbenzidine;
  • the stop solution is 1-2 mol/L sulfuric acid or hydrochloric acid solution.
  • the concentrated washing liquid is 1.0 to 1.5 wt% Tween 20 and 0.5 wt% sulfur willow.
  • the mercury preservative has a pH of 7.3 O.lmol/L phosphate buffer; the concentrated complex solution is a 0.2 mol/L phosphate buffer having a pH of 7.8; the coating buffer is 0.2 at pH 9.0. Mol/L boric acid buffer; the blocking solution contains 8-12 wt% calf serum and 0.03-0.07 wt%.
  • Sodium azide has a pH of 7.4 of 0.05 mol/L phosphate buffer.
  • the lincomycin antibody of the present invention may be a lincomycin monoclonal antibody or a lincomycin polyclonal antibody, which is obtained by using a conjugate of a lincomycin hapten and a carrier protein as an immunogen;
  • the carrier protein may be murine serum albumin, thyroprotein, bovine serum albumin, rabbit serum albumin, human serum albumin, ovalbumin, hemocyanin or fibrinogen.
  • the labeling enzyme used for the enzyme label in the kit of the present invention is horseradish peroxidase or alkaline phosphatase.
  • the color developing agent is composed of the coloring liquid A liquid and the color developing liquid liquid B, and the color developing liquid liquid A is hydrogen peroxide or urea peroxide, and the color developing liquid B liquid
  • the stop solution is 1 ⁇ 2mol/L sulfuric acid or hydrochloric acid solution
  • the color developer is nitrophosphate buffer solution
  • the stop solution is 1 ⁇ 2mol/L sodium hydroxide solution.
  • the anti-antibody in the kit of the present invention is preferably a goat anti-mouse anti-antibody.
  • the present invention also provides lincomycin monoclonal hybridoma cells C-1-3 having the accession number CGMCC No. 2398, and lincomycin monoclonal antibodies secreted therefrom.
  • Lincomycin is a small molecule, only immunoreactive, non-immunogenic, and can not induce immune response in the body.
  • a lincomycin-specific antibody In order to prepare a lincomycin-specific antibody, it must first be structurally modified to obtain a derivative similar to the lincomycin parent structure (ie, lincomycin hapten), and the derivative has a macromolecular carrier protein.
  • the functional group is coupled and finally coupled to obtain a lincomycin immunogen or a coating.
  • the lincomycin hapten can be obtained by reacting lincomycin with an organic small molecule containing a reactive group such as a carboxyl group, an amino group or an aldehyde group, and the purpose thereof is to highlight characteristic groups in the lincomycin parent structure. Providing a reactive group covalently bound to a macromolecular carrier protein to enhance the immunogenicity of lincomycin.
  • the above hapten synthesis method is structurally modified for the active group in the molecular structure of lincomycin, and the specificity and binding force of the finally produced antibody are different depending on the selected group and the binding site.
  • Lincomycin molecular structure modification site and hapten structure are shown in Figure 2.
  • the 3', 4', 5, and 2 on the ring in the molecular structure of lincomycin are hydroxyl groups, 4 is an imino group, and X is a small molecule branch in the hapten structure. Its purpose is to highlight
  • lincomycin drug characteristic structure for example, X may be a branch having 3 to 6 carbon atoms, and R is a carboxyl group or an amino group.
  • the present invention selects a hydroxyl group as a hapten modification site in view of practical operation difficulty.
  • the 3', 4', and 5' hydroxyl groups are located on the ring, and the reaction characteristics and antigen modification and final antibody preparation theory are not much different.
  • the hydroxyl group on the branch has a large difference from the above three hydroxyl groups.
  • the present invention employs two technical schemes: hapten modification of the hydroxyl groups on the ring and branches in the molecular structure of lincomycin, respectively, and selection of the preferred scheme based on the difference in the final preparation of the antibody.
  • Figures 3 and 4 show two lincomycin hemisuccinate haptens, respectively.
  • lincomycin is directly reacted with succinic anhydride. Due to the difference in steric hindrance of each hydroxyl group, succinic anhydride will bind to the hydroxyl group No. 2 on the branched chain to obtain the hapten LIN-S1.
  • succinic anhydride due to the presence of a protecting group Group, succinic anhydride binds to the hydroxyl group on the ring and eventually hydrolyzes The reaction reduces the branched hydroxyl group to give the hapten LIN-S2.
  • the difference in the final antibody preparation effect of the two haptens can be embodied in the later description of the present invention.
  • the carrier protein to which the above hapten binds may be a common carrier protein selected from the group consisting of murine serum albumin, thyroxin, bovine serum albumin, rabbit serum albumin, human serum albumin, ovalbumin, hemocyanin, and fibrinogen.
  • the carrier protein is rich in functional groups such as a carboxyl group, an amino group and the like which are covalently bonded to the lincomycin hapten.
  • the invention combines the lincomycin hapten with the carrier protein by the mixed acid anhydride method or the carbodiimide method, highlights the characteristic structure of the lincomycin and increases the immunogenicity of the lincomycin hapten.
  • the binding molar ratio of the lincomycin semi-antigen to the carrier protein such as bovine serum albumin in the present invention is preferably 12 to 15:1.
  • the lincomycin-specific antibody of the present invention may be a lincomycin monoclonal antibody or a lincomycin polyclonal antibody.
  • the lincomycin monoclonal antibody is preferably a lincomycin mouse monoclonal antibody, more preferably a monoclonal antibody secreted by the hybridoma cell line C-1-3CGMCC No. 2398.
  • the lincomycin monoclonal hybridoma cell line C-1-3CGMCC No. 2398 (category designation: monoclonal hybridoma cell line for lincomycin drug) has been preserved in Chinese microorganisms on March 12, 2008. Common Microbiology Center of the Culture Collection Management Committee (CGMCC, Address: Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101).
  • the lincomycin polyclonal antibody may be a murine, equine, sheep, rabbit or guinea pig antibody, preferably a lincomycin rabbit polyclonal antibody.
  • lincomycin can be used to obtain the hapten LIN-S1 by succinic anhydride method.
  • the synthetic route is shown in Fig. 3.
  • the number of hydroxyl groups on the branch is the least, firstly protected with tert-butyl-dimethylchlorosilane, and then acylated with the 4' hydroxyl group on the ring by succinic anhydride method, then p-toluenesulfonic acid In the presence of hydrolysis, the protecting group is removed to obtain the hapten LIN-S2.
  • the synthetic route is shown in Fig. 4.
  • the lincomycin hapten is coupled with a carrier protein by a mixed acid anhydride method to obtain an immunogen.
  • mice were used as immunized animals, and the lincomycin hapten and carrier protein conjugate were used as immunogens. After the serum titer was greater than 1:3000, the spleen was taken out for cell fusion.
  • New Zealand white rabbits were used as immunized animals, New Zealand white rabbits were immunized with lincomycin hapten and carrier protein conjugate as immunogens, and serum antibody titers were determined after multiple immunizations. After the titer is greater than 1:10000, the rabbit is sacrificed and serum is collected to obtain a polyclonal antibody.
  • the goat anti-mouse anti-antibody is a sheep immunized animal, and the murine antibody is used as an immunogen to immunize the disease-free pathogenic sheep to obtain a goat anti-mouse anti-antibody;
  • the goat anti-rabbit anti-antibody is a sheep immunized animal, and the rabbit-derived antibody is used as an immunogen to immunize the disease-free pathogenic sheep to obtain a goat anti-rabbit anti-antibody.
  • the lincomycin test method and test kit provided by the present invention can be used for detecting lincomycin drugs in samples such as animal tissues and honey.
  • the kit includes a coating precursor and an enzyme label, and may further include a lincomycin standard solution, a substrate color developing solution, a finishing solution, a concentrated washing solution, and a concentrated complex solution.
  • the coating is originally a conjugate of a lincomycin hapten and a carrier protein, a lincomycin antibody or an anti-antibody;
  • the enzyme label is an enzyme-labeled antibody, an enzyme-labeled lincomycin hapten and a carrier protein Conjugate (herein, the conjugate of lincomycin hapten and carrier protein is also called lincomycin-conjugated antigen) or enzyme-labeled anti-antibody; when the coating is originally lincomycin half
  • the enzyme label is an enzyme-labeled anti-antibody;
  • the enzyme label is an enzyme-labeled lincomycin-conjugated antigen;
  • the enzyme label is an enzyme-labeled lincomycin-conjugated antigen.
  • the lincomycin standard solution may be a solution having concentrations of 0, 0.2, 0.6, 1.8, 5.4 and 16.2 g/L, respectively.
  • the labeling enzyme of the enzyme label is horseradish peroxidase or alkaline phosphatase, wherein horseradish peroxidase is preferred; enzyme-labeled goat anti-mouse anti-antibody or goat anti-rabbit anti-antibody is glutaraldehyde
  • the labeling enzyme is coupled with an anti-antibody by a method or a sodium periodate method.
  • the color developing agent is composed of the coloring liquid A liquid and the color developing liquid liquid B, and the color developing liquid A liquid is hydrogen peroxide or urea peroxide, and the color developing liquid B liquid
  • the stop solution is 1 ⁇ 2mol/L of sulfuric acid or hydrochloric acid buffer; when the labeling enzyme is alkaline phosphatase, the color developing solution is p-nitrophosphate buffer, The stop solution is 1 ⁇ 2mol/L sodium hydroxide solution.
  • the concentrated washing liquid may be a pH value of 7.3-7.9, a 0.1-0.3 mol/L phosphate buffer solution containing 0.8-1.3 wt% Tween-20 and 0.1-0.6 wt% thimerosal; the concentrated complex solution may be pH A value of 7.6-8.0 of 0.3-0.7 mol/L phosphate buffer.
  • the detection principle of the present invention is:
  • the lincomycin-conjugated antigen When the lincomycin-conjugated antigen is pre-coated on the microplate, after adding the sample solution or the standard solution, the lincomycin-specific antibody solution is added, and the lincomycin drug and the enzyme label remaining in the sample are added.
  • the lincomycin-conjugated antigen coated on the plate competes with the lincomycin-specific antibody, and the enzyme-labeled anti-antibody is added for amplification, and the color developing solution is used for color development, and the sample is absorbed.
  • the value is inversely related to the content of lincomycin drug, and the residual amount of lincomycin in the sample can be obtained by comparison with the standard curve.
  • the color concentration of the lincomycin residue in the sample can be roughly judged by comparing the color of the series of lincomycin standard solution.
  • the enzyme-labeled lincomycin-conjugated antigen solution is added, and the residual lincomycin drug in the sample is
  • the enzyme-labeled antigen competes for the lincomycin-specific antibody coated on the ELISA plate, and the color is developed with a color developing solution.
  • the absorbance of the sample is negatively correlated with the content of the lincomycin drug, which is obtained by comparison with the standard curve.
  • the residual content of lincomycin in the sample At the same time, according to the color depth of the microplate, the color concentration of the lincomycin residue in the sample can be roughly judged by comparing the color of the series of lincomycin standard solution.
  • the enzyme-labeled lincomycin-specific antibody solution is added, and the lincomycin drug remaining in the sample is
  • the lincomycin-conjugated antigen coated on the plate was competitive with lincomycin-specific antibody, and the color was developed with a color developing solution.
  • the absorbance of the sample was negatively correlated with the content of lincomycin, which was compared with the standard curve.
  • the residual content of lincomycin in the sample can be obtained.
  • the color concentration of the lincomycin residue in the sample can be roughly judged by comparing the color of the series of lincomycin standard solution.
  • the sample solution or the standard solution is added, and then the enzyme-labeled lincomycin-conjugated antigen solution is added, and the remaining forest in the sample can be
  • the cytomycin-conjugated antigen competes with the lincomycin-conjugated antigen for the lincomycin-specific antibody, and the color is developed with a color developing solution.
  • the absorbance of the sample is negatively correlated with the lincomycin drug content, and can be compared with the standard curve.
  • the residual content of lincomycin in the sample is obtained.
  • the color concentration of the lincomycin residue in the sample can be roughly judged by comparing the color of the series of lincomycin standard solution.
  • the invention also provides a method for detecting a lincomycin drug, which comprises the steps of:
  • the pretreatment of the sample is primarily to obtain a lincomycin solution from the sample for subsequent testing.
  • a kit in the present invention When detecting with a kit in the present invention: When the coating is originally lincomycin-conjugated antigen, a standard solution or a sample solution is added to the microplate of the microplate, and then the antibody is added, and after washing, the hole is removed.
  • the absorbance value is measured by a microplate reader; when the original anti-antibody is coated, the lincomycin antibody is added to the microplate of the microplate, washed after washing, and the liquid in the well is removed. And absorb the residual water with absorbent paper, add the standard solution or sample solution, add the enzyme labeled lincomycin hapten, wash after washing, remove the liquid in the hole, and absorb residual water with absorbent paper. Color, termination, absorbance values were measured with a microplate reader.
  • the analysis result of the detection result in the present invention is: using the average absorbance of the standard solution obtained for each concentration
  • the semi-logarithmic value of the concentration (g/L) of the lincomycin standard solution is the X-axis, and the percent absorbance is the ⁇ axis, and a standard curve is drawn. The same method is used to calculate the percent absorbance of the sample solution.
  • the concentration of lincomycin in the sample can be read from the standard curve corresponding to the concentration of each sample.
  • the analysis of the detection results in the present invention can also be carried out by using the regression equation method to calculate the concentration of the sample solution.
  • Figure 1 Chemical structural formula of lincosamide antibiotics
  • Figure 2 Lincomycin hapten engineered structural site
  • Figure 4 Synthetic route of lincomycin hapten LIN-S2;
  • Lincomycin is obtained by a succinic anhydride method to obtain a hapten having a carboxyl functional group.
  • hapten LIN-S1 Weigh 2.3g lincomycin hydrochloride (purchased from Dr., Germany, article number C14635000) and 0.5 g of succinic anhydride were placed in a 50 ml round bottom flask, and anhydrous pyridine was added thereto to completely dissolve, and the reaction was stirred under heating at 70 ° C for 24 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was washed several times with acetone, and the residue was washed with 2 to 3 times the volume of ethyl acetate-n-hexane (2:1, volume ratio) and shaken thoroughly. Crystallization gave the hapten lincomycin hemisuccinate.
  • the lincomycin hapten and bovine serum albumin are coupled by a mixed acid anhydride method to obtain an immunogen.
  • lincomycin hapten was dissolved in 0.1 ml of hydrazine, ⁇ '-dimethylformamide (DMF), cooled to 10 ° C, 2 ⁇ l of isobutyl chloroformate was added, and the reaction was stirred at 10 ° C for 30 minutes, 60 mg.
  • Bovine serum albumin was dissolved in 2 ml of 50 mmol/L Na 2 CO 3 , reacted at 10 ° C for 4 hours, then at 4 ° C overnight, after passing through Sephadex G-25 column, with buffer (pH 7.4, 0.2 mol/L phosphate).
  • Buffer equilibrate and elute, combine the liquid in the elution tube containing bovine serum albumin, place it in a dialysis bag and place it in pH 7.4, 0.2 mol/L phosphate buffer for 7 days, change the solution every day. ⁇ 4 times. Finally, the immunogen is stored frozen. The binding molar ratio of the lincomycin hapten to the carrier protein was determined to be 13:1.
  • the following is an illustrative illustration of coupling a lincomycin hapten to ovalbumin to give a coating.
  • the antibody prepared by the half antibody LIN-S2 has cross-reaction with various antibiotics, and the specificity is poor, and its IC 5Q value is 489 g/kg, which cannot satisfy the detection of lincomycin drug residue. demand.
  • the present invention employs a lincomycin monoclonal antibody prepared by the half antibody LIN-S1 as a preferred antibody, which is composed of a single gram.
  • the monoclonal hybridoma cell line of lincomycin was prepared into a cell suspension of 6 cells/ml with a cryopreservation solution, and stored for a long time in liquid nitrogen. When resuscitation, remove the cryotube and immediately put it into a 37-inch water bath for rapid fusion. After centrifugation to remove the frozen solution, transfer it to a culture flask for cultivation.
  • New Zealand white rabbits were used as immunized animals, and the lincomycin hapten and bovine serum albumin conjugate were used as immunogens.
  • the immunization dose was 1.5 mg/kg.
  • After 10 days of the last immunization blood was collected, serum antibody titer was measured, blood was collected from the heart, and polyclonal antibody was purified by octanoic acid-saturated ammonium sulfate method.
  • Solution preparation coating buffer pH 8.6, 0.2 mol/L boric acid buffer. 10.49 g of Na 2 B 4 O 7 .10H 2 O and 5.57 g of H 3 B0 3 were weighed and dissolved in 1 L of deionized water.
  • Blocking solution containing 10% by weight of calf serum and 0.05% by weight.
  • Sodium azide, pH 7.2, 0.05 mol/L phosphate buffer. Weigh 12.90 g of Na 2 HPO 4 _12H 2 O, 2.18 g of ⁇ 2 ⁇ 0 4 ⁇ 2 ⁇ 2 0, 5 g of azide dissolved in deionized water, and then add 100 ml of calf serum to a volume of 1 L.
  • Concentrated washing solution pH 7.4, containing 1.0 wt% Tween-20, 0.5% thimerosal, 0.2 mol/L of caprate buffer. Weigh 14.51g Na 2 HP0 4 '12H 2 0, 1.48g NaH 2 P0 4 -2H 2 0 dissolved in deionized water, add Tween-20 10ml and After 5 g of thimerosal, make up to 1 L with deionized water. Dilute the washing solution: Take an appropriate amount of concentrated washing solution and mix with deionized water in a volume ratio of 1:19 for use. Concentrated complex solution: 0.2 mol/L phosphate buffer containing 5% bovine serum albumin, pH 7.4.
  • the horseradish peroxidase-labeled goat anti-mouse anti-antibody used in the present invention is obtained by coupling a labeling enzyme with an anti-antibody by a glutaraldehyde method or a sodium periodate method.
  • a labeling enzyme with an anti-antibody by a glutaraldehyde method or a sodium periodate method.
  • An enzyme-linked immunoassay kit for detecting lincomycin is provided to include the following components -
  • the substrate coloring solution is composed of liquid A and liquid B, and the substrate coloring liquid A is urea peroxide, and the substrate coloring liquid B liquid tetramethylbenzidine;
  • the stop solution is 2 mol/L hydrochloric acid
  • the concentrated washing solution was pH 7.4, containing 1.0 wt% Tween-20, 0.5 wt% thimerosal, 0.2 mol/L phosphate buffer.
  • the concentrated complex solution was a 0.2 mol/L phosphate buffer containing 5% by weight of bovine serum albumin containing 5% by weight of bovine serum albumin and pH 7.4.
  • Example 3 Detection of lincomycin in actual samples 1. Sample preparation a) Solution preparation
  • hydrochloric acid solution for animal tissue samples: 1.25 ml of concentrated hydrochloric acid was added to a container containing deionized water to a volume of 1 L.
  • Methanol-hydrochloric acid solution (for animal tissue samples): Mix 10 ml of methanol and 60 ml of 0.015 mol/L hydrochloric acid solution and mix well.
  • the concentrated solution is diluted with deionized water in a volume ratio of 1:1, mixed and used.
  • animal tissue chicken, chicken liver, pork, pig liver, etc.
  • the absorbance value (B) of the standard solution obtained for each concentration was divided by the absorbance value (B Q ) of the first standard solution (0 standard) and multiplied by 100% to obtain a percent absorbance value.
  • the semi-logarithmic value of the lincomycin standard concentration ( ⁇ /L) is the X-axis, and the percent absorbance is the Y-axis, and a standard curve is drawn. The same method is used to calculate the percent absorbance of the sample solution, and the residual amount of lincomycin can be read from the standard curve corresponding to the concentration of each sample.
  • Example 4 Determination of kit quality
  • the muscle, liver and honey were added at a concentration of 2 ( ⁇ g/L), and five kits of five different batches were taken, each of which was repeated 5 times, and the coefficient of variation was calculated separately. See Tables 3 to 5.
  • the kit is stored at 2 ⁇ 8°C. After 6 months of measurement, the maximum absorbance of the kit (zero standard),
  • the 50% inhibitory concentration and the actual measured value of lincomycin addition were within the normal range. It is considered that during the transportation and use, abnormal storage conditions will occur.
  • the kit is placed under the storage condition of 37 ° C for 6 days, and the accelerated aging test is carried out. The results show that the indicators of the kit fully meet the requirements. Taking into account the freezing of the kit, the kit was frozen in a refrigerator at -20 °C for 5 days, and the results also showed that the kits were completely normal. From the above results, it can be concluded that the kit can be stored for at least 6 months at 2 ⁇ 8 °C.
  • Sample No. 1 2 3 4 5 6 7 8 Measured value 2.32 0.84 0.79 1.88 2.16 0.07 2.45 2.31
  • Sample No. 9 10 11 12 13 14 15 16 Measured value 1.64 1.00 1.14 1.99 2.68 1.86 2.78 1.14
  • Sample No. 17 18 19 20 Mean standard deviation Minimum detection limit 1.25 2.54 1.90 1.50 1.71 0.71 3.85 Table 8
  • Negative honey sample measurement results g/kg Sample number 1 2 3 4 5 6 7 8 Measured value 0.14 0.84 0.89 0.18 0.32 0.45 0.68 0.40
  • Sample number 9 10 11 12 13 14 15 16 Measured value 0.21 1.03 0.41 0.86 1.42 0.32 0.25 0.36 Sample No.

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Description

检测林可霉素的酶联免疫试剂盒 技术领域
本发明涉及酶联免疫检测技术， 具体涉及一种用于检测林可霉素的酶联免疫试剂盒 及其应用。
背景技术
林可霉素（ lincomycin )又称洁霉素或林肯霉素，是由链霉菌发酵产生的林可酰胺类抗 生素 （结构式如图 1)，有较强的抑菌活性。其残留通过食物链进入人体后累积可以导致细 菌耐药性， 因此， 各国都对其作出限量要求， 中国农业部 235 号文件规定其残留限量为 0.05ppm (鸡蛋)， 日本肯定列表规定其残留限量为 0.02ppm (鸡可食用内脏)。
林可霉素残留量的常规检测方法主要有高效液相色谱（HPLC)、 液相色谱-串联质谱 法 （LC-MS/MS )、 纸色谱等， 由于复杂的仪器设备和繁琐的过程以及对检验人员的高技 能要求， 不适合现场监控和大量样本的筛查。
发明概述
本发明的一个目的是提供一种用于林可霉素检测的酶联免疫试剂盒。使用该试剂盒进 行检测， 操作简单， 耗时少， 可用于如动物组织、 蜂蜜、 牛奶等样本中林可霉素药物的快 速检测， 尤其适合现场大批量样品的筛选检测。
本发明的另一目的是提供林可霉素抗体和生产制备该抗体的方法。所述的林可霉素抗 体对游离的或者与动物组织或蛋白等结合的林可霉素有结合特异性。所述的林可霉素抗体 可为单克隆抗体或多克隆抗体。
本发明的再一目的是提供一种林可霉素半抗原及其合成方法，并用所述半抗原与大分 子载体蛋白偶联得到林可霉素免疫原。
为实现本发明的目的， 本发明提供了一种检测林可霉素的酶联免疫试剂盒， 其包括 包被原和酶标记物，所述包被原选自林可霉素半抗原与载体蛋白的偶联物、林可霉素抗体 和抗抗体； 当所述包被原为林可霉素半抗原与载体蛋白的偶联物时，所述酶标记物为酶标 抗抗体； 当所述包被原为林可霉素抗体时，所述酶标记物为酶标林可霉素偶联抗原； 当所 述包被原为抗抗体时，所述酶标记物为酶标林可霉素偶联抗原；所述林可霉素半抗原是将 林可霉素和琥珀酸酐通过缩合反应得到的。上述试剂盒还可包括林可霉素标准溶液、显色 液、 浓缩洗涤液、 终止液和浓缩复溶液； 所述浓缩洗涤液为 pH值为 7.3-7.9、 含有 0.8-1.3 wt%吐温 -20、 0.1-0.6 wt%¾柳汞的 0.1-0.3mol/L磷酸盐缓冲液； 所述浓缩复溶液为 pH值 7.6-8.0的 0.3-0.7mol/L磷酸盐缓冲液； 所述包被缓冲液为 pH值 8.6-9.0的 0.1-0.3mol/L硼 酸缓冲液；所述封闭液为含有 8-12 wt%小牛血清和 0.03-0.07 wt%。叠氮化钠、 pH值 7.2-7.4 的 0.05mol/L磷酸盐缓冲液；所述底物显色液由显色液 A液和显色液 B液组成，显色液 A 液为过氧化氢或过氧化脲， 显色液 B 液为邻苯二胺或四甲基联苯胺； 所述终止液为 l~2mol/L硫酸或盐酸溶液。 优选地， 浓缩洗涤液为含 1.0~1.5wt%吐温 20和 0.5wt%硫柳 汞防腐剂的 pH值为 7.3的 O.lmol/L磷酸盐缓冲液；所述浓缩复溶液为 pH值 7.8的 0.2mol/L 磷酸盐缓冲液； 所述包被缓冲液为 pH值 9.0的 0.2mol/L硼酸缓冲液； 所述封闭液为含有 8-12wt%小牛血清和 0.03-0.07wt%。叠氮化钠的 pH值为 7.4的 0.05mol/L磷酸盐缓冲液。
本发明的林可霉素抗体可以是林可霉素单克隆抗体也可以是林可霉素多克隆抗体， 其是以林可霉素半抗原与载体蛋白的偶联物作为免疫原得到的；所述载体蛋白可以是鼠血 清蛋白、 甲状腺蛋白、 牛血清蛋白、 兔血清蛋白、 人血清蛋白、 卵清蛋白、 血蓝蛋白或纤 维蛋白原。
本发明试剂盒中的酶标记物所用的标记酶为辣根过氧化物酶或碱性磷酸酯酶。 当标 记酶为辣根过氧化物酶时， 所述显色剂由显色液 A液和显色液 B液组成， 显色液 A液为 过氧化氢或过氧化脲， 显色液 B液为邻苯二胺或四甲基联苯胺， 终止液为 l〜2mol/L硫 酸或盐酸溶液； 当标记酶为碱性磷酸酯酶时， 显色剂为硝基磷酸盐缓冲液， 终止液为 1〜 2mol/L氢氧化钠溶液。
本发明试剂盒中的抗抗体优选为羊抗鼠抗抗体。
本发明还提供了保藏号为 CGMCC No.2398的林可霉素单克隆杂交瘤细胞 C-1-3, 以 及由其分泌产生的林可霉素单克隆抗体。
发明详述
林可霉素是小分子物质， 只有免疫反应性， 没有免疫原性， 不能诱发机体产生免疫应 答。为了制备林可霉素特异性抗体，必须首先将其结构改造得到与林可霉素母体结构类似 的衍生物（即林可霉素半抗原），且使该衍生物具有能与大分子载体蛋白偶联的官能基团， 并最终偶联得到林可霉素免疫原或包被原。
所述林可霉素半抗原可通过将林可霉素与含有羧基、氨基、醛基等活泼基团的有机小 分子进行反应得到，其目的是突出林可霉素母体结构中的特征基团，提供与大分子载体蛋 白共价结合的活泼基团， 增强林可霉素的免疫原性。
上述半抗原的合成方法是针对林可霉素分子结构中的活泼基团进行结构改造， 根据 所选基团及结合位点的不同，最终所产生的抗体的特异性和结合力也不同。林可霉素分子 结构改造位点及半抗原结构通式如图 2所示。 林可霉素分子结构中的环上的 3'， 4'， 5, 及支链上的 2为羟基， 4为亚氨基， 半抗原结构通式中 X为小分子支链， 其目的是突出林 可霉素药物特征结构， 例如 X可为含有 3~6个碳原子的支链， R为羧基或氨基。
由于羟基较亚氨基活泼， 考虑到实际操作难度， 本发明选用羟基作为半抗原改造位 点。 图 2中 3'， 4'， 5'位羟基位于环上， 其反应特性及抗原改造和最终抗体制备理论差异 不大， 支链上 2号羟基， 与前述 3个羟基差异较大。 固本发明采用两种技术方案： 即分别 就林可霉素分子结构中环上和支链上的羟基进行半抗原改造，并根据最终制备抗体的差异 选取优选方案。
图 3和图 4分别示出两种林可霉素半琥珀酸酯半抗原。 图 3中， 将林可霉素直接与 琥珀酸酐进行反应， 由于各个羟基位阻差异，琥珀酸酐会与支链上 2号羟基结合而得到半 抗原 LIN-S1 ; 图 4中， 由于存在保护基团， 琥珀酸酐与环上羟基结合， 并最终通过水解 反应还原支链羟基而得到半抗原 LIN-S2。 两种半抗原最终制备抗体效果的差异在本发明 后述内容中可以体现。
与上述半抗原结合的载体蛋白可为选自鼠血清蛋白、 甲状腺蛋白、 牛血清蛋白、 兔 血清蛋白、人血清蛋白、 卵清蛋白、血蓝蛋白和纤维蛋白原等中的常用载体蛋白， 这些载 体蛋白含有丰富的羧基、 氨基等可与林可霉素半抗原共价结合的官能基团。
本发明采用混合酸酐法或碳化二亚胺法将林可霉素半抗原与载体蛋白偶联， 突出了 林可霉素的特征结构，增加了林可霉素半抗原的免疫原性。经测定本发明中林可霉素半抗 原与载体蛋白如牛血清白蛋白的结合摩尔比优选为 12~15:1。
本发明的林可霉素特异性抗体可为林可霉素单克隆抗体或林可霉素多克隆抗体。。林 可霉素单克隆抗体优选为林可霉素鼠单克隆抗体， 更优选为杂交瘤细胞株 C-1-3CGMCC No.2398分泌的单克隆抗体。
所述林可霉素单克隆杂交瘤细胞株 C-1-3CGMCC No.2398 (分类命名： 对林可霉素 药物的单克隆杂交瘤细胞株） 己于 2008年 03月 12日保藏于中国微生物菌种保藏管理委 员会普通微生物中心（简称 CGMCC, 地址： 北京市朝阳区大屯路， 中国科学院微生物研 究所， 邮编 100101 )。
林可霉素多克隆抗体可为鼠源、 马源、 羊源、 兔源或豚鼠源抗体， 优选为林可霉素 兔多克隆抗体。
以下对本发明的主要技术内容进行描述。
本发明所描述的实验方法可由本领域技术人员参考 《酶免疫测定技术》 （杨利国， 胡 少昶， 魏平华等. 南京大学出版社， 南京： 1998.) 而得以实施。
1. 半抗原的合成
由于四个羟基位阻差异， 可将林可霉素采用琥珀酸酐法得到半抗原 LIN-S1 , 合成路 线如图 3。
支链上 2号羟基位阻最小， 首先用叔丁基 -二甲基氯硅垸将其选择性保护， 然后采用 琥珀酸酐法将环上的 4'号羟基酰化， 然后在对甲苯磺酸存在下水解脱去保护基， 得到半 抗原 LIN-S2， 合成路线如图 4。
2. 林可霉素抗体的制备
(1) 免疫原制备
将林可霉素半抗原与载体蛋白采用混合酸酐法进行偶联得到免疫原。
(2) 林可霉素鼠单克隆抗体的制备
a) 动物免疫程序：采用 Balb/c小鼠作为免疫动物， 以林可霉素半抗原与载体蛋白偶联 物为免疫原， 待血清效价大于 1:3000后， 取出脾脏进行细胞融合。
b) 细胞融合与克隆化：取免疫 Balb/c小鼠脾细胞与 SP2/0骨髓瘤细胞融合，筛选细胞 株， 直到得到稳定分泌单克隆抗体的杂交瘤细胞株。
c) 优选单克隆抗体的确定： 测定两种技术方案制备的单克隆抗体性质， 由 LIN-S1得 到的抗体特异性、 灵敏度优于由 LIN-S2所得抗体。 (3) 林可霉素兔多克隆抗体的制备
： 采用新西兰大白兔作为免疫动物， 以林可霉素半抗原与载体蛋白偶联物为免疫原对 新西兰大白兔进行免疫， ί多次免疫后， 测定血清抗体效价。 待效价大于 1:10000后， 处死 兔子， 采集血清， 得到多克隆抗体。
3. 抗抗体的制备
(1) 羊抗鼠抗抗体是以羊作为免疫动物，以鼠源抗体为免疫原对无病菌体羊进行免疫， 得到羊抗鼠抗抗体；
(2) 羊抗兔抗抗体是以羊作为免疫动物，以兔源抗体为免疫原对无病菌体羊进行免疫， 得到羊抗兔抗抗体。
4. 本发明所提供的林可霉素检测方法及其检测试剂盒可以用于检测动物组织和蜂蜜等样 品中的林可霉素药物。
所述试剂盒包括包被原和酶标记物， 还可包括林可霉素标准品溶液、 底物显色液、 终 止液、 浓缩洗涤液和浓缩复溶液。
所述包被原为林可霉素半抗原与载体蛋白的偶联物、林可霉素抗体或抗抗体； 所述酶 标记物为酶标抗体、酶标林可霉素半抗原与载体蛋白的偶联物（本文中， 林可霉素半抗原 与载体蛋白的偶联物也称作林可霉素偶联抗原）或酶标记抗抗体； 当所述包被原为林可霉 素半抗原与载体蛋白的偶联物时， 所述酶标记物为酶标抗抗体； 当所述包被原为抗体时， 所述酶标记物为酶标林可霉素偶联抗原； 当所述包被原为抗抗体时，所述酶标记物为酶标 林可霉素偶联抗原。
所述林可霉素标准品溶液可以是浓度分别为 0， 0.2， 0.6， 1.8， 5.4和 16.2 g/L的溶液。 所述酶标记物的标记酶为辣根过氧化物酶或碱性磷酸酯酶， 其中优选辣根过氧化物 酶;酶标记的羊抗鼠抗抗体或羊抗兔抗抗体是采用戊二醛法或过碘酸钠法将标记酶与抗抗 体进行偶联得到的。
当标记酶为辣根过氧化物酶时， 显色剂由显色液 A液和显色液 B液组成， 显色液 A 液为过氧化氢或过氧化脲， 所述显色液 B 液为邻苯二胺或四甲基联苯胺， 终止液为 1~ 2mol/L 的硫酸或盐酸缓冲液； 当标记酶为碱性磷酸酯酶时， 显色液为对硝基磷酸盐缓冲 液， 终止液为 l~2mol/L氢氧化钠溶液。
所述浓缩洗涤液可以为 pH值 7.3-7.9、 含有 0.8-1.3 wt%吐温 -20和 0.1-0.6 wt%硫柳汞 的 0.1-0.3mol/L磷酸盐缓冲液； 所述浓缩复溶液可以为 pH值 7.6-8.0的 0.3-0.7mol/L磷酸 盐缓冲液。
5. 本发明的检测原理为：
当在酶标板上预包被林可霉素偶联抗原时， 加入样本溶液或标准品溶液后， 再加入 林可霉素特异性抗体溶液，样本中残留的林可霉素药物与酶标板上包被的林可霉素偶联抗 原竞争林可霉素特异性抗体， 加入酶标记抗抗体进行放大作用， 用显色液显色， 样本吸光 值与林可霉素药物的含量呈负相关， 与标准曲线比较即可得出样本中林可霉素的残留量。 同时根据酶标板上颜色的深浅，与系列浓度的林可霉素标准品溶液颜色的比较可粗略判断 样品中林可霉素残留量的浓度范围。
当在酶标板上预包被林可霉素特异性抗体时， 加入样本溶液或标准品溶液后， 再加入 酶标记林可霉素偶联抗原溶液，样本中残留的林可霉素药物与酶标记抗原竞争包被在酶标 板上的林可霉素特异性抗体，用显色液显色，样本吸光值与林可霉素药物的含量呈负相关， 与标准曲线比较即可得出样本中林可霉素的残留含量。同时根据酶标板上颜色的深浅，与 系列浓度的林可霉素标准品溶液颜色的比较可粗略判断样品中林可霉素残留量的浓度范 围。
当在酶标板上预包被林可霉素偶联抗原时， 加入样本溶液或标准品溶液后， 再加入 酶标记林可霉素特异性抗体溶液，样本中残留的林可霉素药物与酶标板上包被的林可霉素 偶联抗原竞争林可霉素特异性抗体，用显色液显色，样本吸光值与林可霉素药物的含量呈 负相关，与标准曲线比较即可得出样本中林可霉素的残留含量。同时根据酶标板上颜色的 深浅，与系列浓度的林可霉素标准品溶液颜色的比较可粗略判断样品中林可霉素残留量的 浓度范围。
当在酶标板上预包被抗抗体时， 加入林可霉素抗体孵育后， 加入样本溶液或标准品 溶液后，再加入酶标记林可霉素偶联抗原溶液，样本中残留的林可霉素药物与酶标记林可 霉素偶联抗原竞争林可霉素特异性抗体，用显色液显色，样本吸光值与林可霉素药物的含 量呈负相关，与标准曲线比较即可得出样本中林可霉素的残留含量。同时根据酶标板上颜 色的深浅，与系列浓度的林可霉素标准品溶液颜色的比较可粗略判断样品中林可霉素残留 量的浓度范围。
6. 本发明还提供了一种检测林可霉素药物的方法， 它包括步骤：
(1) 样品前处理；
(2) 用前述试剂盒进行检测；
(3) 分析检测结果。
样品的前处理主要是为了从样品中获得林可霉素溶液， 从而用于后续的检测。
本发明中用试剂盒检测时： 当包被原为林可霉素偶联抗原时， 向酶标板微孔中加入标 准品溶液或样本溶液再加入抗体， 温育后洗涤后， 甩掉孔中液体， 并以吸水纸吸除残余水 分， 再加入酶标抗抗体， 温育后洗涤， 甩掉孔中液体， 并以吸水纸吸除残余水分， 显色、 终止， 用酶标仪测定吸光度值； 当包被原为林可霉素偶联抗原时， 向酶标板微孔中加入标 准品溶液或样本溶液再加入酶标记抗体， 温育后洗涤， 甩掉孔中液体， 并以吸水纸吸除残 余水分， 显色、 终止， 用酶标仪测定吸光度值； 当包被原为林可霉素特异性抗体时， 向酶 标板微孔中加入标准品溶液或样本溶液再加入酶标记林可霉素半抗原，温育后洗涤，甩掉 孔中液体， 并以吸水纸吸除残余水分， 显色、 终止， 用酶标仪测定吸光度值； 当包被原为 抗抗体时， 向酶标板微孔中加入林可霉素抗体， 温育后洗涤， 甩掉孔中液体， 并以吸水纸 吸除残余水分， 再加入标准品溶液或样品溶液后加入酶标林可霉素半抗原， 温育后洗涤， 甩掉孔中液体， 并以吸水纸吸除残余水分， 显色、 终止， 用酶标仪测定吸光度值。
本发明中检测结果分析过程为： 用所获得的每个浓度的标准品溶液的吸光度平均值
(B) 除以第一个标准溶液 （0标准） 的吸光度值 （Bo) 再乘以 100%， 即百分吸光度值。 计算公式为- 百分吸光度值 （％) = (B/Bo) l00%
以林可霉素标准品溶液的浓度（ g/L)的半对数值为 X轴， 百分吸光度值为 Υ轴， 绘 制标准曲线图。用同样的办法计算样品溶液的百分吸光度值，相对应每一个样品的浓度则 可从标准曲线上读出样本中林可霉素的残留量。
本发明中检测结果的分析也可以采用回归方程法， 计算出样品溶液浓度。 附图说明
图 1 : 林可酰胺类抗生素化学结构通式；
图 2: 林可霉素半抗原改造结构位点；
图 3 : 林可霉素半抗原 LIN-S1的合成路线；
图 4: 林可霉素半抗原 LIN-S2的合成路线；
图 5: 试剂盒标准曲线。 具体实施方式
下面结合具体的实施例来进一步阐述本发明。 应理解， 这些实施例仅用于说明本发 明， 而非用来限制本发明的范围。 实施例 1 试剂盒组分的制备
1.抗原的合成
a. 半抗原的合成
将林可霉素采用琥珀酸酐法得到具有羧基官能团的半抗原。
半抗原 LIN-S1 的具体步骤： 称取 2.3g 盐酸林可霉素 （购自德国 Dr公司， 货号 C14635000)和 0.5g琥珀酸酐放入 50ml圆底烧瓶中加无水吡啶至完全溶解， 70°C加热搅 拌反应 24h。 反应结束后， 减压蒸馏去溶剂， 残余物用丙酮洗涤数次， 用 2~3倍残余物体 积的乙酸乙酯-正己烷 （2:1， 体积比）洗涤残余物， 充分振荡， 使其结晶， 得半抗原林可 霉素半琥珀酸酯。
半抗原 LIN-S2的具体步骤：
称取林可霉素 1.59g置于 25ml圆底烧瓶中， 加 6mlN,N-二甲基甲酰胺溶解 (DMF)， 搅拌条件下依次加入 1.41g咪唑和 1.56g叔丁基二甲基氯硅垸（t-BuMe2Si-Cl)， 30°C下搅 拌 2h，产物用乙酸乙酯提取，提取液用旋转蒸发仪浓缩后真空干燥 24h，得 4-O-t- BuMe2Si -LIN。
取 4-O-t- BuMe2Si-LIN 0.56g和 O.lg琥珀酸酐放入置于 50ml圆底烧瓶中，加无水吡 啶至完全溶解， 70°C加热搅拌反应 24h。 反应结束后， 减压蒸馏去溶剂， 残余物用丙酮洗 涤数次， 用 2~3 倍残余物体积的乙酸乙酯洗涤残余物， 充分振荡， 乙酸乙酯清液用旋转 蒸发仪浓缩后真空干燥 24h得 4-O-t- BuMe2Si-2-0-林可霉素半琥珀酸酯。
取 4-O-t- BuMe2Si-2-0-林可霉素半琥珀酸酯 0.3g置于 50ml圆底烧瓶中， 加 17ml甲 醇溶解，室温搅拌下逐滴加入 10ml含 0.26g水合对甲苯磺酸的甲醇溶液，继续搅拌 25min， 旋蒸除去甲醇，产物在水和乙酸乙酯层之间进行分配， 乙酸乙酯层用旋转蒸发仪浓缩后真 空干燥 24h,得半抗原 LIN-S2。 b. 免疫原的合成
以下为示例性说明， 将林可霉素半抗原与牛血清白蛋白采用混合酸酐法进行偶联得到 免疫原。
具体操如下：
取 5.8mg林可霉素半抗原用 0.1ml Ν， Ν'-二甲基甲酰胺 (DMF)溶解， 冷却至 10°C， 加 2μ1氯甲酸异丁酯， 10°C搅拌反应 30分钟， 60mg牛血清白蛋白用 2ml 50mmol/L Na2C03 溶解， 10°C反应 4小时，然后 4°C过夜，过 Sephadex G-25柱后，用缓冲液（pH7.4、 0.2mol/L 磷酸盐缓冲液）平衡和洗脱， 合并含有牛血清白蛋白的洗脱管内液体， 将其用透析袋装好 放入 pH7.4、 0.2mol/L磷酸盐缓冲液中透析 7天， 每天换液 3〜4次。 最后将免疫原冻干 保存。 经测定林可霉素半抗原与载体蛋白的结合摩尔比为 13:1。 c 包被原林可霉素偶联抗原的制备
以下为示例性说明， 将林可霉素半抗原与卵清蛋白偶联得到包被原。
具体操作如下： 取 5.8mg林可霉素半抗原用 O.lml DMF溶解， 冷却至 10°C， 加 2μ1氯甲酸异丁酯， 10°C搅拌反应 30分钟， 30mg卵清蛋白用 2ml 50mmol/L Na2C03溶解， 10°C反应 4小时， 然后 4°C过夜， 过 Sephadex G-25柱后， 用缓冲液（pH7.4、 0.2mol/L磷酸盐缓冲液）平衡 和洗脱， 合并含有卵清蛋白的洗脱管内液体， 将其用透析袋装好放入 pH7.4、 0.2mol/L磷 酸盐缓冲液中透析 7天， 每天换液 3〜4次。 最后将免疫原冻干保存。 经测定林可霉素半 抗原与载体蛋白的结合摩尔比为 15:1。
2. 单克隆抗体的制备
a. 动物免疫
选取 6-8周龄健康 Balb/c小鼠， 按照免疫剂量为 100μ§/只进行免疫。 首次免疫时取等 量弗氏完全佐剂（购自 Sigma公司， 货号 F5881 )与免疫原均勾混合， 后续免疫时与等量 弗氏不完全佐剂 （购自 Sigma公司， 货号 F5506) 混合。
b. 细胞融合和克隆化
小鼠血清测定结果较高后， 取其脾细胞， 按 7: 1比例 （数量比） 与 SP2/0骨髓瘤细胞 融合， 釆用间接竞争 ELISA测定细胞上清液， 筛选阳性孔。 利用有限稀释法对阳性孔进 行克隆化， 直到得到分泌单克隆抗体的杂交瘤细胞株。 c优选单克隆抗体细胞株的确定 得到采取两种半抗原技术方案制备的单克隆抗体后， 测定其 50%抑制浓度及特异性， 结果如下表所示。 两种半抗原技术方案所得抗体性质测定结果
Figure imgf000010_0001
由表 1可知， 由半抗体 LIN-S2所制备的抗体对多种抗生素均有交叉反应， 特异性较 差， 且其 IC5Q值为 489 g/kg， 无法满足林可霉素药物残留检测的需求。
固本发明采用半抗体 LIN-S1制备的林可霉素单克隆抗体作为优选抗体， 其是由单克 隆杂交瘤细胞株 C-1-3CGMCC N0.2398分泌的，其针对林可霉素特异性良好 (如表 1)， IC50 能达到 0.4 g/L。
d. 细胞冻存和复苏
将林可霉素的单克隆杂交瘤细胞株用冻存液制成 Ι χΙΟ6个 /ml的细胞悬液， 在液氮中 长期保存。 复苏时取出冻存管， 立即放入 37Ό水浴中速融， 离心去除冻存液后， 移入培 养瓶内培养。
e. 单克隆抗体的生产与纯化
取 6-8周龄 Balb/c小鼠若干只， 腹腔注入灭菌石蜡油 0.5ml/只， 7天后腹腔注射林可 霉素的单克隆杂交瘤细胞株 5χ107个 /只， 7天后采集腹水。 用辛酸-饱和硫酸铵法进行腹 水纯化， -20°C保存。
2. 多克隆抗体的制备
采用新西兰大白兔作为免疫动物， 以林可霉素半抗原与牛血清白蛋白偶联物为免疫 原， 免疫剂量为 1.5mg/kg， 首免时将免疫原与等量的弗氏完全佐剂 （来源同上） 混合制 成乳化剂， 颈背部皮下多点注射， 间隔 3~4周取相同剂量免疫原加等量弗氏不完全佐剂 (来源同上）混合乳化， 加强免疫一次， 共免疫 5次， 最后一次不加佐剂。 最后一次免疫 10天后采血，测定血清抗体效价，心脏采血，用辛酸-饱和硫酸铵法纯化得到多克隆抗体。
3. 羊抗鼠抗抗体的制备过程： 以羊作为免疫动物， 以鼠源抗体为免疫原对无病原体羊进 行免疫， 得到羊抗 抗抗体； 羊抗兔抗抗体的制备： 以羊作为免疫动物， 以兔源抗体为免 疫原对无病原体羊进行免疫， 得到羊抗兔抗抗体。
4. 酶标板的制备 a. 溶液配制 包被缓冲液： pH8.6、 0.2mol/L的硼酸缓冲液。 称取 10.49g Na2B4O7.10H2O、 5.57g H3B03 溶解于 1L去离子水中。 封闭液： 含 10wt%小牛血清和 0.05wt%。的叠氮化钠、 pH 7.2、 0.05mol/L的磷酸盐缓冲液。 称取 12.90g Na2HPO4 _12H2O、 2.18g ΝαΗ2Ρ04·2Η20、 5g叠氮纳用去离子水溶解后， 再加 入小牛血清 100ml, 定容至 1L。 浓缩洗涤液： pH7.4、 含有 1.0 wt%吐温 -20、 0.5%硫柳汞、 0.2mol/L的磯酸盐缓冲液。 称 取 14.51g Na2HP04'12H20、 1.48g NaH2P04-2H20用去离子水溶解， 加入吐温 -20 10ml和 5g硫柳汞后， 用去离子水定容至 1L。 稀释洗涤液： 取适量浓缩洗涤液用去离子水按照 1:19的体积比例混匀后备用。 浓缩复溶液： 含有 5%牛血清白蛋白、 pH7.4 的 0.2mol/L的磷酸盐缓冲液。 称取 14.51g Na2HP04 12H20 1.48g ΝαΗ2Ρ04·2Η20用去离子水溶解， 加入 50g牛血清白蛋白后， 用去 离子水定容至 1L。 b. 酶标板的包被
用包被缓冲液将林可霉素偶联抗原、 抗体或抗抗体稀释成 0.05-0.2 g/ml， 每孔加入 ΙΟΟμΙ, 37°C温育 2h或.4°C过夜， 倾去包被液， 用稀释 20倍的浓缩洗涤液洗涤 2次， 每 次 30秒， 甩掉孔中液体， 以吸水纸吸除残余水分后， 向每孔中加入 200μ1封闭液， 37°C 温育 2h， 倾去孔内液体， 干燥后用铝膜真空密封， 4°C干燥处保存备用。
5. 酶标记羊抗鼠抗抗体
本发明中所用辣根过氧化物酶酶标记的羊抗鼠抗抗体是采用戊二醛法或过碘酸钠法 将标记酶与抗抗体进行偶联得到的。 实施例 2 检测林可霉素的嗨联免疫试剂盒的组建
组建检测林可霉素的酶联免疫试剂盒， 使其包含下述组分-
(1) 包被林可霉素偶联抗原的酶标板；
(2) 用辣根过氧化物酶标记的羊抗鼠抗抗体；
(3) 林可霉素单克隆抗体工作液；
(4) 林可霉素标准品溶液 6瓶，浓度分别为 (Vg/L、0.2 g/L、0.6 g/L、1.8 g/L、5.4 g/L、 16.2 g/L;
(5) 底物显色液由 A液和 B液组成，底物显色液 A液为过氧化脲，底物显色液 B液 四甲基联苯胺；
(6) 终止液为 2mol/L盐酸；
(7) 浓缩洗涤液为 pH7.4、 含有 1.0 wt %吐温 -20、 0.5 wt %硫柳汞、 0.2mol/L的磷酸 盐缓冲液。
(8) 浓缩复溶液为含有 5 wt %牛血清白蛋白的含有 5 ^ %牛血清白蛋白、 pH7.4的 0.2mol/L的磷酸盐缓冲液。 实施例 3 实际样品中林可霉素的检测 1. 样品前处理 a) 溶液配制
0.015mol/L盐酸溶液 （动物组织样本专用）： 量取 1.25ml浓盐酸加入到盛有去离子 水的容器中定容至 1L。
甲醇一盐酸溶液 （动物组织样本专用）： 量取 10ml甲醇和 60ml 0.015mol/L盐酸溶 液混合均匀。
复溶液： 取浓缩复溶液用去离子水按照 1 : 1的体积比例进行稀释， 混匀后备用。 b) 动物组织 （鸡肉、 鸡肝、 猪肉、 猪肝等）
称取 2.0g匀浆后的样本至 50ml聚苯乙烯离心管中， 加入 10ml甲醇一盐酸溶液， 振荡 5min， 3000g以上， 室温离心 5min， 移取 200μ1上清液， 加入 600μ1复溶液充分 混匀， 取 50μ1用于分析。
c) 蜂蜜样本
称取 l.Og蜂蜜至 50ml聚苯乙烯离心管中， 加入 5ml去离子水，用涡旋仪涡动至 蜂蜜全部溶解， 再使用振荡器振荡 5min， 3000g以上， 室温离心 5min， 取上层清液 lml, 加入 lml复溶液用涡旋仪涡动 30s， 取 50μ1用于分析。
2. 用试剂盒检测
向包被有林可霉素偶联抗原的酶标板微孔中加入林可霉素标准品溶液或样品溶液 50μ1,再加入林可霉素单克隆抗体工作液 50μ1，用盖板模封板， 25°C恒温箱中反应 30min， 倒出孔内液体， 每孔加入 250μ1洗涤液， 30s后倒出孔内液体， 如此重复操作共洗板 5次， 最后用吸水纸除去残余水分。加入辣根过氧化物酶标记的羊抗鼠抗抗体工作液 100μ1，25°〇 恒温箱中反应 30min， 倒出孔内液体， 重复洗板步骤， 每孔加入底物显色液 A液过氧化 脲， 底物显色液 B液四甲基联苯胺（TMB)， 轻轻振荡混匀， 25°C恒温箱避光显色 15min， 每孔加入 2mol/L终止液盐酸 50μ1， 轻轻振荡混匀， 用酶标仪波长设定在 450nm处， 测定 每孔吸光度值 （OD值）。
3. 检测结果分析
用所获得的每个浓度的标准品溶液的吸光度平均值 （B) 除以第一个标准品溶液 （0 标准）的吸光度值（BQ)再乘以 100%，得到百分吸光度值。以林可霉素标准品浓度（μΒ/L) 的半对数值为 X轴， 百分吸光度值为 Y轴， 绘制标准曲线图。 用同样的办法计算样品溶 液的百分吸光度值，相对应每一个样品的浓度，则可从标准曲线上读出林可霉素的残留量。 实施例 4 试剂盒质量的测定
1 标准品精密度试验：
分别从三个不同的时间段制备的酶标板中各抽出一批酶标板，每批各抽取 10个试剂 盒， 每板各抽出 20个微孔， 测定 1.8 g/L标准溶液的吸光度值， 计算变异系数。 表 2 标准可重复性试验（CV%)
1 2 3 4 5 6 7 8 9 10 01批 5.3 4.2 15.7 6.9 8.6 11.5 13.7 12.4 9.4 7.5 CV% 03批 7.3 12.5 8.4 7.6 9.7 11.4 10.8 5.9 7.3 10.5
06批 11.7 6.7 8.3 14.1 8.2 12.6 6.4 13.6 7.8 13.5 通过上述试验结果可以得出， 每批试剂盒各 10次标准品变异系数在 4.2%-15.7%之 间。
2 样本精密度和准确度试验 a) 样品精密度试验：
以 2(^g/L浓度的林可霉素对肌肉、 肝脏、 蜂蜜进行添加测定， 分别取三个不同批次 的试剂盒各五个， 每个浓度重复 5次， 分别计算变异系数， 结果见表 3〜5。
表 3 肌肉样本可重复性试验
批号 实测值 g/L) 变异系数 CV%
16.5 19.8 15.3 17.2 14.9 11.6
01 17.3 14.6 19.5 18.7 15.1 12.7
18.9 16.2 17.9 16.3 18.7 7.3
17.5 15.6 16.9 18.2 14.6 8.8
03 18.5 16.4 19.8 15.3 16.7 10.3
19.7 19.5 16.4 18.3 15.8 9.9
18.5 17.3 18.1 19.9 14.3 11.8
06 16.7 17.4 16.2 14.7 18.9 9.2
19.5 14.6 15.4 18.2 19.7 13.5 表 4 肝脏样本可重复性试验
批号 实测值 （ g/L) 变异系数 cv%
14.2 16.7 19.8 18.5 14.6 14.5
01 16.7 15.8 16.4 19.7 18.7 9.5
16.2 18.4 17.3 18.9 18.5 6.2
19.7 19.6 14.3 14.7 15.7 15.8
03 17.2 16.7 19.4 18.2 15.4 8.7
19.0 18.4 16.8 14.2 19.5 12.2
16.9 17.2 19.3 18.5 16.4 6.8
06 18.3 17.4 16.2 14.7 18.9 9.8
17.5 16.3 18.4 14.5 17.7 9.1
蜂蜜样本可重复性试验
批号 实测值 （μ /L) 变异系数 cv%
18.9 20.2 21.8 18.4 19.2 6.8
01 18.3 19.5 20.7 21.5 18.6 6.9
18.3 19.6 20.3 21.7 21.9 7.4
20.7 21.8 21.4 17.9 18.3 9.0
03 21.6 18.0 17.6 21.7 21.9 10.7
18.6 19.1 19.6 20.5 21.4 5.6
19.2 18.6 18.4 21.2 21.8 7.9
06 19.7 20.4 21.6 20.5 21.3 3.7
18.9 19.2 18.3 20.4 21.7 6.9 结果表明肌肉、 肝脏、 蜂蜜样本的变异系数均在 3.7%-15.8%之间。
b) 样本准确度试验
取两个浓度的林可霉素标准品溶液分别为 2(^g/kg (L)、 4( g/kg (L)， 分别对样 进行添加回收试验， 每个浓度做 4个平行， 分别计算准确度。 试剂盒的准确度
Figure imgf000016_0001
结果表明肌肉样本添加回收率在 78.2%-98.3%之间， 肝脏样本的添加回收率在 75.6%-97.2%之间， 蜂蜜样本添加回收率在 86.7%-107.4%之间。
3 试剂盒保存实验
试剂盒保存条件为 2~8°C， 经过 6个月的测定， 试剂盒的最大吸光度值 （零标准）、
50%抑制浓度、 林可霉素添加实际测定值均在正常范围之内。 考虑在运输和使用过程中， 会有非正常保存条件出现， 将试剂盒在 37°C保存条件下放置 6天， 进行加速老化实验， 结果表明该试剂盒各项指标完全符合要求。 考虑到试剂盒冷冻情况发生， 将试剂盒放入 -20°C冰箱冷冻 5天， 测定结果也表明试剂盒各项指标完全正常。 从以上结果可得出试剂 盒可以在 2~8°C至少可以保存 6个月以上。
4 试剂盒最低检测限测定 取不含林可霉素药物的阴性鸡肉、阴性蜂蜜样本用本发明所制试剂盒分别进行 20次 检测， 测定结果的平均值加上 3倍标准差作为试剂盒的最低检测限。 阴性鸡肉样本测定结果统计表
样品号 1 2 3 4 5 6 7 8 测定值 2.32 0.84 0.79 1.88 2.16 0.07 2.45 2.31 样品号 9 10 11 12 13 14 15 16 测定值 1.64 1.00 1.14 1.99 2.68 1.86 2.78 1.14 样品号 17 18 19 20 平均值 标准差 最低检测限 测定值 1.25 2.54 1.90 1.50 1.71 0.71 3.85 表 8 阴性蜂蜜样本测定结果统计表 g/kg 样品号 1 2 3 4 5 6 7 8 测定值 0.14 0.84 0.89 0.18 0.32 0.45 0.68 0.40 样品号 9 10 11 12 13 14 15 16 测定值 0.21 1.03 0.41 0.86 1.42 0.32 0.25 0.36 样品号 17 18 19 20 平均值 标准差 最低检测限 测定值 1.49 1.19 1.16 0.59 0.66 0.41 1.90 由表 7、 表 8可知， 本发明所研制的试剂盒鸡肉样本最低检测限为 3.85 g/kg， 蜂蜜 样本最低检测限为 1.90 g/kg。

Claims

权利要求
1、 一种检测林可霉素的酶联免疫试剂盒， 包括包被原和酶标记物， 所述包被原选 自林可霉素半抗原与载体蛋白的偶联物、林可霉素抗体和抗抗体； 当所述包被原为林可霉 素半抗原与载体蛋白的偶联物时，所述酶标记物为酶标抗抗体； 当所述包被原为林可霉素 抗体时， 所述酶标记物为酶标林可霉素偶联抗原； 当所述包被原为抗抗体时， 所述酶标记 物为酶标林可霉素偶联抗原；所述林可霉素半抗原是将林可霉素和琥珀酸酐通过缩合反应 得到的。
2、 根据权利要求 1所述的酶联免疫试剂盒， 其特征在于： 所述试剂盒还包括林可霉 素标准溶液、 显色液、 浓缩洗涤液、 终止液和浓缩复溶液； 所述浓缩洗涤液为 pH 值为 7.3-7.9、 含有 0.8-1.3 wt%吐温 -20、 0.1-0.6 ^%硫柳汞的 0.1-0.3mol/L的磷酸盐缓冲液； 所述浓缩复溶液为 pH值为 7.6-8.0、 0.3-0.7mol/L的磷酸盐缓冲液； 所述包被缓冲液为 pH 值为 8.6-9.0、 0.1-0.3mol/L 的硼酸缓冲液； 所述封闭液为含有 8-12 wt%小牛血清和 0.03-0.07 wt%。叠氮化钠、 pH值 7.2-7.4、 0.05mol/L的磷酸盐缓冲液； 所述底物显色液由 显色液 A液和显色液 B液组成， 显色液 A液为过氧化氢或过氧化脲， 显色液 B液为邻苯 二胺或四甲基联苯胺； 所述终止液为 l~2mol L硫酸或盐酸溶液。
3、 根据权利要求 1或 2所述的酶联免疫试剂盒， 其特征在于： 所述林可霉素抗体是 以林可霉素半抗原与载体蛋白的偶联物作为免疫原得到的； 所述载体蛋白为鼠血清蛋白、 甲状腺蛋白、牛血清蛋白、兔血清蛋白、人血清蛋白、卵清蛋白、血蓝蛋白或纤维蛋白原。
4、 根据权利要求 3所述的试剂盒， 其特征在于所述林可霉素抗体为单克隆抗体， 其 由杂交瘤细胞株 C-1-3 CGMCC No.2398分泌产生。
5、 根据权利要求 1或 2所述的酶联免疫试剂盒， 其特征在于： 所述酶标记物的标记 酶为辣根过氧化物酶或碱性磷酸酯酶。
6、 根据权利要求 1或 2所述的酶联免疫试剂盒， 其特征在于： 所述抗抗体为羊抗鼠 抗抗体。
7、 根据权利要求 2所述的酶联免疫试剂盒， 所述浓缩洗涤液为 1.0~1.5wt%吐温 20 和 0.5wt%硫柳汞防腐剂的 pH值为 7.3、 0.1mol/L磷酸盐缓冲液； 所述浓缩复溶液为 pH 值为 7.8、 0.2mol/L的磷酸盐缓冲液； 所述包被缓冲液为 pH值为 9.0、 0.2mol/L的硼酸缓 冲液；所述封闭液为含有 8-12wt%小牛血清和 0.03-0.07wt%。叠氮化钠、 pH值 7.4、0.05mol/L 的磷酸盐缓冲液。
8、 根据权利要求 2所述的酶联免疫试剂盒， 其特征在于： 当标记酶为辣根过氧化物 酶时，所述显色剂由显色液 A液和显色液 B液组成，显色液 A液为过氧化氢或过氧化脲， 显色液 B液为邻苯二胺或四甲基联苯胺， 终止液为 l〜2mol/L硫酸或盐酸溶液； 当标记 酶为碱性磷酸酯酶时， 显色剂为硝基磷酸盐缓冲液， 终止液为 l〜2m0l/L氢氧化钠溶液。
9、 保藏号为 CGMCC No.2398的林可霉素单克隆杂交瘤细胞 C-l-3。
10、由保藏号为 CGMCC No.2398的林可霉素单克隆杂交瘤细胞 C-1-3分泌产生的林 可霉素单克隆抗体。
PCT/CN2008/001668 2008-04-16 2008-09-27 检测林可霉素的酶联免疫试剂盒 WO2009127094A1 (zh)

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