CN103675286B - A kind of lincomycin chemiluminescent enzyme-linked immunosorbent immunity quick detection kit - Google Patents

A kind of lincomycin chemiluminescent enzyme-linked immunosorbent immunity quick detection kit Download PDF

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CN103675286B
CN103675286B CN201310673629.8A CN201310673629A CN103675286B CN 103675286 B CN103675286 B CN 103675286B CN 201310673629 A CN201310673629 A CN 201310673629A CN 103675286 B CN103675286 B CN 103675286B
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lincomycin
solution
hole
elisa plate
antibody
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CN103675286A (en
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王善普
李秀梅
王兴兵
靳婉霞
王宇东
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LUOYANG LAIPSON INFORMATION TECHNOLOGY CO., LTD.
Luoyang Modern Biotechnology Research Institute 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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G01N21/763—Bioluminescence
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

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Abstract

The present invention relates to a kind of detection kit, specifically a kind of lincomycin chemiluminescent enzyme-linked immunosorbent immunity quick detection kit.Comprise box body, be located at the ELISA Plate in box body and be located at reagent in box body, each hole of described ELISA Plate is coated with envelope antigen, and wherein envelope antigen is made up of the metabolin of lincomycin and ovalbumin coupling; Described reagent comprises: antibody, lincomycin series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, the luminescent solution of the sheep anti mouse of the monoclonal antibody of lincomycin, ELIAS secondary antibody and horseradish peroxidase mark.Utilize mentioned reagent box to detect remaining lincomycin, adopt application of sample, wash, add ELIAS secondary antibody, wash, add luminescent solution inspection, survey the steps such as result judgement, the remnants of lincomycin can be measured accurately.

Description

A kind of lincomycin chemiluminescent enzyme-linked immunosorbent immunity quick detection kit
Technical field
The present invention relates to a kind of detection kit, specifically a kind of lincomycin chemiluminescent enzyme-linked immunosorbent immunity quick detection kit.
Background technology
In current detection cow's milk, the technology of lincomycin antibiotic residue mainly contains following several method:
(1) microorganism is obstructed detection method, also known as Antimicrobial experiment (micmorganisminhibitorytest, be called for short MIT), coming qualitative according to the inhibiting effect of microbiotic to the physiological function of specific microorganism, breeding metabolism or quantitatively determine antimicrobial agents residual quantity in sample, is detect the more classical and application method more widely of Ruzhong microbiotic.Antibiotic residue test stone (GB4789.27-2003) detection method in China's sweet milk, the inhibition zone abroad generally adopted the 1970s and 1980s test, turbidity test, the color changing type detection kit of selling in the market in addition all belongs to this type of.Variety classes indicator bacteria is different to variety classes antibiotic sensitive degree, selects suitable detection indicator bacteria to reduce detectability, improves detection sensitivity.But microorganism detection operation is wasted time and energy, and is not suitable for doing large-scale production.
(2) Physico-chemical tests method
After the nineties in 20th century, most Physico-chemical tests method measuring food lincomycin medicament residue mainly relies on liquid chromatography technology to be separated, next is look/mass spectrometric hyphenated technique, the detection method such as vapor-phase chromatography, high performance thin layer chromatography, because of its distinctive performance separately, slightly apply in antibiotics leftover detection.Chromatography detects medicament residue in cow's milk will through sample preparation (comprising the steps such as the extraction of sample, de-albumen, centrifugal, chromatographic column purification, derivatization), the separation of left drug and the detection of left drug.The special reaction that Physico-chemical tests method utilizes the group in antibiotic molecule to have or character, to measure its content, can carry out qualitative and quantitative analysis and drug identification, can be used as the confirmation method that Ruzhong microbiotic detects.This method detection sensitivity is higher, but instrument and testing cost is high, trace routine is complicated, comparatively time-consuming etc., and these unfavorable factors make this method be only limitted to laboratory milk sample to measure.
(3) immune analysis method
Immune analysis method is the analytical approach based on the specificity of antigen and antibody, reversibility association reaction, and current medicament residue immuno analytical method mainly divides three major types: method, the immunity receptor method of relatively independent analytical approach, immuno analytical method and the coupling of conventional physical and chemical analysis technology.Dutch vanweeman, schurrs in 1973 and Sweden Engvall, Perlmann propose enzyme linked immunosorbent assay respectively, and over more than 30 year, Chinese scholars has carried out large quantity research to microbiotic in immunological method detection food.But because microbiotic is haptens, antigen constructing technology difficulty is large, immunologic opsonin is strong, testing cost is high, the companies such as current Idexx Lab Inc., Cha coral Sciences are to exempt from the research in analytic approach comparatively ripe, and this technology domestic is still in research and development.Currently mainly use enzyme linked immunosorbent assay, radioimmunoassay luminescence method, fluorescent immune method, chemoluminescence method etc., the known enzyme of more several immuno-labelling technique sensitivity is exempted from compared with luminescence, and it is lower that its enzyme exempts from sensitivity.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of chemical luminescence ELISA detection kit of quick, highly sensitive detection lincomycin, sensing range is wide, easy and simple to handle fast, nontoxic pollution-free and the chemiluminescent enzyme-linked immunosorbent immune analytic reagent kit of the detection lincomycin of instrument simple economy, provide its preparation method simultaneously, and detect the method for lincomycin antibiotics in sample.
The present invention for the adopted technical scheme that solves the problem is: a kind of lincomycin chemiluminescent enzyme-linked immunosorbent immunity quick detection kit, comprise box body, be located at the ELISA Plate in box body and be located at reagent in box body, it is characterized in that: each hole of described ELISA Plate is coated with envelope antigen, wherein envelope antigen is made up of the metabolin of lincomycin and ovalbumin coupling; Described reagent comprises: antibody, lincomycin series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, the luminescent solution of the sheep anti mouse of the monoclonal antibody of lincomycin, ELIAS secondary antibody and horseradish peroxidase mark.
Described ELISA Plate is 96 hole chemiluminescence ELISA Plate of opaque polystyrene 8 hole × 12.
Described envelope antigen concentration is 5 μ g/mL.
The monoclonal antibody of described lincomycin is the monoclonal antibody that conjugate that the bovine serum albumin coupling being 6.7KDa ~ 6.8KDa by the metabolin of lincomycin and molecular weight ranges is made prepares as immunogen immune mouse, and its working concentration is 1:15000.
A kind of above-mentioned lincomycin chemiluminescent enzyme-linked immunosorbent immunity quick detection kit that uses detects the residual antibiotic method of lincomycin, comprises the following steps:
1) application of sample: according to the addition in 50 μ L/ holes, lincomycin series standard strength solution and sample solution is added respectively in the hole of ELISA Plate, then according to the addition in 50 μ L/ holes, in each hole, lincomycin antibody working fluid is added, afterwards room temperature constant-temperature incubation 2.5h;
2) wash: incline the middle liquid that portals, and adds the cleansing solution in 280 μ L/ holes in ELISA Plate, pat dry after leaving standstill 5min, in triplicate;
3) ELIAS secondary antibody is added: every hole adds ELIAS secondary antibody working fluid 100 μ L, room temperature constant-temperature incubation 1.5h;
4) wash: incline the middle liquid that portals, and adds the cleansing solution in 280 μ L/ holes in ELISA Plate, pat dry after leaving standstill 5min, in triplicate;
5) luminescent solution is added: every hole adds luminescent solution 100 μ L;
6) detect: the luminous intensity measuring every hole with chemical illumination immunity analysis instrument, use lincomycin series standard solution concentration to be respectively: 0.1ng/mL(0 standard), 0.5ng/mL, 0.8ng/mL, lng/mL, 5ng/mL, 10ng/mL;
7) result judges: with the luminous value in hole corresponding to measured lincomycin series standard strength solution, luminous value divided by first standard (0 standard) is multiplied by 100 again, be inhibited rate B/B0%, take inhibiting rate as ordinate, the logarithm of each lincomycin series standard strength solution concentration is that horizontal ordinate makes typical curve, the luminous value in the hole per sample corresponding to solution and gained typical curve, calculate the concentration of sample solution.
Beneficial effect: highly sensitive is the superiority of chemiluminescence immune assay key, it is 10-12mol/L that its sensitivity of lincomycin chemiluminescent enzyme-linked immunosorbent immunity quick detection kit involved in the present invention can reach 22-50ng/mL(RIA), the present invention simultaneously also has wide linear dynamics scope, the light signal duration is long, analytical approach is fast easy, result is stablized, error is little, security is good and the operating period is long, chemiluminescence immune assay can detect the material that the method such as radiommunoassay and enzyme-linked immuno assay cannot detect, early diagnosis tool is clinically of great significance.
Embodiment
A kind of lincomycin chemiluminescent enzyme-linked immunosorbent immunity quick detection kit, comprise box body, be located at the ELISA Plate in box body and be located at reagent in box body, each hole of described ELISA Plate is coated with envelope antigen, and wherein envelope antigen is made up of the metabolin of lincomycin and ovalbumin coupling; Described reagent comprises: antibody, lincomycin series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, the luminescent solution of the sheep anti mouse of the monoclonal antibody of lincomycin, ELIAS secondary antibody and horseradish peroxidase mark.
Described ELISA Plate is 96 hole chemiluminescence ELISA Plate of opaque polystyrene 8 hole × 12, and described envelope antigen concentration is 5 μ g/mL.
The monoclonal antibody of described lincomycin is that the conjugate that the bovine serum albumin coupling being 6.7KDa ~ 6.8KDa by the metabolin of lincomycin and molecular weight ranges is made prepares as immunogen immune mouse, and its working concentration is 1:15000.
Lincomycin chemiluminescent enzyme-linked immunosorbent immunity quick detection kit detects the residual antibiotic method of lincomycin, comprises following detecting step:
1) application of sample: the lincomycin series standard strength solution or the sample solution that add 50 μ L/ holes in ELISA Plate, then add lincomycin antibody working fluid 50 μ L/ hole, room temperature (25 DEG C) constant-temperature incubation 2.5h;
2) wash: incline the middle liquid that portals, and adds the cleansing solution in 280 μ L/ holes in ELISA Plate, pat dry after leaving standstill 5min, in triplicate;
3) ELIAS secondary antibody is added: every hole adds ELIAS secondary antibody working fluid 100 μ L, room temperature constant-temperature incubation 1.5h;
4) wash: incline the middle liquid that portals, and adds the cleansing solution in 280 μ L/ holes in ELISA Plate, pat dry after leaving standstill 5min, in triplicate;
5) luminescent solution is added: every hole adds luminescent solution l00 μ L;
6) detect: the luminous intensity measuring every hole with chemical illumination immunity analysis instrument;
7) result judges: with measured standard items luminous value, luminous value divided by first standard (0 standard) is multiplied by 100 again, be inhibited rate B/B0%, take inhibiting rate as ordinate, the logarithm of lincomycin concentration is that horizontal ordinate makes typical curve, and the concentration of each sample can read from typical curve.
1 operation steps:
The required reagent of 1.1 preparation
Prepare concentrated cleaning solution:
Containing volume fraction 0.05% polysorbas20 mmol/l, the phosphate buffer of pH8.0.
Prepare calibration solution:
Described in the preferred 1:15000 of working concentration of the antibody of the sheep anti mouse of described horseradish peroxidase mark, lincomycin series standard solution concentration is respectively: 0.1ng/mL, 0.5ng/mL, 0.8ng/mL, lng/mL, 5ng/mL, 10ng/mL.
Described envelope antigen concentration is 4.5 μ g/mL preferably.The preparation of described envelope antigen is specifically completed according to ovalbumin (cOVA) coupling that the molecular weight ranges of glutaraldehyde method and activation is 6.7KDa ~ 6.8KDa by 3-amino-2-oxazolone (AOZ).
Chemical luminescence ELISA detection kit lowest detectable limit of the present invention can reach 0.1ng/mL, and linear detection range is at 0.1-10ng/mL, and in plate, the coefficient of variation is within 20%, and in water sample, the recovery is between 78%-118%.
Preparation luminous substrate liquid
Luminescent solution is three (methylol) the amino first protective embankment solution of 0.01M luminol and pH=8.80.001M p-cresol and volume by volume concentration is 3/10000H 2o 2mixed liquor.Described luminol is luminous substrate, and p-cresol is luminescence enhancer.
The foundation of 1.2 lincomycin chemiluminescent enzyme-linked immunosorbent immunodetections
1. antibody and envelope antigen concentration is preferred:
ELISA Plate longitudinally uses 100mL/ hole, concentration gradient is by the envelope antigen solution bag quilt of 80.0 μ g/mL, 40.0 μ g/mL, 20.0g/mL, 9.0 μ g/mL, 4.5 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL and 0.625 μ g/mL, 4 DEG C of placements are spent the night, plate is washed three times with the cleansing solution in 280 μ L/ holes, close with 250 μ L/ hole confining liquids again, room temperature places 2.5 hours, washs three times; Laterally add 100 μ L/ holes, dilution gradient presses the antibody-solutions of l:100 ~ 1:500, and room temperature places 2 hours, washs three times; Add the sheep anti-mouse antibody of the horseradish peroxidase mark of the l:10000 in 100 μ L/ holes, room temperature places l hour, washs three times; Add the luminescent solution in 100 μ L/ holes, measure luminous value.The envelope antigen concentration of obvious graded and antibody dilution is had to carry out specific assay for optium concentration with luminous value with the concentration of envelope antigen.
The mensuration of antibody sensitivity
Preferred through above-mentioned square formation method, applicant selects envelope antigen concentration to be 4.5 μ g/mL, and antibody concentration is 1:5000, carries out the mensuration of the sensitivity of antibody:
(1) bag is by process: with the carbonate buffer solution of 0.05MpH9.6, the envelope antigen of lincomycin is made into the solution of 4.5 μ g/mL, adds 100 μ L in the reacting hole of each polystyrene board, 4 DEG C are spent the night.Next day, discard solution in hole, wash 3 times with lavation buffer solution, 280 μ L/ holes, each 5 minutes, pat dry.
(2) closed process: close above-mentioned ELISA Plate of having wrapped quilt with the confining liquid in 250 μ L/ holes, incubated at room 2.5 hours, washing;
(3) competition process: add the medicine 50 μ L/ hole of dilution antibody (1:2000) 50 μ L/ hole and variable concentrations in the above-mentioned reacting hole closed, incubated at room 2-4 hour, washing;
(4) enzyme mark process: antibody (1:5000) the 100 μ L/ hole adding the sheep anti mouse of diluted fresh horseradish peroxidase mark in each reacting hole, incubated at room 1.5 hours, washing;
(5) luminescence process: the luminescent solution adding 100 μ L/ hole Extemporaneous in each reacting hole, detects with chemical illumination immunity analysis instrument immediately;
(6) computation process: testing result calculates with inhibiting rate, % inhibiting rate=%B/B0, B is the luminous value adding rival, and B0 is the luminous value not having rival.The concentration calculating medicine during 50% inhibiting rate is the sensitivity of this antibody.
The assembling of the chemiluminescence enzyme linked immunoassay reagent kit of 2.3 detection lincomycins of the present invention
(1) composition of the chemiluminescence enzyme linked immunoassay reagent kit of lincomycin is detected
A, be coated with the solid phase carrier (milky opaque polystyrene 96 hole chemiluminescence ELISA Plate) of envelope antigen (conjugate of AOZ and ovalbumin);
B, lincomycin antibody working fluid (volume by volume concentration is 1:5000);
C, lincomycin standard solution 6 bottles, concentration is respectively 0.lng/mL, 0.5ng/mL, 0.8ng/mL, lng/mL, 5ng/mL and 10ng/mL;
The sheep anti-mouse igg antibody working fluid (working concentration is 1:5000) of d, horseradish peroxidase-labeled;
E concentrated phosphoric acid salt buffer solution: the KH of the NaCl of 80g, 2.0g 2pO 4, the Na of 229.0g 2hPO 412H 2the KC1 of O, 2.0g is dissolved in 1000mL distilled water;
F, concentrated cleaning solution: above-mentioned concentrated phosphoric acid salt buffer adds the polysorbas20 (Tween20) that volume by volume concentration is 0.05mol/L;
G, luminescent solution: three (methylol) amino first protective embankment solution (pH8.8)+volume by volume concentration of 0.01M luminol ten 0.001M p-cresol is the H of 3/10000 2o 2.
The preparation of 2.4 ELISA Plate
Be dissolved in by envelope antigen in coating buffer, be made into the solution of 4.5ng/mL, every hole of ELISA Plate adds 10 μ L, 4 DEG C of overnight incubation, incline coating buffer, and every hole adds 280 μ L washings and removes liquid in hole, cleansing solution washs 3 times, pats dry, and preserves at 4 DEG C with masking foil vacuum seal.
The mensuration program of the enzyme linked immunological kit of 2.5 lincomycins of the present invention.
The preparation of reagent
1) sample diluting liquid: use after the concentrated phosphoric acid salt buffer solution distilled water diluting 10 times provided in kit;
2) cleansing solution: use after the concentrated cleaning solution distilled water diluting 10 times provided in kit;
3) luminescent solution: three (methylol) aminomethane solution (pH8.8) 3/10000(volume ratio of 0.01M luminol+0.001M p-cresol).
4.2 sample pre-treatments
(1) water sample is by the filter membrane suction filtration of water sample through 0.45 μm, then adds the Tween-20 that volume by volume concentration is 0.05%, obtains testing sample;
(2) milk by milk sample 4 DEG C, 10000 revs/min centrifugal 15 minutes, remove fat deposit; The milk cleansing solution of degreasing is diluted to 1:10, obtains testing sample, urine sample is by urine samples at 4 DEG C, and 10000 revs/min centrifugal 15 minutes, removes precipitation, remaining pig urine cleansing solution is diluted to 1:10, obtains testing sample;
(3) mixed in hydrochloric acid of animal tissue's sample thief and 4mL0.1M, and put together with the extraction of excusing from death ripple 20min, then 10000 revs/min of centrifugal 15min, get supernatant 10MNaOH and adjust pH to 9.5 ± 0.5, vortex vibration 5min, then 10000 revs/min of centrifugal 15min.Get supernatant and add 5mL isobutyl alcohol vibration 2min, potpourri standing at room temperature 15min, then 3000 revs/min of centrifugal 10min, separate organic phase, aqueous phase uses isobutyl alcohol (each 10mL) extracting twice again, the organic phase of three extractions combines, 50-6TC water-bath evaporated under reduced pressure, residue cleansing solution dissolves the solution being made into l:10 again, obtains testing sample.
4.3 detecting step
1) application of sample: the lincomycin series standard strength solution or the sample solution that add 50 μ L/ holes in ELISA Plate, then add lincomycin antibody working fluid 50 μ L/ hole, room temperature (25 DEG C) constant-temperature incubation 2.5h;
2) wash: incline the middle liquid that portals, and adds the cleansing solution in 280 μ L/ holes in ELISA Plate, pat dry after leaving standstill 5min, in triplicate;
3) ELIAS secondary antibody is added: every hole adds ELIAS secondary antibody working fluid 100 μ L, room temperature constant-temperature incubation 1.5h;
4) wash: incline the middle liquid that portals, and adds the cleansing solution in 280 μ L/ holes in ELISA Plate, pat dry after leaving standstill 5min, in triplicate;
5) luminescent solution is added: every hole adds luminescent solution l00 μ L;
6) detect: the luminous intensity measuring every hole with chemical illumination immunity analysis instrument.
4.4 results judge
With measured standard items luminous value, luminous value divided by first standard (0 standard) is multiplied by 100 again, the rate that is inhibited (B/B0%), take inhibiting rate as ordinate, the logarithm of lincomycin concentration is that horizontal ordinate makes typical curve, and the concentration of each sample can read from typical curve.
% inhibiting rate=% standard items luminous value (or sample)/0 standard items luminous value.
Effect explanation
1) highly sensitive
Highly sensitive is the superiority of chemiluminescence immune assay key, existing several immuno-labelling technique sensitivity more known, lincomycin chemiluminescent enzyme-linked immunosorbent immunity quick detection kit prepared by the present invention detects remaining lincomycin, it is 10-12mol/L that its sensitivity can reach 22-50ng/mL(RIA), chemiluminescence immune assay can detect the material that the method such as radiommunoassay and enzyme-linked immuno assay cannot detect, and is of great significance early diagnosis tool clinically.
(2) wide linear dynamics scope
The comparison of existing several immuno-labelling technique range of linearity, luminous intensity is between 4-6 magnitude and measure between material concentration linear, compared with this is the scope of 2.0 with the EIA enzyme immunoassay absorbance (OD value) of colour developing, with the obvious advantage. although RIA also has wider linear dynamics scope, and radioactivity limits its application.
(3) the light signal duration is long
The light signal duration that the CLIA of wide variety of glow-type produces can reach even one day a few hours.Simplify experimental implementation and measurement.
(4) analytical approach is fast easy
Most mensuration of analyzing is the step mode only needing to add a kind of reagent (or complex reagent).
(5) result is stable, error is little
Sample system is oneself luminescence directly, without any need for light source irradiation, eliminating various possible factor (light source stability, light scattering, light wave selector switch etc.) to analyzing the impact brought, making analysis result sensitive reliable and stable.
(6) security is good and the operating period is long
Eliminate use radiomaterial, up to the present, also do not find its harmfulness; Stable reagent, storage life can reach six months to more than 1 year.

Claims (1)

1. a lincomycin chemiluminescent enzyme-linked immunosorbent immunity quick detection kit, comprise box body, be located at the ELISA Plate in box body and be located at reagent in box body, it is characterized in that: each hole of described ELISA Plate is coated with envelope antigen, wherein envelope antigen is made up of the metabolin of lincomycin and ovalbumin coupling; Described reagent comprises: the monoclonal antibody of lincomycin, ELIAS secondary antibody, lincomycin series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution and luminescent solution, and described ELIAS secondary antibody is the antibody of the sheep anti mouse of horseradish peroxidase mark; Described ELISA Plate is 96 hole chemiluminescence ELISA Plate of opaque polystyrene 8 hole × 12,
Described envelope antigen concentration is 5 μ g/ml;
The monoclonal antibody of described lincomycin is the monoclonal antibody that conjugate that the bovine serum albumin coupling being 6.7KDa ~ 6.8KDa by the metabolin of lincomycin and molecular weight ranges is made prepares as immunogen immune mouse, and its working concentration is 1:15000;
Detection method is as follows: use lincomycin series standard solution concentration to be respectively: 0.1ng/mL, 0.5ng/mL, 0.8ng/mL, lng/mL, 5ng/mL, 10ng/mL, comprise the following steps:
1) application of sample: according to the addition in 50 μ L/ holes, lincomycin series standard strength solution and sample solution is added respectively in the hole of ELISA Plate, then according to the addition in 50 μ L/ holes, in each hole, lincomycin antibody working fluid is added, afterwards room temperature constant-temperature incubation 2.5h;
2) wash: incline the middle liquid that portals, and adds the cleansing solution in 280 μ L/ holes in ELISA Plate, pat dry after leaving standstill 5min, in triplicate;
3) ELIAS secondary antibody is added: every hole adds ELIAS secondary antibody working fluid 100 μ L, room temperature constant-temperature incubation 1.5h;
4) wash: incline the middle liquid that portals, and adds the cleansing solution in 280 μ L/ holes in ELISA Plate, pat dry after leaving standstill 5min, in triplicate;
5) luminescent solution is added: every hole adds luminescent solution 100 μ L;
6) detect: the luminous intensity measuring every hole with chemical illumination immunity analysis instrument;
7) result judges: with the luminous value in hole corresponding to measured lincomycin series standard strength solution, luminous value divided by first standard is multiplied by 100 again, be inhibited rate B/B0%, take inhibiting rate as ordinate, the logarithm of each lincomycin series standard strength solution concentration is that horizontal ordinate makes typical curve, the luminous value in the hole per sample corresponding to solution and gained typical curve, calculate the concentration of sample solution.
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