CN1707267A - Enzyme-linked immunologic reagent kit for detecting streptomycin drug - Google Patents
Enzyme-linked immunologic reagent kit for detecting streptomycin drug Download PDFInfo
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- CN1707267A CN1707267A CN 200410046568 CN200410046568A CN1707267A CN 1707267 A CN1707267 A CN 1707267A CN 200410046568 CN200410046568 CN 200410046568 CN 200410046568 A CN200410046568 A CN 200410046568A CN 1707267 A CN1707267 A CN 1707267A
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- streptomysin
- dihydrostreptomycin
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Abstract
The present invention discloses one kind of enzyme-linked immunological reagent kit for detecting streptomycin and similar medicine. The enzyme-linked immunological reagent kit for detecting streptomycin and similar medicine is multiple residue one. It has streptomycin and dihydrostreptomycin crossing rate of 100 %, simple sample pre-treatment including water dilution of milk sample and extraction with buffering phosphoric acid solution and pH regulation of pork and kidney sample, fast detection, high detection sensitivity up to 11.2 microgram/L and other advantages, and may be used in detection of residual streptomycin and similar medicines in animal food products.
Description
Technical field
The present invention relates to a kind of enzyme linked immunological kit that detects streptomycin medicament in enzyme linked immunological and the detection of veterinary drugs in food analysis technical field.
Background technology
In Milk Production, the veterinary drug improper use will cause milk or dairy produce veterinary drug residue to exceed standard.And milk is the important composition of our daily bread and infant's main food, and high concentration is residual to impair one's health.Streptomysin is as aminoglycosides antibiotics, can be used for prevention and infection such as treatment Gram-negative bacteria and tubercle bacillus, in the common disease mammitis of milk cow, tuberculosis etc. all need the streptomysin partner treatment, if but incorrect use can cause that streptomysin is residual to exceed standard.Many studies have shown that, streptomysin can cause irreversible infringement to kidney and auditory nerve, the residual milk of long-term drinking streptomysin equals to take for a long time low dose of streptomysin beyond doubt, allergic reaction can take place after having the people of allergic constitution to take the milk of residual streptomysin.Even some the conditionality pathogenic bacteria in the normal drinking person, body easily produce drug resistance, in case illly be difficult to prove effective with antibiotic therapy of the same race again.Streptomysin is the conventional medicine of cattle farm treatment mammitis, because long-term a large amount of the use increases antibody-resistant bacterium, some mammitis cases become and are difficult to cure with conventional method.Therefore the streptomysin maximum residue limit(MRL) of China's formulation is 200 μ g/L in milk.
Traditional method of inspection of streptomysin adopts microbial method and high performance liquid chromatography at present.The microorganism detection method is to use method more widely, commonly used have paper disk method (PD) and a TTC method, but the microbial method round of visits is long, poor specificity, complicated operation, and the residual limit of identification of streptomysin only is 1mg/L and 0.5mg/L in PD method and the TTC method detection milk, and this does not satisfy the streptomysin maximum residue limit(MRL) requirement that China formulates far away.High performance liquid chromatography also is the important method of residue detection, because operation was more loaded down with trivial details during the milk sample was handled, and the streptomysin drug residue is few in the milk, and background interference is often very serious, also all needs to react by column front derivation or post-column derivation to improve UV-detector and detect residual sensitivity.
The innovation and creation content
The purpose of this invention is to provide a kind of enzyme linked immunological kit that detects streptomycin medicament.
The enzyme linked immunological kit of detection streptomycin medicament provided by the present invention comprises that two of quilt are anti-, specific antibody and the enzyme mark streptomysin or the dihydrostreptomycin of streptomysin or dihydrostreptomycin.
Wherein, described streptomycin medicament is streptomysin or dihydrostreptomycin.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises A, B, C, D, E, F reagent solution, G reagent solution, H reagent solution, I reagent solution, J reagent solution, K reagent solution, L reagent solution.
Described A, B, C, D, E, F reagent solution are streptomysin or dihydrostreptomycin series concentration standard solution.
Described G reagent solution is the enzyme conjugates concentrate, contains enzyme mark streptomysin or the dihydrostreptomycin of 0.1-100mg/L.
Described H reagent solution is an antibody concentrated solution, and containing protein concentration is the streptomysin of 0.1-100mg/L or the specific antibody of dihydrostreptomycin.
Described I reagent solution is a tetramethyl benzidine.
Described J reagent solution is a phosphate buffer.
Described K reagent solution is a citrate buffer solution.
Described L reagent solution is a stop buffer.
The specific antibody of described streptomysin or dihydrostreptomycin can be streptomysin or dihydrostreptomycin monoclonal antibody or polyclonal antibody; Described streptomysin or dihydrostreptomycin monoclonal antibody or polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, pig source, rabbit source or cavy source antibody, described streptomysin or dihydrostreptomycin monoclonal antibody are preferably streptomysin or dihydrostreptomycin mouse monoclonal antibody, and described streptomysin or dihydrostreptomycin polyclonal antibody are preferably streptomysin or dihydrostreptomycin rabbit polyclonal antibody.
The conjugate of all available streptomysin of above antibody or dihydrostreptomycin and carrier protein prepares according to a conventional method as immunogene.
Described carrier protein can be bovine serum albumin(BSA) (BSA), human serum albumins (HSA), ovalbumin (OVA), albumin rabbit serum (RSA), thyroglobulin (TG) or hemocyanin common carrier albumen such as (KLH); The conjugate of described streptomysin or dihydrostreptomycin and carrier protein can adopt formaldehyde method, glutaraldehyde method, diazotising method, carbodlimide method, ethyloic azanol-carbodlimide method or multi-anhydride method synthetic.
Described two anti-can be anti-mouse or anti-rabbit antiantibody, are preferably goat anti-rabbit igg or sheep anti-mouse igg.
Described marker enzyme can be horseradish peroxidase or alkaline phosphatase, is preferably horseradish peroxidase, and horseradish peroxidase can be marked on streptomysin or dihydrostreptomycin by glutaraldehyde method, sodium periodate method or ethyloic hydroxylamine assay.
The carrier mass that can be used as fixing described two specific antibodies that resist is a lot, as polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber, Ago-Gel etc.The form of this carrier can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc.
Under preferred condition, the enzyme linked immunological kit of detection streptomycin medicament of the present invention comprises that two of quilt are anti-, streptomysin specific antibody and enzyme mark streptomysin.
Detection principle of the present invention is adsorbed on the solid phase carrier for resisting two, add sample, streptomysin or dihydrostreptomycin specific antibody and enzyme mark streptomysin or dihydrostreptomycin, residual streptomysin or dihydrostreptomycin and enzyme mark streptomysin or dihydrostreptomycin competition streptomysin or dihydrostreptomycin specific antibody in the testing sample, the colour developing back stops, the working sample light absorption value, streptomysin or dihydrostreptomycin residual quantity are negative correlation in this value and the sample, relatively can draw the content of streptomysin or dihydrostreptomycin with typical curve.Simultaneously according to the depth of the color sample on the ELISA Plate, but with the concentration range of the comparison judgement sample of the streptomysin of series concentration or dihydrostreptomycin standard solution color.
The enzyme linked immunological kit of detection streptomycin medicament of the present invention is many residue detection kit, and the crossing-over rate of streptomysin and dihydrostreptomycin is 100%; The main residual quantity that adopts streptomycin medicament in the qualitative or samples such as detection by quantitative animal tissue (musculature of pig, chicken, ox, sheep and liver, kidney etc.), milk of ELISA competing method; Pre-treatment requirement to sample is low, and sample pretreatment process is simple, can directly measure behind the milk sample dilute with water, and pork and kidney transfer pH can go up sample after the phosphate buffer extracting, and energy while fast detecting gross sample; Assay method is simple, saves time, and milk sample can draw measurement result within an hour; Lowest detectable limit reaches 11.2 μ g/L, has characteristics such as high specific, high sensitivity, pinpoint accuracy, pin-point accuracy, can play a significant role in animal food streptomycin medicament residue detection.
Embodiment
The preparation of embodiment 1, antigen and antibody
(1) streptomysin or dihydrostreptomycin antigen is synthetic
With oralbumin or bovine serum albumin(BSA) or human serum albumins is carrier, streptomysin or dihydrostreptomycin are haptens, with formaldehyde method or glutaraldehyde method or diazotising method or carbodlimide method or ethyloic azanol-carbodlimide method or multi-anhydride method, streptomysin or dihydrostreptomycin are coupled on oralbumin or bovine serum albumin(BSA) or the human serum albumins, obtain streptomysin or dihydrostreptomycin antigen.
(2) preparation of streptomysin or dihydrostreptomycin rabbit polyclonal antibody
Adopting the big ear rabbit of Japan as immune animal, is immunogene with antigen synthetic in the step (1), and immunizing dose is the 2mg/mL immunizing antigen at every turn, and first immunisation is with the complete freund adjuvant emulsification of 2mL, and booster immunization is with the incomplete freund adjuvant emulsification of 2mL.In each immune 2 weeks at interval, immune 5-10 time altogether, for the last time not with the direct intramuscular injection of immunologic adjuvant, blood sampling detects after 7 days, and behind the mensuration serum antibody titer, serum is extracted in the arteria carotis bloodletting, obtains the polyclonal antibody of purifying through ammonium sulfate precipitation.
(3) streptomysin or dihydrostreptomycin mouse monoclonal antibody preparation
Adopting BALB/C mice as immune animal, is immunogene with antigen synthetic in the step (1), and dosage is that 50 μ g (0.1mL) add the complete freund adjuvant emulsification of equal-volume, carries out subcutaneous first multi-point injection immunity.After January, get same amount immunizing antigen and add incomplete freund adjuvant, booster immunization is carried out in emulsification, and immunizing antigen does not add the adjuvant lumbar injection and carries out booster immunization after January, and antibody titer is measured in blood sampling in 5 days afterwards.Extracting spleen cell carries out Fusion of Cells in 4: 1 ratios and myeloma cell SP2/0.Adopt limiting dilution or soft agar flat band method screening hybridoma, obtain the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain.
Cell cryopreservation and recovery are got the hybridoma that is in exponential phase and are made 1 * 10 with cryopreserving liquid
6-5 * 10
6The cell suspension of individual/mL is sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.Make regular check on the stability of cell activity and secretory antibody.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
Adopt in the body and induce method, BALB/c mouse (8 age in week) abdominal cavity is only injected sterilization paraffin oil 0.5ml/, 7-14 days pneumoretroperitoneum injection hybridomas 5 * 10
5-10
6Individual/as only, to gather ascites after 7-10 days.Carry out the ascites purifying through sad-ammonium sulfate precipitation method, bottle packing ,-20 ℃ of preservations.
(4) enzyme labeling streptomysin or dihydrostreptomycin is synthetic
Streptomysin or dihydrostreptomycin are marked on the horseradish peroxidase synzyme mark streptomysin with glutaraldehyde method or sodium periodate method or ethyloic hydroxylamine assay.
(5) two anti-preparations
As immune animal, is that immunogene carry out immunity with mouse or rabbit igg with sheep, obtains sheep anti mouse or goat-anti rabbit antiantibody.Concrete grammar is as follows: will be behind the rabbit or the emulsification of rat immune globulin usefulness equivalent Fu Shi Freund's complete adjuvant of ammonium sulfate precipitation method purifying, muscle, subcutaneous, the adult sheep of intracutaneous multiple spot immunity, immunizing dose is a 10mg albumen/only, after January, rabbit or rat immune globulin with half amount add freund 's incomplete adjuvant muscle, subcutaneous or intracutaneous injection sheep, afterwards, per two all booster immunizations once.Fine jade expands to tire and reaches 1: 32 when above, blood sampling, and separation of serum after slightly carrying with ammonium sulfate precipitation method, with immune affinity column chromatography extraction goat anti-rabbit igg or sheep anti-mouse igg, obtains the goat anti-rabbit igg or the sheep anti-mouse igg of purifying.
The enzyme linked immunological kit of embodiment 2, detection streptomycin medicament
1, detects the structure of the enzyme linked immunological kit of streptomycin medicament
This kit is mainly by box body, ELISA Plate, A reagent bottle (standard solution 1), B reagent bottle (standard solution 2), C reagent bottle (standard solution 3), D reagent bottle (standard solution 4), E reagent bottle (standard solution 5), F reagent bottle (standard solution 6), G reagent bottle (enzyme conjugates concentrate), H reagent bottle (antibody concentrated solution), I reagent bottle (tetramethyl benzidine), J reagent bottle (phosphate buffer), K reagent bottle (citrate buffer solution), L reagent bottle (stop buffer), the cover plate film, carriage is formed.Above-mentioned A is housed, B, C, D, E, F, G, H, I reagent bottle in the carriage.The foam carriage, ELISA Plate, J reagent bottle (phosphate buffer), K reagent bottle (citrate buffer solution), L reagent bottle (stop buffer) and cover plate film are installed in the box body.ELISA Plate is made up of plastic stent and the plastic strip with holes that separates separately.
2, the preparation of agents useful for same
A, B, C, D, E, F reagent solution are streptomysin or dihydrostreptomycin series concentration standard solution, its concentration is respectively 0,0.4,2,6,20,80 μ g/L.
The G reagent solution is the enzyme conjugates concentrate, contains horseradish peroxidase-streptomysin or the dihydrostreptomycin label of 0.1-100mg/L.
The H reagent solution is an antibody concentrated solution, contains the streptomysin that protein concentration is 0.1-100mg/L or the mouse monoclonal antibody or the rabbit polyclonal antibody of dihydrostreptomycin.
The I reagent solution is a 0.1-10mg/ml tetramethyl biphenyl amine aqueous solution.
The J reagent solution is phosphate buffer (0.01M pH7.2).
The K reagent solution is citrate buffer solution (0.1M pH5.0).
The L reagent solution is a stop buffer, 1-2mol/L sulfuric acid or hydrochloric acid.
3, the preparation of ELISA Plate
Be cushioned liquid (0.05M pH9.6 carbonate buffer solution) with goat anti-rabbit igg or sheep anti-mouse igg dilution or 0.1-5 μ g/ml with bag, every hole adds 100 μ l, 4 ℃ are spent the night, and the bag that inclines is cushioned liquid, wash 3 times with the 0.05M pH7.2 phosphate buffer of 0.05% tween, each 30 seconds, pat dry, in every hole, add 5% skim milk 0.05M phosphate buffer, 250 μ l then, room temperature incubation 1-2h, the liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
The pre-treatment of embodiment 3, sample and detection
1, milk sample: get the milk of fresh or freeze thawing, direct behind the dilute with water as supplying test agent;
2, tissue sample: get pork, each 1g of renal tissue, add 0.2M phosphate buffer (pH9.2) 9mL, behind the mixing, vibration 30min, 5000rpm, 10min gets supernatant 0.5mL with 0.2M phosphate buffer (pH6) 0.5mL dilution, as supplying test agent.
3, detect
Before detecting streptomycin medicament kit is recovered room temperature (18 ℃-30 ℃), with G reagent solution and J reagent solution preparation enzyme mark streptomysin or dihydrostreptomycin solution (0.01-10mg/L), with H reagent solution and J reagent solution preparation antibody-solutions (0.01-10mg/L).In the ELISA Plate aperture, add 20 μ l sample solution or standard solution, 20 μ l enzymes mark streptomysin or dihydrostreptomycin solution and 100 μ l antibody-solutions, be covered with the cover plate film and place 30min in 19 ℃-30 ℃, wash ELISA Plate with water 3 times, thieving paper pats dry, with I reagent solution and K reagent solution preparation zymolyte mixed liquor (citrate buffer solution of 0.1-1% tetramethyl benzidine), get 100 μ l and add mixing behind the ELISA Plate aperture, the about 30min of lucifuge colour developing, add 100 μ l L stop buffers at last, measure A with microplate reader
450Value.Be calculated as follows the percentage absorbance:
(B-is the mean light absorbency value of standard solution or sample; B
0-be the standard solution mean light absorbency value of 0 concentration.)
Logarithm with streptomysin in the standard solution or dihydrostreptomycin concentration is an X-axis, and the percentage absorbance is a Y-axis, and the drawing standard curve calculates C (concentration of streptomysin or dihydrostreptomycin the sample solution) from typical curve.
Embodiment 5, kit sensitivity, specificity, precision, accuracy and critical value (cut-off) test
The enzyme-labelled antigen of this kit is a horseradish peroxidase mark streptomysin, and specific antibody is the streptomysin rabbit polyclonal antibody.
According to the relevant regulations of animal-derived food veterinary drug residue examination criteria establishment rule, respectively sensitivity, specificity, accuracy, precision and the critical value (cut-off) of this kit to be measured, concrete outcome is as follows.
1, kit sensitivity test
Carried out the kit sensitivity test according to a conventional method, the result shows that the typical curve minimum point of this kit is 0.4 μ g/L, and standard curve range is 0.4 μ g/L-80 μ g/L; In milk sample, detect and be limited to about 11.2 μ g/L.
2, specific assay
Select and the similar 7 kinds of medicines of streptomysin 26S Proteasome Structure and Function, streptomysin 50% inhibition concentration is compared with each medicine 50% inhibition concentration, draw a series of cross reacting rates, the result is as shown in table 1, test shows that dihydrostreptomycin and streptomysin are close with zymophorous group unity structure in enzyme linked immunosorbent assay (ELISA), crossing-over rate is approaching, and other 6 kinds of medicines and streptomysin almost do not have cross reaction.
The cross reaction of table 1. streptomysin ELISA detection method
| Medicine name | Cross reacting rate % |
| Dihydrostreptomycin | ??100.7 |
| Gentamicin | ??＜0.1 |
| Kanamycins | ??＜0.1 |
| Amikacin | ??＜0.1 |
| Neomycin | ??＜0.1 |
| Sisomicin | ??＜0.1 |
| Tobramycin | ??＜0.1 |
3, accuracy, precision are measured
In blank milk sample, add streptomysin or dihydrostreptomycin standard solution respectively, making its concentration is 100 μ g/L, 200 μ g/L and 400 μ g/L, each concentration prepares 5 parts in sample respectively, use its content of streptomysin standard curve determination then, replication 3 times, the coefficient of variation between calculate recovery rate, plate within variance coefficient and plate respectively.The result shows that in milk the recovery that 100 μ g/L streptomysins add concentration is 70%-120%, and the recovery that 200 μ g/L streptomysins add concentration is 60%-110%, and the recovery that 400 μ g/L streptomysins add concentration is 45%-110%; Variation within batch coefficient CV≤20%, interassay coefficient of variation CV≤25%.100 μ g/L, the interpolation recovery of 200 μ g/L and 400 μ g/L dihydrostreptomycins is 40%-120%, variation within batch coefficient CV≤20%, interassay coefficient of variation CV≤25%.
4, the mensuration of critical value (cut-off value)
By in milk, adding streptomysin to maximum residue limit(MRL) 200 μ g/L, measure 20 times, repeat 3 times, calculate the cut-off value.The result shows that the cut-off mean value of this kit is 116.1 μ g/L.
5, actual sample is measured
Use kit milk sample is detected, result such as following table 2 show that this milk sample meets standard for drinking.
Table 2.
| The sample title | Total quantity | Number positive | Positive rate |
| Milk | ??20 | ??0 | ??0％ |
Claims (10)
1, a kind of enzyme linked immunological kit that detects streptomycin medicament comprises that two of quilt are anti-, specific antibody and the enzyme mark streptomysin or the dihydrostreptomycin of streptomysin or dihydrostreptomycin.
2, enzyme linked immunological kit according to claim 1 is characterized in that: described kit also comprises A, B, C, D, E, F reagent solution, G reagent solution, H reagent solution, I reagent solution, J reagent solution, K reagent solution and L reagent solution.
3, enzyme linked immunological kit according to claim 1 and 2, it is characterized in that: the specific antibody of described streptomysin or dihydrostreptomycin is streptomysin or dihydrostreptomycin monoclonal antibody or polyclonal antibody, and they are that conjugate with streptomysin or dihydrostreptomycin and carrier protein obtains as immunogen preparing.
4, enzyme linked immunological kit according to claim 3 is characterized in that: described streptomysin or dihydrostreptomycin monoclonal antibody or polyclonal antibody are mouse source, Ma Yuan, Yang Yuan, pig source, rabbit source or cavy source antibody.
5, enzyme linked immunological kit according to claim 4 is characterized in that: described streptomysin or dihydrostreptomycin monoclonal antibody are streptomysin or dihydrostreptomycin mouse monoclonal antibody.
6, enzyme linked immunological kit according to claim 4 is characterized in that: described streptomysin or dihydrostreptomycin polyclonal antibody are streptomysin or dihydrostreptomycin rabbit polyclonal antibody.
7, enzyme linked immunological kit according to claim 3 is characterized in that: described carrier protein is bovine serum albumin(BSA), human serum albumins, ovalbumin, albumin rabbit serum, thyroglobulin or hemocyanin.
8, enzyme linked immunological kit according to claim 1 and 2 is characterized in that: described marker enzyme is horseradish peroxidase or alkaline phosphatase.
9, enzyme linked immunological kit according to claim 1 and 2 is characterized in that: described two anti-are anti-mouse or anti-rabbit antiantibody; Described two anti-goat anti-rabbit igg or the sheep anti-mouse iggs of being preferably.
10, enzyme linked immunological kit according to claim 1 is characterized in that: the enzyme linked immunological kit of described detection streptomycin medicament comprises that two of quilt are anti-, streptomysin specific antibody and enzyme mark streptomysin.
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| CNB2004100465683A CN100348979C (en) | 2004-06-11 | 2004-06-11 | Enzyme-linked immunologic reagent kit for detecting streptomycin drug |
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| CNB2004100465683A CN100348979C (en) | 2004-06-11 | 2004-06-11 | Enzyme-linked immunologic reagent kit for detecting streptomycin drug |
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| CN103374048A (en) * | 2012-04-26 | 2013-10-30 | 北京勤邦生物技术有限公司 | Streptomycin hapten, as well as preparation method and application thereof |
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