CN104614529A - Ampicillin penicilloic acid detection kit and detection method - Google Patents
Ampicillin penicilloic acid detection kit and detection method Download PDFInfo
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- CN104614529A CN104614529A CN201510018846.2A CN201510018846A CN104614529A CN 104614529 A CN104614529 A CN 104614529A CN 201510018846 A CN201510018846 A CN 201510018846A CN 104614529 A CN104614529 A CN 104614529A
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- 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
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Abstract
The invention relates to a hybridoma cell strain, an ampicillin penicilloic acid monoclonal antibody secreted by the hybridoma cell strain and an application of the monoclonal antibody in preparation of a kit for detecting ampicillin penicilloic acid. The kit for detecting ampicillin penicilloic acid comprises an elisa plate, a confining liquid, an ampicillin penicilloic acid standard substance, an antibody working solution, an enzyme labelled antibody, a developing solution and a stop solution, wherein the antibody working solution is a diluent for the ampicillin penicilloic acid monoclonal antibody secreted by the hybridoma cell strain. The invention further relates to the hybridoma cell strain and the ampicillin penicilloic acid monoclonal antibody secreted by the hybridoma cell strain. The hybridoma cell strain, the ampicillin penicilloic acid monoclonal antibody secreted by the hybridoma cell strain and the application of the monoclonal antibody in preparation of the kit for detecting ampicillin penicilloic acid have the benefits as follows: the specific ampicillin penicilloic acid monoclonal antibody is adopted to quantitatively detect residues of part of penicillins and metabolite products of penicillins in animal foodstuff; a detection result is fast to obtain, the accuracy is high, the reproducibility is good, and the detection sensitivity is high.
Description
Technical field
The invention belongs to enzyme linked immunological kit field, be specifically related to a kind of ammonia benzyl penicilloic acid detection kit and detection method.
Background technology
PCs has the features such as bactericidal activity is strong, toxic and side effect is little, applies widely in animal feeding, is mainly used in treating Animal diseases, and portioned product has the effect promoting growth of animal simultaneously.But, after microbiotic residual in animal food enters human body, there is certain damaging effect.In general, residual in animal food microbiotic does not show as Acute Toxicity to people.People take in the food of low dosage residual antibiotic for a long time, and As time goes on, microbiotic residual in human body is accumulated gradually, thus causes various chronic toxic effect.The people of some allergic constitution also can cause allergy (metamorphosis) to react after the microbiotic that contact is residual.The impact of antibiotic residue in animal food on health is extensive, human gastrointestinal tract flora imbalance can be caused, cause the possibility of opportunistic infections to increase, also can show as the many aspects such as allergy, toxic action, bacterial drug resistance, teratogenesis, mutagenesis and carcinogenesis.
Ampicillin is Beta-lactam medicine, and the beta-lactam nucleus in its structure is unstable, deposits easily rupture in case at alkaline environment and beta-lactamase, generates ammonia benzyl penicilloic acid, is not easily detected by corresponding ampicillin antibody.And use other detection methods, as thin-layered chromatography (TLC) and high performance liquid chromatography (HPLC) detect time, testing result is subject to the impact of mobile phase pH value, temperature and other factors and open loop, cause the stability of TLC and HPLC method and reappearance bad, the recovery is not high.
Summary of the invention
In order to solve the problems referred to above that prior art exists, the invention provides a kind of ammonia benzyl penicilloic acid detection kit and detection method, having rapidly and efficiently, the feature of highly sensitive, favorable reproducibility.
The technical solution adopted in the present invention is:
A kind of hybridoma cell strain, its deposit number is CGMCC NO.10112, preservation date is on Dec 5th, 2014, and depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
A kind of ammonia benzyl penicilloic acid monoclonal antibody of being secreted by hybridoma cell strain.
Ammonia benzyl penicilloic acid monoclonal antibody detects the application in the kit of ammonia benzyl penicilloic acid in preparation.
For detecting a kit for ammonia benzyl penicilloic acid, comprise ELISA Plate, confining liquid, ammonia benzyl penicilloic acid standard items, antibody working fluid, enzymic-labelled antibody, nitrite ion and stop buffer; Described antibody working fluid is the dilution of the ammonia benzyl penicilloic acid monoclonal antibody of described hybridoma cell strain secretion.
For detecting a kit for ammonia benzyl penicilloic acid, also comprise sample diluting liquid and concentrated cleaning solution, described sample diluting liquid is the phosphate buffer of pH6.5-8.5, and described concentrated cleaning solution is the mixed liquor of sample diluting liquid and polysorbas20.
A kind of ammonia benzyl penicilloic acid kit is detecting the application in animal food.
Beneficial effect of the present invention is: adopt specific ammonia benzyl penicilloic acid monoclonal antibody, quantitatively detects penicillin medicine and metabolic product remaining in animal food thereof; Testing result is quick, accuracy is high, favorable reproducibility, detection sensitivity are high.
Accompanying drawing explanation
Fig. 1 is the experimental data that the present invention carries out Precision Experiment.
Embodiment
This experiment adopts ammonia benzyl penicilloic acid coupling protein as immunogene, thus obtains ammonia benzyl penicilloic acid specific antibody.Ampicillin structure is as shown in (formula 1), and the structure of penicillin metabolic product penicilloic acid is as shown in (formula 2), and ammonia benzyl penicilloic acid structural formula is for shown in (formula 3).
Formula 1 ampicillin structural formula
Formula 2 penicilloic acid structural formula
Formula 3 ammonia benzyl penicilloic acid structural formula
Below by specific embodiment, the present invention is further detailed.
The preparation of embodiment 1 hybridoma cell strain and monoclonal antibody
Adopt carbodiimide method, ammonia benzyl penicilloic acid and keyhole limpet hemacyanin (mcKLH) are carried out coupling, synthetic immunogen.Carry out immunity to the Balb/c mouse in 56 week ages, carry out 6 routine immunizations and a booster immunization altogether, dosage is 100 μ g/, and immunization time is spaced apart 2 weeks, and the time of booster immunization is three days after the 6th routine immunization.After immunity terminates, obtain the immune spleen cell of mouse.Myeloma cell takes SP2/0 cell, because it can not grow in HAT selective medium, and itself can not synthetic immunoglobulin, therefore carry out with it the hybridoma that Fusion of Cells obtains and only secrete antibody molecule that is homogeneous, that come from splenocyte completely, be the comparatively ideal myeloma cell for merging, and there is easy cultivation, fusion rate advantages of higher.Application cell integration technology sets up hybridoma, and adopting indirect elisa method to filter out can the positive hybridoma cell strain of stably excreting ammonia benzyl penicilloic acid monoclonal antibody, carries out going down to posterity, frozen.
The positive hybridoma cell purge from cell bottle growth conditions being in logarithmic phase gets off, and with the centrifugation l0min of 1000rpm/min, abandons supernatant, and collecting cell precipitates, and is suspended by cell precipitation with DMEM basic culture solution, and mixing cell number is adjusted to 10
6/ mL carries out lumbar injection to the Balb/c mouse that 6 week age is large, every mouse peritoneal injection 0.5mL.Within 5 ~ 10 days, obtain the ascites containing ammonia benzyl penicilloic acid monoclonal antibody afterwards, sad-saturated ammonium sulfate method (sad consumption: 100 μ l/mL ascites) is adopted to carry out purifying to the ascites collected, to remove the impurity such as the lipoprotein, lipid or the cell fragment that contain in ascites, thus obtain the required ammonia benzyl penicilloic acid monoclonal antibody of experiment.
The Cleaning Principle of embodiment 2 described kit of the present invention
This kit adopts indirect competitive ELISA method, pre-coated coupled antigen on capillary strip.Wherein, coupled antigen adopts carbodiimides (EDC, DIC, DCC) method, ammonia benzyl penicilloic acid standard items and carrier protein (adopting bovine serum albumin(BSA), human albumin, mouse haemocyanin, rabbit serum proteins or ovalbumin) are carried out coupling, forms the coating antigen that this kit uses.Coupled antigen pre-coated on residual ammonia benzyl penicilloic acid in sample and capillary strip competes ammonia benzyl penicilloic acid monoclonal antibody, after abundant reaction, add enzymic-labelled antibody, enzymic-labelled antibody and residual coating antigen specific binding, then with the colour developing of tmb substrate nitrite ion, be marked at enzyme on coating antigen and nitrite ion reacts, after adding stop buffer, solution is by blue yellowing.Detect the light absorption value of sample under 450nm visible ray, absorbance becomes negative correlation with the content of its contained residual ammonia benzyl penicilloic acid, is calculated, can draw the content of ammonia benzyl penicilloic acid residual in respective sample by drawing standard curve.
Embodiment 3 detects the preparation of the kit of ammonia benzyl penicilloic acid
One, kit ingredient: ELISA Plate, confining liquid, enzymic-labelled antibody, antibody working fluid, nitrite ion, stop buffer, concentrated cleaning solution, sample diluting liquid, ammonia benzyl penicilloic acid standard items.
Each solution in reagent preparation box
1. sample diluting liquid (phosphate buffer): get potassium dihydrogen phosphate and disodium hydrogen phosphate, be mixed with the phosphate buffer of pH6.5 ~ 8.5.
2. confining liquid: get haemocyanin (bovine serum albumin(BSA), human albumin, mouse haemocyanin, rabbit serum proteins or ovalbumin), make the confining liquid that concentration is 0.8 ~ 1.5%.
3. concentrated cleaning solution (10X): measure 100ml sample diluting liquid (1X), adds the polysorbas20 of 40 ~ 60 μ L, and mixing, to obtain final product.
4. antibody working fluid: get ammonia benzyl penicilloic acid monoclonal antibody dilution 100000 ~ 150000 times, to obtain final product.
5. enzymic-labelled antibody: the marker enzyme in enzyme labeling is horseradish peroxidase or alkaline phosphatase, wherein preferred horseradish peroxidase; Enzyme marker is that sheep anti mouse two is anti-or goat-anti rabbit two is anti-.Enzymic-labelled antibody obtains by market purchasing, by the enzymic-labelled antibody of buying dilution 10000 ~ 20000 times.
6. substrate nitrite ion: colour developing A liquid, colour developing B liquid
Colour developing A liquid: the aqueous solution being the hydrogen peroxide of 1.5 ~ 2.5%: colour developing B liquid: the aqueous solution being the tetramethyl benzidine of 0.5 ~ 2.5%;
7. series standard solution: get ammonia benzyl thiazole acid standard items, make the standard solution that concentration is 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb, 24.3ppb, 72.9ppb, 218.7ppb, 500ppb respectively.
8. stop buffer: the sulfuric acid solution selecting 1.5 ~ 2.5M.
Two, kit package
| 96 hole ELISA Plate | 2 pieces |
| Confining liquid | 50mL |
| Sample diluting liquid (10X) | 100mL |
| Concentrated cleaning solution (10X) | 100mL |
| Antibody working fluid | 20mL |
| Enzymic-labelled antibody | 30mL |
| Colour developing A liquid | 15mL |
| Colour developing B liquid | 15mL |
| Stop buffer | 15mL |
| Series standard solution | The each 500 μ L of each concentration |
Three, the operation steps of ammonia benzyl penicilloic acid detection kit
1, taken out from 4 DEG C of cold storage environment by kit, use after being placed in equilibrium at room temperature about 30min, all reagent should fully shake up before using;
2, the lath experimentally required for demand taking-up, puts into valve bag by no lath, is placed in 4 DEG C of preservations;
3, cleansing solution and sample diluting liquid is prepared: concentrated cleaning solution (10X) and sample diluting liquid (10X) are diluted according to 1:9 with deionized water respectively;
4, liquid in taken ELISA Plate mesopore is dried, add cleansing solution 200 μ l hole, wash 3 times, each 2min, pat dry (bubble be not eliminated after patting dry can puncture with clean rifle head) with thieving paper stand-by;
5, number: sample and the corresponding micropore of standard items are numbered and the position at record standard hole and sample aperture place according to the order of sequence; 6, sample pre-treatments: get 2 ~ 5ml testing sample (as milk, or other animal foods), removes wherein after animal protein, dilutes 10 times, obtain sample liquid to be measured with sample diluting liquid;
If for Beta-lactam medicine prototype residual in indirect detection animal food and metabolic product thereof, then add according to 1:1 ratio by the sample stoste after 0.2mol/L NaOH and process, abundant mixing, with 1mol/LNaOH solution adjust pH to being greater than 12, fully 30min is stirred under room temperature, about regulating solution ph to 8 with 1mol/L HCL, final volume after record sample preparation, Beta-lactam medicine prototype residual in sample is converted to relative medicine metabolic product after 30 DEG C of water-bath 48h, dilute 10 times with sample diluting liquid, obtain sample liquid to be measured;
7, application of sample: each concentrations control solution pipetting series standard solution respectively, the sample handled well, in the micropore (i.e. 50 μ l/ holes) of respective label, then add antibody working fluid 50 μ L/ hole, with cover plate film shrouding, mixing of vibrating gently.30min is reacted in temperature 37 DEG C of environment;
8, take out ELISA Plate, liquid in hole is dried, adds cleansing solution 200 μ L/ hole, wash 3 times, each 2min, pat dry (bubble be not eliminated after patting dry can puncture with clean rifle head) with thieving paper;
9, add enzymic-labelled antibody: in each micropore, add enzymic-labelled antibody 100 μ L respectively, after cover plate membrane cover plate, put in 37 DEG C of environment and react 30min.Then dried by liquid in micropore, add cleansing solution 200 μ L/ hole, wash 3 times, each 2min, pats dry with thieving paper;
10, develop the color: colour developing A liquid is fully mixed according to 1:1 ratio with colour developing B liquid, adds the nitrite ion 100 μ L after mixing in each micropore, mixing of vibrating gently, lucifuge colour developing 20min in 25 DEG C of environment;
11, measure: add stop buffer 50 μ L in each micropore, mixing of vibrating gently, setting microplate reader, in 450nm place, measures every hole absorption photometric value (OD value); Derived data calculates.
The testing result of embodiment 4 ammonia benzyl penicilloic acid kit
One, precision test
Test method:
Indirect competitive ELISA method is adopted to measure the OD value of variable concentrations (0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 72.9ppb, 218.7ppb, 500ppb) ammonia benzyl penicilloic acid standard items, method is with the detection method of this kit, with standard items light absorption value number percent for ordinate, with the semilog of ammonia benzyl penicilloic acid standard concentration (ng/mL) for horizontal ordinate, drawing standard curve map, verifies the precision of this detection method.Test figure is shown in accompanying drawing 1.
Conclusion: find out from experimental data, this detection method precision is higher, typical curve linear correlation R
2value is 0.9779 (>0.95), meets detection basic demand.
Two, accuracy experiment
Experimental technique:
Get blank milk, add ammonia benzyl penicilloic acid by the concentration of 4ng/mL, 10ng/mL, extract sample liquid according to sample treatment, carry out indirect competitive ELISA detection, each interpolation concentration is done 5 times and 10 times respectively and is diluted and do three parallel repeating holes.Read OD
450calculate according to inhibiting rate accounting equation after light absorption value and add ammonia benzyl penicilloic acid content in sample extracting solution, and calculate its TIANZHU XINGNAO Capsul.
Experimental data:
Conclusion: this kit testing result is all greater than 87%, and the recovery is higher.
Three, minimum quantitative limit: adopt indirect competitive ELISA method to measure the OD of variable concentrations ammonia benzyl penicilloic acid standard items
450, when concentration is 0.1ppb, the data of result favorable reproducibility can be obtained, therefore, push up most and be quantitatively limited to 0.1ppb.
Four, cross reacting rate test
Experimental technique:
Employing indirect competitive ELISA method measures and calculates the IC50 value of relevant Beta-lactam medicine and metabolic product (concrete detection of drugs is as follows) thereof, calculates according to cross reacting rate accounting equation.
The disposal route of medicine:
Get Beta-lactam medicine standard items 0.2g, add 0.2mol/L NaOH 2mL, after abundant mixed dissolution, adjust solution ph to being greater than 12 with 1mol/L NaOH, after fully stirring 30min under room temperature, regulate solution ph to 8.7 with 1mol/L HCL, generate relative medicine metabolic product after 30 DEG C of water-bath 48h, 4 DEG C of preservations.
Experimental result:
Cross reacting rate between medicine prototype:
The direct cross reacting rate of drug metabolite:
Five, stability test
The storage stability test test period is 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 24 weeks, 48 weeks.Carry out the foundation of typical curve respectively and add recovery experiment, the validity of reagent and the storage stability of kit in detection kit.Result shows, the storage stability time is 48 weeks.
Six, result measures
1, the calculating of percentage absorptance, the percentage absorptance of standard items or sample equals the absorbance of mean value (diplopore) divided by first standard (negative control) of the percentage absorbance of standard items or sample, then is multiplied by 100%, to obtain final product,
The mean absorbance values of B-standard solution or sample solution
The mean absorbance values of B0-0ng/ml standard solution
2, the drafting of typical curve and calculating
With standard items percentage absorptance for ordinate, with the semilog of ammonia benzyl penicilloic acid standard concentration (ng/mL) for horizontal ordinate, drawing standard curve map.The percentage absorptance of sample substituted in typical curve, read the concentration corresponding to sample from typical curve, the extension rate being multiplied by its correspondence is residual ammonia benzyl penicilloic acid actual concentrations in sample.
The present invention adopts specific ammonia benzyl penicilloic acid monoclonal antibody, adopts indirect competitive ELISA method quantitatively to detect penicillin medicine and metabolic product remaining in animal food thereof; Detect while can completing some batch samples at short notice, and testing result is quick, accuracy is high, favorable reproducibility, detection sensitivity are high.
The present invention is not limited to above-mentioned preferred forms; anyone can draw other various forms of products under enlightenment of the present invention; no matter but any change is done in its method or structure; every have identical with the application or akin technical scheme, all drops within protection scope of the present invention.
Claims (6)
1. a hybridoma cell strain, its deposit number is CGMCC NO.10112.
2. an ammonia benzyl penicilloic acid monoclonal antibody of being secreted by hybridoma cell strain according to claim 1.
3. monoclonal antibody according to claim 2 detects the application in the kit of ammonia benzyl penicilloic acid in preparation.
4. for detecting a kit for ammonia benzyl penicilloic acid, it is characterized in that: comprise ELISA Plate, confining liquid, ammonia benzyl penicilloic acid standard items, antibody working fluid, enzymic-labelled antibody, nitrite ion and stop buffer; Described antibody working fluid is the dilution of the ammonia benzyl penicilloic acid monoclonal antibody of hybridoma cell strain as claimed in claim 1 secretion.
5. a kind of kit for detecting ammonia benzyl penicilloic acid according to claim 4, it is characterized in that: also comprise sample diluting liquid and concentrated cleaning solution, described sample diluting liquid is the phosphate buffer of pH6.5 ~ 8.5, and described concentrated cleaning solution is the mixed liquor of sample diluting liquid and polysorbas20.
6. one kind utilizes the application of kit in detection animal food for detecting ammonia benzyl penicilloic acid described in any one of claim 4-5.
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Cited By (1)
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| CN107904210A (en) * | 2017-12-27 | 2018-04-13 | 江南大学 | One plant of procaine monoclonal antibody hybridoma cell strain 4G6 and its application |
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Cited By (1)
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| CN107904210A (en) * | 2017-12-27 | 2018-04-13 | 江南大学 | One plant of procaine monoclonal antibody hybridoma cell strain 4G6 and its application |
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