CN102608319A - Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting gentamicin and sisomicin - Google Patents

Abstract

The invention discloses a specific monoclonal antibody capable of recognizing gentamicin and sisomicin simultaneously. The monoclonal antibody of the invention is secreted by hybridoma DEC/2D5 which is preserved in China Center for Type Culture Collection with the preservation number of CCTCC NO: C201145. The invention also discloses a preparation method of the specific monoclonal antibody, coating antigen and immunogen as well as an enzyme linked immunosorbent assay kit. Compared with the prior art, the monoclonal antibody prepared according to the invention can recognize gentamicin and sisomicin simultaneously, therefore, the detection efficiency of the prior art is improved. The kit and the method of the invention have the advantages of simplicity and convenience in operation, sensitivity, accuracy and the like.

Description

Be used to detect monoclonal antibody and the enzyme-linked immunoassay method and the kit of gentamicin and Sisomicin

Technical field

The present invention relates to a kind of can discern the monoclonal antibody of gentamicin and Sisomicin and be used to detect gentamicin and Sisomicin enzyme-linked immune analytic method and kit.

Background technology

Gentamicin and Sisomicin belong to aminoglycoside antibiotics (AMGs); Owing to being prone to accumulate at cortex renis and inner ear perilymph, this type microbiotic causes ototoxicity and renal toxicity; And, so its residue detection paid more and more attention in animal derived food comparatively serious by the microorganism persister of aminoglycoside inactive enzyme generation.ELISA (ELISA) method is because advantage such as simple, quick, sensitive, special and be widely used in the quick residue detection field of AMGs gradually.But present ELISA method mainly concentrates on single residue detection aspect, with the ELISA method of a kind of antibody AMGs that detection architecture is different simultaneously seldom.

Application number is that 200710064346.8 patent of invention discloses a kind of enzyme linked immunological kit and method that detects the gentamicin medicine; This patent is with gentamicin and carrier protein; Carrier protein is: mouse haemocyanin, thyroprotein, bovine serum albumin(BSA), albumin rabbit serum, human serum albumins, ovalbumin or hemocyanin; Adopt glutaraldehyde method or carbodlimide method to carry out coupling and obtain immunogene and coating antigen; Prepared polyclonal antibody and monoclonal antibody can only be discerned gentamicin, and the detection time of its sample is longer.Application number is that 200920246849.1 patent of invention discloses a kind of gentamicin ELISA detection kit; This patent adopts the method coupling gentamicin and bovine serum albumin(BSA) (bovine serum albu minute of direct activation albumen; BSA) obtain immunogene, same method obtains coating antigen with gentamicin and thyroprotein coupling, and prepared monoclonal antibody equally only can be discerned gentamicin; And its sample preparation time is longer, and reagent is not optimized.

Summary of the invention

First purpose of the present invention provides a kind of monoclonal antibody that can discern gentamicin and Sisomicin.

Second purpose of the present invention is to utilize this monoclonal antibody, sets up a kind of enzyme-linked immunoassay method that can be used for gentamicin and Sisomicin detection.

The 3rd purpose of the present invention provides a kind of kit that is used for gentamicin and Sisomicin detection.

The 4th purpose of the present invention provides the application of said monoclonal antibody in the enzyme linked immunological kit of preparation detection gentamicin and Sisomicin.

The 5th purpose of the present invention provides the application of kit in animal tissue's gentamicin and Sisomicin residue detection that contains said monoclonal antibody.

The present invention realizes through following technical scheme:

A kind of monoclonal antibody that can discern gentamicin and Sisomicin, it is to be that the hybridoma EDC/2D5 of CCTCC NO:C201145 is secreted by preserving number.

Above-mentioned hybridoma EDC/2D5 is deposited in the Chinese typical culture collection center (CCTCC) that is positioned at Wuhan City, Hubei Province Wuhan University, and preserving number is CCTCC NO:C201145.

Used immunogene is by haptens gentamicin and bovine serum albumin(BSA) coupling preparation.

Further, the invention provides a kind of enzyme-linked immunoassay method that gentamicin and Sisomicin detect that is used for, this method comprises the steps such as processing and detection of preparation and the sample of immunogene, coating antigen, antibody, and is specific as follows:

(1) haptens gentamicin and bovine serum albumin(BSA) (BSA) coupling are obtained immunogene;

(2) haptens gentamicin and ovalbumin (OVA) coupling are obtained coating antigen;

(3) utilize the immunogen immune mouse of step (1), obtain the hybridoma EDC/2D5 that preserving number is CCTCC NO:C201145 through Fusion of Cells and screening;

(4) use preserving number to prepare monoclonal antibody as the hybridoma EDC/2D5 of CCTCC NO:C201145;

(5) coating antigen with step (2) encapsulates solid phase carrier (like ELISA Plate);

(6) testing sample is handled with 2% trichloroacetic acid, the centrifuging and taking supernatant, adjust pH is determinand solution;

(7) the determinand solution to step (6) carries out enzyme linked immunosorbent detection.

The component of 2% trichloroacetic acid and proportioning are in the step (6): accurately take by weighing trichloroacetic acid 20.0g, add a small amount of distilled water dissolving earlier, be settled to 1000mL again.

The present invention with said monoclonal antibody and coating antigen as core reagent and other conventional agent combination; Processed the enzyme linked immunological kit that can detect gentamicin and Sisomicin; In conjunction with above-mentioned enzyme-linked immunoassay method, realized enzyme linked immunosorbent detection to gentamicin and Sisomicin.

Major advantage of the present invention is:

1, the monoclonal anti physical efficiency of the present invention's preparation is discerned gentamicin and Sisomicin simultaneously, and prior art only can single identification gentamicin.

2, the ELISA method of the present invention's foundation is short detection time, and detection efficiency is high.

3, the tissue sample disposal route that the present invention relates to is simple, need not expensive instrument, need not organic reagent, the operator is not had health hazard, and only need generic centrifuge to get final product, and is adapted at basic unit's operation.

Description of drawings

Fig. 1 is the mass spectrogram of the substance assistant laser desorpted method detection of carrier protein BSA among the present invention.

Fig. 2 is the mass spectrogram that immunogenic substance assistant laser desorpted method among the present invention detects, and is used to explain the coupling effect of haptens gentamicin and BSA in conjunction with Fig. 1.

Accompanying drawing 3 is the indirect competitive ELISA response curve of monoclonal antibody of the present invention and gentamicin (GEN) standard items; The X axle is gentamicin (GEN) concentration of standard solution logarithm value, and the Y axle is that the OD value of gentamicin standard solution is divided by " zero " hole OD value (B/B0).

Embodiment

Through embodiment the present invention is described further below.

The preparation of embodiment 1 immunogene and coating antigen

1.1 gentamicin-BSA's is synthetic

Take by weighing gentamicin 164.0mg and BSA200.0mg and be dissolved in (pH7.4) in the 15mL pure water, stir, dropwise adding is dissolved in carbodiimide (EDC) 430.00mg in the 5mL pure water then, and stirring reaction is 8 hours under the room temperature.This reactant liquor changes in the bag filter at last, in PBS liquid (pH7.4), dialyses centrifugal postlyophilization 4 days for 4 ℃.Shown in Fig. 1-2, identify the coupling success through substance assistant laser desorpted method (MALDI-TOF-MS), it is subsequent use to put-20 ℃ of preservations.

1.2 gentamicin-OVA's is synthetic

Take by weighing gentamicin 148.0mg and OVA240.0mg and be dissolved in (pH7.4) in the 12mL pure water, stir, dropwise adding is dissolved in the EDC430.00mg in the 4mL pure water then, and stirring reaction is 8 hours under the room temperature.This reactant liquor changes in the bag filter at last, in PBS liquid (pH7.4), dialyses 4 days for 4 ℃.Centrifugal postlyophilization, it is subsequent use to put-20 ℃ of preservations.

Embodiment 2 MONOCLONAL ANTIBODIES SPECIFIC FOR

The preparation of hybridoma: with reference to Yang Hanchun " animal immunology "; Immunogene gentamicin-BSA immunity Balb/C mouse (available from Disease Prevention Control Center, Hubei Prov's Experimental Animal Center) with embodiment 1 preparation; Immune programme for children is: fundamental immunity with immunogene and isopyknic Freund's complete adjuvant emulsification after; In the subcutaneous multi-point injection of mouse back, later every interval 2 all booster immunizations are once used Freund emulsification instead.At last in merging first three day (be better than most immunity finish the back rest and reorganize January) lumbar injection, reinforced immunological, the antigen amount doubles, and does not add adjuvant.

During fusion, one of the Balb/C mouse of the last reinforced immunological of learning from else's experience, eye socket sacrificed by exsanguination (collect serum, be positive serum) is soaked sterilization in 5 minutes in 75% alcohol.Aseptic taking-up mouse spleen; Isolate splenocyte; With the SP2/0 myeloma cell (SP2/0 myeloma cell is from this laboratory) of prepared fresh in the ratio of 1-2 * 107 SP2/0 and 108 immune spleen cells (1: 10～1: 15) in the 50mL centrifuge tube; With 15mLRPMI-1640 basal liquid re-suspended cell, 1500r/ minute centrifugal 5 minutes, wash cell 1 time.The nutrient culture media that temperature is bathed in centrifugal gap, the water that temperature is bathed, the PEG that temperature is bathed etc. puts into super-clean bench.Take out the thieving paper of sterilization then, after emptying to the greatest extent on the centrifuge tube that myeloma cell and immune spleen cell are housed, tip upside down on that the control solid carbon dioxide drips on the thieving paper, rap the pipe end cell is become flexible.Open timer, draw 0.8mLPEG with the 1mL suction pipe, the hand-held centrifuge tube that cell mixing is housed places it in a moment in the water-bath, and PEG is added drop-wise on the cell mixing slowly, and the limit edged stirs gently, adds in 1 minute, continues to stir 30 seconds.Get the 10mL basal liquid with suction pipe, slowly be added on the fused cell along tube wall, the limit edged shakes (can not blow and beat) gently, adds 1mL respectively in 5 minutes; 2mL, 3mL, 4mL adds basal liquid at last to 40mL; After adding, cover lid is put upside down several times repeatedly, makes the cell mixing.800r/ minute 5 minutes centrifugal, abandons supernatant.The HAT nutrient culture media that absorption contains feeder cells stirs the fused cell in the centrifuge tube with suction pipe gently, dropwise splashes near the liquid level in the serum bottle that contains feeder cells, and stirring and evenly mixing, action will gently be stirred cell gently, must not blow and beat.Put upside down mixing.Then with cell inoculation on Tissue Culture Plate, two in every hole places incubator to cultivate.Once merge and to inoculate 4～6 96 orifice plates.Also can plant less as required, generally press the cell number of SP2/0 and calculate, every hole inoculum concentration contains about 104 SP2/0 cells approximately.In 37 ℃, cultivate in the 5%CO2 incubator.

Counted 0 day the same day of merging, and the preceding 3 days kinetocyte plates of trying not keep the incubator homeostasis.1 HAT complete medium was added in every hole in the 3rd day; The 5th day every hole sucking-off 1/2 culture supernatant (100 μ L) adds 1 HT complete medium again; Later on every at a distance from 2 days the same method suction go 1/2～3/4 culture supernatant, after 7 days, change to the HT complete medium.

Treat that the fused cell colony grows to culture hole 1/10～1/5, screen with indirect ELISA method and the indirect competitive ELISA method set up simultaneously.Compare with zero medicine hole, medicine hole OD value can the repressed positive that is judged to.According to inhibiting rate and cell colony upgrowth situation, select the cell hole that 1-2 single colony only arranged of 2～6 strong positives, adopt the limiting dilution method to clone.

Through 3～4 time clonings, be 100% until clone's positive rate, finishing screen is selected the hybridoma of anti-gentamicin of secretion and Sisomicin, and the chromosome average of this cell line is 101.The applicant is this hybridoma called after EDC/2D5, and delivers the Chinese typical culture collection center preservation that is positioned at Wuhan City, Hubei Province Wuhan University on June 29th, 2011, and its preserving number is CCTCC NO:C201145.

Preparation of ascites monoclonal antibody and evaluation: only got the Balb/c number of mice in preceding 7 days in inoculation, every mouse peritoneal injection 0.5ml incomplete Freund carries out pre-service.Use RPMI 1640 basal mediums to suspend, and cell number is transferred to 1 * 10 by the cell of preserving number as the hybridoma EDC/2D5 enlarged culture of CCTCC NO:C201145 ⁶Individual/mL, every mouse peritoneal inoculation 0.5ml.Treat that mouse web portion obviously expands, spiritual variation, the dying ascites of gathering when motionless.According to literature method (Zhu Liping, Chen Xueqing. immunology common experimental method. Beijing: People's Medical Officer Press, 2000), purifying obtains monoclonal antibody.Employing is carried out the hypotype evaluation available from the mouse source monoclonal antibody hypotype identification kit (Mouse Mab Isotyping Test Kit) of ROCKLAND company to the resulting monoclonal antibody of the present invention, and the result is a mouse IgG1 hypotype.

The foundation of embodiment 3 gentamicin racing ELISA detecting methods

3.1 the preparation of reagent (reagent that present embodiment uses all adopts following method preparation except that other indicates)

Carbonate buffer solution (pH9.6): accurately take by weighing Na ₂CO ₃1.59g, NaHCO ₃2.93g a small amount of ultrapure water dissolving is settled to 1000mL.

Cleansing solution (pH7.4): accurately take by weighing NaCl 8.00g, KH ₂PO ₄0.20g, Na ₂HPO ₄12H ₂O 2.90g, KCl 0.20g, a small amount of ultrapure water dissolving adds Tween20 0.50mL, is settled to 1000mL.

Phosphate buffer (PBS) is (pH7.4): accurately take by weighing NaCl8.00g, KH ₂PO ₄0.20g, Na ₂HPO ₄12H ₂O 2.90g, KCl0.20g, a small amount of ultrapure water dissolving is settled to 1000mL.

Confining liquid: accurately take by weighing ovalbumin 10.00g, add the 1000mL phosphate buffer, stirring and evenly mixing dissolves until albumen fully.

Physiological saline: accurately take by weighing NaCl 8.50g, a small amount of ultrapure water dissolving is settled to 1000mL.

Antibody diluent, enzymic-labelled antibody dilution and substrate solution fly Science and Technology Ltd. far away by Wuhan to be provided.

Stop buffer: accurately measure concentrated sulphuric acid 100mL, slowly be added drop-wise in the 800mL ultrapure water.

3.2 the preliminary of coating antigen concentration and antibody working concentration confirmed

It at first is combination through the method initial option coating antigen and the antibody working concentration of square formation titration.Use carbonate buffer solution that GEN-OVA coating antigen doubling dilution is become the horizontal coated elisa plate of 32,16,8,4,2,1,0.5,0.25 μ g/mL; The EDC/2D05 monoclonal antibody is used the phosphate buffer doubling dilution to become 1: 500,1: 1000,1: 2000,1: 4000,1: 8000,1: 16000,1: 32000,1: 64000,1: 128000,1: 512000 etc. is vertically added ELISA Plate.16000) and (8,1: 32000) the square formation titration results is seen table 1, the combination of the following coating antigen concentration of initial option and antibody working concentration: (8,1:.

3.3 confirming of best coating antigen concentration and antibody working concentration

The best encapsulates concentration and confirms: the concentration that encapsulates so that the square formation titration is selected is done respectively to suppress curve with the antibody dilution combination, and gentamicin standard items concentration is set to 0,5,10,20,40,80 μ g/mL, its zero medicine hole and IC ₅₀Value is seen table 2.The ratio of antigen-antibody is the key that influences its sensitivity, if antigen or antibody excess all will cause IC ₅₀Higher, comprehensive IC ₅₀The linearly dependent coefficient of value, zero hole OD value and inhibition curve, it is 8 μ g/mL that the best encapsulates concentration, antibody dilution is tentatively confirmed as 1: 32000.

The titration of table 1EDC/2D5 monoclonal antibody square formation

Table 2 the best encapsulates concentration optimization

Coating antigen concentration (μ g/mL)	Antibody dilution multiple (1: X)	0 hole OD value	IC 50Value (μ g/L)
				8	16000	2.81	27.20
8	32000	2.44	18.55

The optimum antibody dilutability is confirmed: encapsulating the concentration coated elisa plate with the best, was 7 dilutions of centre concentration equal difference design gradient with antibody with 1: 40000, its zero medicine hole and IC ₅₀Value is seen table 3.Along with the increase of antibody dilution, IC ₅₀Value reduces, and zero medicine hole value also reduces.Comprehensive IC ₅₀The linearly dependent coefficient of value, zero hole OD value and inhibition curve, selecting 1: 48000 is the optimum antibody dilutability.

Table 3 optimum antibody dilutability is optimized

Antibody coefficient multiple (1: X)	0 hole OD value	IC50 value (μ g/L)
			16000	2.81	27.20
32000	2.44	18.55
			36000	2.3	17.14
40000	2.25	17.7

44000	2.27	17.12
			48000	2.02	13.09
52000	2.45	17.01

3.4 the foundation of typical curve

Gentamicin standard items concentration is set to 6 concentration such as 0,5,10,20,40,80 μ g/L, measures the drawing standard curve according to top definite indirect competitive ELISA method.As shown in Figure 3, the gentamicin that the present invention sets up and the regression equation and the linear dependence index of the residual indirect competitive ELISA method of Sisomicin are respectively: y=-0.547x+1.166, r=0.996, IC _{50 values}Be 16.33 ± 1.24 μ g/L (n=5).The range of linearity is 5～80 μ g/L.

3.5 specificity

Become gradient concentration to carry out indirect competitive ELISA various aminoglycoside antibiotics doubling dilutions commonly used respectively, calculate IC ₅₀Value is with gentamicin standard items IC ₅₀The value contrast obtains cross reacting rate, and the result sees table 4.The result shows, the indirect competitive ELISA method that the present invention sets up is respectively 100% and 33.80% to the cross reacting rate of gentamicin and Sisomicin, and with the equal no cross reaction of other aminoglycoside antibiotics.

The specificity of the residual ELISA detection method of table 4 gentamicin and Sisomicin

The competition thing	IC 50(μg/L)	Cross reacting rate (%)
			Gentamicin	16.33	100
Sisomicin	48.31	33.80
			Neomycin	＞10000	＜0.02
Amikacin	＞10000	＜0.02
			Paromomycin	＞10000	＜0.02
Neamine	＞10000	＜0.02
			Kanamycins	＞10000	＜0.02
Streptomysin	＞10000	＜0.02
			Dihydrostreptomycin	＞10000	＜0.02
Ribostamycin	＞10000	＜0.02

Spectinomycin	＞10000	＜0.02
			Apramycin	＞10000	＜0.02
Certomycin	＞10000	＜0.02
			TOB	＞10000	＜0.02
Hygromycin	＞10000	＜0.03
			Kasugarnycin	＞10000	＜0.03

The assembling of embodiment 4 gentamicins of the present invention and the how residual ELISA detection kit of Sisomicin

4.1 the composition of ELISA kit of the present invention

1) is coated with the solid phase carrier (ELISA Plate) of coating antigen gentamicin-OVA;

2) the gentamicin standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 5 μ g/L, 10 μ g/L, 20 μ g/L, 40 μ g/L, 80 μ g/L;

3) gentamicin monoclonal antibody working fluid;

4) the sheep anti-mouse igg antibody working fluid of horseradish peroxidase (HRP) mark;

5) concentrated phosphoric acid salt buffer: NaCl 80.0g, KH ₂PO ₄2.0g, Na ₂HPO ₄12H ₂O 29.0g, KCl 2.0g adds ultrapure water water to 1000mL;

6) concentrated cleaning solution: NaCl 80.0g, KH ₂PO ₄2.0g, Na ₂HPO ₄12H ₂O 29.0g, KCl 2.0g, polysorbas20 5mL adds ultrapure water to 1000mL;

7) substrate solution A: flying scientific & technical corporation far away by Wuhan provides;

8) substrate solution B: flying scientific & technical corporation far away by Wuhan provides;

9) stop buffer: 2mol/L sulfuric acid solution.

4.2 the preparation of ELISA Plate

With coating buffer gentamicin-OVA is diluted to 8 μ g/mL, every hole adds 100 μ L, and 4 ℃ encapsulated 12～16 hours; The coating buffer that inclines, every hole adds 250 μ L cleansing solutions, washs 3 times, claps and does, and every then hole adds confining liquid 250 μ L, hatches 2 hours for 37 ℃; The liquid in the hole that inclines, cleansing solution washing 3 times after the thieving paper arsis is done, is inverted in 37 ℃ of incubators with ELISA Plate, leaves standstill oven dry 30 minutes, and ELISA Plate oven dry back and the drying agent aluminium foil bag of packing into together encapsulates with vacuum packaging machine.

The mensuration program of embodiment 5 kits of the present invention

5.1 the preparation of reagent

1) cleansing solution: the cleansing solution that provides in the kit is used with after 10 times of the ultrapure water dilutions.

2) substrate mixed liquor:,, at present join existing usefulness with substrate solution A and by volume 1: 100 mixing of substrate solution B of preparation according to each institute expense.

3) component of 2% trichloroacetic acid and proportioning are: accurately take by weighing trichloroacetic acid 20.0g, add a small amount of distilled water dissolving earlier, be settled to 1000mL again.

5.2 tissue sample pre-treatment

Get 2g homogeneous structure sample, add 2% trichloroacetic acid 8mL, vortex 3 minutes, 4000r/ minute centrifugal 10 minutes again, get the 1mL supernatant, add 1M NaOH 75 μ l and transfer pH, it is for use to get 50 μ l.

Annotate: this method is 4 to the dilution of sample multiple.

5.3 determination step

1) application of sample: in the ELISA Plate micropore, add gentamicin series concentration standard solution or sample solution 50 μ L, add gentamicin monoclonal antibody working fluid 50 μ L then, place wet box, 37 ℃ of constant temperature were hatched 30 minutes;

2) washing: pour out the liquid in the hole, add cleansing solution 250 μ L in every hole, leave standstill 30 seconds after, wash 3 times and claps dried;

3) add the sheep anti-mouse igg antibody working fluid of horseradish peroxidase (HRP) mark: add the sheep anti-mouse igg antibody working fluid 100 μ L of horseradish peroxidase (HRP) mark in every hole, constant temperature was hatched 30 minutes in 37 ℃ of wet boxes;

4) washing: pour out the liquid in the hole, add cleansing solution 250 μ L in every hole, leave standstill 30 seconds after, wash 3 times and claps dried;

5) add substrate: add substrate mixed liquor 100 μ L in every hole, hatched 15 minutes in 37 ℃ of wet boxes;

6) add stop buffer: add stop buffer 50 μ L in every hole;

7) measure: the OD value (OD value) of measuring every hole with ELIASA at the 450nm place.

5.4 the result judges

With the standard items OD value measured divided by " zero " hole OD value (B/B ₀) be ordinate, the logarithm value of gentamicin concentration is that horizontal ordinate is made typical curve, the line linearity of going forward side by side returns, and provides regression equation.According to the inhibiting rate of formula calculation sample, with inhibiting rate substitution regression equation, calculate mensuration concentration, multiply by corresponding extension rate, be the residual concentration of gentamicin and Sisomicin in the sample.

Sensitivity, precision and the accuracy of embodiment 6 kits of the present invention

6.1 the sensitivity of kit of the present invention

IC with typical curve ₅₀Value and organize the sensitivity index of LDL (LOD) as detection kit of the present invention.The gentamicin standard items are diluted to 6 concentration such as 0 μ g/L, 5 μ g/L, 10 μ g/L, 20 μ g/L, 40 μ g/L, 80 μ g/L, 5 repeating holes of each concentration according to indirect competitive ELISA method replication 5 times, are got the IC that measures for 5 times ₅₀Mean value.LOD is through the following steps decision; Measure the OD value of the musculature of 20 parts of blank chickens; Regression equation calculation according to typical curve goes out corresponding gentamicin concentration; Calculate the mean value

and the standard deviation (SD) of gentamicin concentration then,

calculates the LDL in the tissue according to formula.IC of the present invention ₅₀Value is 16.33 ± 1.24 μ g/L, and the lowest detection of gentamicin in chicken muscle is limited to 34.09 μ g/L.

6.2 the precision of kit of the present invention

Respectively that gentamicin standard items concentration such as 5,10,20,40,80 μ g/L is corresponding its typical curve equation of OD value substitution is obtained the measured value that ELISA detects; With the coefficient of variation between the plate inner panel of standard items concentration determination value calculating indirect competitive ELISA typical curve, the result sees table 5.The result shows, reaches Variation Lines number average＜15% between plate in the plate of typical curve, explains that indirect competitive ELISA method of the present invention has better precision.

The coefficient of variation in the plate of table 5 typical curve and between plate

6.3 the accuracy of kit of the present invention

In the homogenate chicken muscle tissue of 2g, add the gentamicin standard solution, make its final concentration be respectively 200 μ g/kg, 100 μ g/kg and 50 μ g/kg; Add the Sisomicin standard solution, make its final concentration be respectively 200 μ g/kg, 100 μ g/kg and 50 μ g/kg; 5 repetitions of each concentration, replication 3 times.The gentamicin in the mensuration interpolation tissue and the concentration of Sisomicin, calculate recovery rate is examined the accuracy of kit according to the following equation; Calculate batch interior and interassay coefficient of variation, the repeatability of examination kit.Accuracy and repeated result see table 6 and table 7, show that this kit has reliable accuracy, and be little with interassay coefficient of variation in batch, good reproducibility.

Gentamicin adds the recovery and the coefficient of variation in table 6 chicken muscle

Sisomicin adds the recovery and the coefficient of variation in table 7 chicken muscle

Claims (7)

1. the monoclonal antibody that can discern gentamicin and Sisomicin is characterized in that, it is to be that the hybridoma EDC/2D5 of CCTCC NO:C201145 is secreted by preserving number.

2. the described hybridoma EDC/2D5 of claim 1 is deposited in Chinese typical culture collection center, and its preserving number is CCTCC NO:C201145.

3. the kit that comprises the said monoclonal antibody of claim 1.

4. kit according to claim 3, this kit are the enzyme linked immunological kits that detects gentamicin and Sisomicin.

5. an enzyme-linked immunoassay method that detects gentamicin and Sisomicin comprises the preparation of immunogene, coating antigen, antibody and the processing and the detection of sample, and its step is following:

(1) haptens gentamicin and bovine serum albumin(BSA) coupling are obtained immunogene;

(2) haptens gentamicin and ovalbumin coupling are obtained coating antigen;

(3) utilize the immunogen immune mouse of step (1), obtain the hybridoma EDC/2D5 that preserving number is CCTCC NO:C201145 through Fusion of Cells and screening;

(4) use preserving number to prepare monoclonal antibody as the hybridoma EDC/2D5 of CCTCC NO:C201145;

(5) coating antigen with step (2) encapsulates solid phase carrier;

(6) testing sample is handled with 2% trichloroacetic acid, the centrifuging and taking supernatant, adjust pH gets determinand solution;

(7) the determinand solution to step (6) carries out enzyme linked immunosorbent detection.

6. the application of the described monoclonal antibody of claim 1 in the enzyme linked immunological kit of preparation detection gentamicin and Sisomicin.

7. claim 3 or the application of 4 described kits in animal tissue's gentamicin and Sisomicin residue detection.

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