CN104558189B - Monoclonal antibody and enzyme-linked immunoassay method and kit for detecting cefalexin, cefadroxil and Cefradine - Google Patents

Abstract

The invention discloses a kind of monoclonal antibody specific that can recognize cefalexin, cefadroxil and Cefradine and it is a kind of be used to detecting the enzyme-linked immunoassay method and kit of cefalexin, cefadroxil and Cefradine, it by preserving number is CCTCC NO that monoclonal antibody of the invention, which is,:Secreted by C201340 hybridoma cell strain 3A6.Compared with prior art, the monoclonal antibody that prepared by the present invention can be while recognize cefalexin, cefadroxil and Cefradine, and enzyme-linked immunoassay method and kit of the invention has detection efficiency and sensitivity high, the advantages of preci-sion and accuracy is good.

Description

For detecting the monoclonal antibody of cefalexin, cefadroxil and Cefradine and enzyme-linked exempting from Epidemic disease method and kit

Technical field

The invention belongs to medicament residue analysis and immunological technique field, and in particular to a kind of anti-cefalexin, cephalo hydroxyl The monoclonal antibody of ammonia benzyl and Cefradine, the invention further relates to for detecting cefalexin, cefadroxil and Cefradine Enzyme-linked immunoassay method and kit.

Background technology

Cefalexin, cefadroxil and Cefradine belong to cephalosporins veterinary drug, the basic knot with beta-lactam Structure, antimicrobial spectrum is wider, is usually used in preventing and treating bacterium infection, is widely applied in veterinary clinic and aquaculture.People In order to pursue economic interests and drug abuse, cause medicine largely to be remained in animal foodstuff.Human consumption contains the food of medicine After product, a series of damaging actions are produced to body, allergic reaction is such as produced, occur to suffer a shock when serious even dead, medicine enters Break colony balance after intestines and stomach, while can also increase the drug resistance of bacterium, cause the failure of clinical application.

Now have been developed a variety of methods for detecting such medicament residue, such as microbial process, instrumental method and enzyme-linked Immunization method.Microbial method is used for the preliminary screening of a large amount of samples, and instrumental method is usually used in the confirmation to positive, ELISA method both can with it is qualitative can also sxemiquantitative.In these residue analysis methods, instrumental method is high due to its required instrument Expensive, complex operation, testing cost are high, and are unfavorable for screening and the Site Detection of batch samples.Though microbial method is sieved in residual Select and good performance is shown on high flux, but method lacks specificity, it is impossible to confirm the species of residue, and bacterium pair used Different types of Susceptibility to antibiotics has differences, and is easily caused the generation of false negative and false positive results.And ELISA method is grasped Make simplicity, high flux, high sensitivity, low expense, the defect of instrumental method and microbial process can be overcome well.

CN101354401A discloses a kind of cefalexin residual enzyme-linked immunologic detection reagent kit and its application, the kit It is only capable of detecting one medicine of cefalexin, can be while detecting that multiple Cephalosporins are remained in food so setting up one kind ELISA method and kit it is very necessary.

The content of the invention

The present invention first purpose be to provide it is a kind of can be while recognizing cefalexin, cefadroxil and Cefradine Monoclonal antibody.

Second object of the present invention is to utilize the monoclonal antibody, is prepared a kind of for detecting cefalexin, cephalo hydroxyl The enzyme linked immunological kit of ammonia benzyl and Cefradine.

Third object of the present invention is to utilize the kit, sets up a kind of for cefalexin, cefadroxil and head Spore draws the enzyme-linked immunoassay method for determining the detection of non-diagnostic purpose.

Above-mentioned purpose is achieved through the following technical solutions：

A kind of to recognize the monoclonal antibody of cefalexin, cefadroxil and Cefradine, it is to be by preserving number CCTCC NO:Secreted by C201340 hybridoma 3A6.

Above-mentioned hybridoma 3A6, is deposited in China typical culture collection center, and deposit number is CCTCC NO: C201340。

Immunogene used is to be coupled to prepare by haptens cefalexin and bovine serum albumin(BSA) (BSA).

Further, the invention provides cefalexin, cefadroxil and Cefradine medicine in one kind detection food are residual The enzyme-linked immunoassay method stayed, comprises the following steps：

(1) cefalexin is obtained into coating antigen using glutaraldehyde method with oralbumin (OVA) coupling；

(2) it is CCTCC NO with preserving number:C201340 hybridoma 3A6 prepares monoclonal antibody；

(3) with the coating primordial covering solid phase carrier of step (1)；

(4) enzyme linked immunosorbent detection is carried out after testing sample is extracted.

Preferably, the extracting method of the testing sample is：

Milk：Milk 1mL, room temperature 4000rpm centrifugation 10min are measured, intermediate layer is taken, is PBS with volume ratio: first Alcohol=9: direct sample detection after 1 40 times of diluted；

Meat：Meat sample homogeneous thing 2g is weighed, PBS 10mL is added, acutely after vibration 5min, room temperature 4000rpm centrifuges 10min；Supernatant intermediate layer is taken, sample detection after 8 times is diluted with PBS.

The present invention is made using said monoclonal antibody and coating antigen as core reagent and conventional other agent combinations The enzyme linked immunological kit of cefalexin, cefadroxil and Cefradine can be detected, with reference to above-mentioned enzyme-linked immunoassay method, is realized To the enzyme linked immunosorbent detection of cefalexin, cefadroxil and Cefradine.

Main advantages of the present invention are：

1. monoclonal antibody prepared by the present invention can be while recognize cefalexin, three kinds of heads of cefadroxil and Cefradine Spore bacteriums medicine, and existing monoclonal antibody can not recognize above medicine simultaneously.

2. monoclonal antibody prepared by the present invention has higher identification sensitivity, head to three of the above Cephalosporins The IC of cefalexin₅₀Only 1.83 μ g/L, the IC of cefadroxil₅₀Only 1.55 μ g/L, Cefradine is 1.78 μ g/L, identification Sensitivity is better than existing cephalosporins monoclonal antibody.And have higher friendship to three of the above Cephalosporins Reactivity is pitched, it is very low to the cross reacting rate of other medicines, illustrate to have preferably specificity.

3. ELISA method and kit that the present invention is set up can be while detect cefalexin, cefadroxil in newborn class, meat Benzyl and Cefradine medicament residue, the method degree of accuracy are high, and precision is good, and once determining to complete, with existing detection method Compare, there is obvious advantage in the species and efficiency of detection medicine, substantial amounts of time and cost can be saved, with more preferable Market value.

4. involved sample treatment is simple in method, easy to operate, the organic reagent used in sample treatment is to operation The healthy harm of person is relatively small.

Brief description of the drawings

Fig. 1 is haptens used in the present invention (cefalexin), bovine serum albumin(BSA) (BSA) and immunogene (cephalo ammonia Benzyl-BSA conjugate) UV scanning collection of illustrative plates.

Fig. 2 be haptens used in the present invention (cefalexin), oralbumin (OVA) and coating antigen (cefalexin- OVA conjugates) UV scanning collection of illustrative plates.

Fig. 3 is the monoclonal antibody of the present invention and the indirect competitive ELISA response curve of cefalexin standard items, and X-axis is Cefalexin (CFE) concentration of standard solution logarithm value, Y-axis is the OD value divided by " zero " Kong Guang of cefalexin standard solution Density value (B/B0).

Embodiment

Below by embodiment, the invention will be further described, but does not limit the present invention.

The preparation of the immunogene of embodiment 1 and coating antigen

The preparation of 1.1 immunogenes

Cefalexin 36.5mg accurately is weighed, it is A liquid to be dissolved completely in 4mL PBS.BSA 68mg accurately are weighed, are made It is B liquid that it, which is substantially dissolved in 6mLPBS,.Separately take carbodiimide (EDC) 120.0mg and n-hydroxysuccinimide (NHS) 60mg Be dissolved in 1mLPBS is C liquid.C liquid is added dropwise in B liquid under ice bath, magnetic agitation, ice bath reaction 4h then will mixing Liquid is added dropwise in A liquid, ice bath reaction 12h.After the completion of reaction, reaction solution is transferred in bag filter, dialyse 3d in 4 DEG C of PBS, often 4~6h changes a dialyzate, is freeze-dried sample after having dialysed, produces conjugate cefalexin-BSA, puts -20 DEG C of guarantors Deposit.

The preparation of 1.2 coating antigens

Take OVA 120mg to be dissolved in 8mL phosphate buffers (PBS), be A liquid.Cefalexin 36.5mg is dissolved in 2mL PBS In, it is B liquid.Under room temperature condition, A liquid and B liquid are mixed, 0.1mL glutaraldehydes (GA) solution is then slowly added into, reacted at room temperature 6h.The 3d that dialysed in supernatant, 4 DEG C of PBS, every 4~6h is taken to change a dialyzate, be freeze-dried sample after having dialysed after centrifugation, Conjugate cefalexin-OVA is produced, -20 DEG C of preservations are put.

The preparation of the monoclonal antibody of embodiment 2

The preparation of hybridoma：With reference to Yang Hanchun《Animal immunology》, the immunogene cephalo ammonia prepared with embodiment 1 Balb/C mouse (being purchased from Hubei Prov. Academy of Medical Sciences's Experimental Animal Center) are immunized in benzyl-bovine serum albumin(BSA) conjugate.Immune journey Sequence is：Fundamental immunity is injected in mouse with the protein emulsion containing 100 μ g immunogenes emulsified with isometric Freund's complete adjuvant Nape part it is subcutaneous；The protein emulsion containing 100 μ g immunogenes emulsified later every 15 days with incomplete Freund's adjuvant is added It is strong immune.From immune three times, tail blood is adopted within the 8th day after being immunized every time, serum, indirect elisa method detection serum antibody effect is separated Valency.The mouse (potency is high, sensitivity is good) of immuno-competent stops immune in case fusion.During fusion, last reinforced immunological of learning from else's experience Balb/C mouse one, eye socket sacrificed by exsanguination (collects serum, as positive serum), and 5min sterilizations are soaked in 75% alcohol.Nothing Bacterium takes out mouse spleen, isolates splenocyte, and (SP2/0 myeloma cell is from this with freshly prepared SP2/0 myeloma cell Laboratory) press 1~2 × 10⁷Individual SP2/0 and 10⁸Individual immune spleen cell (1:10~1:15) ratio is used in 50mL centrifuge tubes Cell is resuspended in 15mLRPMI-1640 basal liquids, and 1500r/min centrifugation 5min wash cell 1 time.The gap of centrifugation is by the training of warm bath Base is supported, the water of warm bath, the polyethylene glycol (PEG) of warm bath etc. is put into super-clean bench.The blotting paper of sterilizing is then taken out, marrow is will be equipped with After being emptied on the centrifuge tube of oncocyte and immune spleen cell to the greatest extent, tip upside down on and water droplet is drained on blotting paper, rapping ttom of pipe makes cell pine It is dynamic.Timer is opened, 0.8mLPEG is drawn with 1mL suction pipes, the hand-held centrifuge tube equipped with cell mixing is placed it in water-bath For a moment, PEG is slowly added drop-wise on cell mixing, side edged is gently mixed, and is added in 1min, persistently stirs 30s.Use suction pipe 10mL basal liquids are taken, are added slowly to along tube wall on fused cell, side edged gently shakes and (can not blown and beaten), are added respectively in 5min 1mL, 2mL, 3mL, 4mL, finally add basal liquid to 40mL, after adding, close the lid, and overturn several times repeatedly, mix cell. 800r/min 5min are centrifuged, and abandon supernatant.The HAT culture mediums containing feeder cells are drawn, it is with suction pipe that the fusion in centrifuge tube is thin Born of the same parents are gently mixed, and are added dropwise in the serum bottle containing feeder cells, stir and evenly mix dropwise near liquid level, and action is light by cell It is gently mixed, must not blows and beats.It is reverse to mix.Then cell is seeded on Tissue Culture Plate, drips, be placed in per hole two Cultivated in incubator.Single cell fusion can be inoculated with 4~6 piece of 96 orifice plate.It can also plant less as needed, the general cell number for pressing SP2/0 Calculate, per hole inoculum concentration containing about 10⁴Left and right SP2/0 cells.In 37 DEG C, 5%CO₂Cultivated in incubator.

It is calculated as 0d on the day of fusion, preceding 3d tries not kinetocyte plate, keeps incubator homeostasis.3d is mended per hole Plus 1 and drip HAT complete mediums；5d suctions out l/2 culture supernatants (100 μ L) per hole, adds 1 drop HT complete mediums；After Every 2d, ibid method sucks the culture supernatant of l/2~3/4, and HT complete mediums are changed to after 7d.

Cell colony length to be fused is to culture hole 1/10~1/5, while being screened with the indirect ELISA method set up. Compared with zero medicine hole, medicine hole OD values repressed can be judged to the positive.According to inhibiting rate and cell colony upgrowth situation, selection The cell hole of only 1-2 single colony of 2~6 strong positives, cloning is carried out using limited dilution method.

By 3~4 time clonings, the monoclonal for secreting anti-cefalexin, cefadroxil and Cefradine is finally filtered out The hybridoma cell strain of antibody, dyed body is counted, and the chromosome number of the hybridoma is 103.6.Applicant is ordered Entitled hybridoma 3A6, and the Chinese Typical Representative training sent in 2013 in the Wuhan University of Wuhan City, Hubei Province for 28th for 03 month Thing collection (CCTCC) preservation is supported, deposit number is CCTCC NO:C201340.

Ascites monoclonal antibody is prepared and identification：By cell line 3A6 through Balb/C mouse are injected intraperitoneally, monoclonal antibody is produced. The operation requirement of Thermo Scientific companies mouse monoclonal antibody Rapid ELISA homotype detection kit, to institute of the present invention Obtained monoclonal antibody carries out hypotype identification, is as a result mouse IgG₁Hypotype.

The foundation of the racing ELISA detecting method of embodiment 3

The preparation of 3.1 reagents (reagent that the present embodiment is used is prepared in addition to another indicate using following methods)

Phosphate buffer：NaCl 8.0g, KH₂PO₄0.2g, Na₂HPO₄·12H₂O 2.9g, KCl 0.2g, plus double steamings Water adjusts pH to 7.4 to 1000mL；

Coating buffer：Take Na₂CO₃1.59g, NaHCO₃2.93g, plus tri-distilled water is to 1000mL, adjusts pH value to 9.6；

Cleaning solution：NaCl 8.0g, KH₂PO₄0.2g, Na₂HPO₄·12H₂O 2.9g, KCl 0.2g, Tween 20 0.5mL, plus distilled water is to 1000mL, adjusts pH to 7.4；

Confining liquid：Ovalbumin 1g is dissolved in 100mL phosphate buffers；

Substrate solution A：3,3', 5', 5- tetramethyl biphenyl diamines (TMB) 200mg, absolute ethyl alcohol 100mL, plus distilled water is extremely 1000mL；

Substrate solution B：Na₂HPO₄14.6g, citric acid 9.3g, 0.75% carbamide peroxide 6.4mL, plus distilled water is extremely 1000mL；

Substrate cocktail：By A liquid and B liquid by volume 1:1 mixing is produced, now with the current；

Terminate liquid：2mol/L sulfuric acid solutions.

3.2 coating original contents and antibody working concentration are primarily determined that

Cefalexin-the OVA of above-mentioned synthesis is selected as coating antigen, be diluted to coating buffer 3.2mg/L, 1.6mg/L, 6 concentration such as 0.8mg/L, 0.4mg/L, 0.2mg/L, 0.1mg/L, in 96 hole elisa Plates, the from first to the 6th row are sequentially added, 4 DEG C overnight；Washing 3 times, is patted dry, and adds confining liquid 250 μ L, 37 DEG C of closing 120min；Washing 3 times, is patted dry, in the every of ELISA Plate Individual hole adds 50 μ L phosphate buffers (PBS), and then to sequentially add 50 μ L phosphate buffers dilute for the first row to the 6th row The extension rate released is 500,1000,2000,4000,16000,32000,64000 monoclonal antibody, and 37 DEG C are incubated 30min, Washing 3 times, is patted dry；Each hole adds 1:The sheep anti-mouse igg antibody (referred to as two of the HRP marks of 5000 times of phosphate buffer dilutions Anti-, signified secondary antibody is the sheep anti-mouse igg antibody of HRP marks below, purchased from Wuhan Fei Yi Bioisystech Co., Ltd) 100 μ L, 37 DEG C of incubation 30min, wash 3 times, pat dry；Each hole adds 100 μ L Substrate cocktails, and lucifuge colour developing 15min adds 50 μ L and terminated Liquid, determines OD value (OD values) at 450nm wavelength with automatic ELIASA, the results are shown in Table 1.

As a result show, the coating concentration for primarily determining that coating antigen cefalexin-OVA is 0.2mg/L, and antibody working concentration is 1:16000。

Table 1 is coated with primarily determining that for original content and antibody working concentration

The determination of 3.3 optimal coating original contents and antibody working concentration

Concentration is coated with the coating antigen cefalexin-OVA primarily determined that, with 0.2mg/L coated elisa plates.By cefalexin 7.2 μ g/L, 3.6 μ g/L, 1.8 μ g/L, 0.9 μ g/L, 0.45 μ g/L, 0 μ g/L, 6 concentration are diluted to phosphate buffer, are resisted Body is with 1:16000 be intermediate concentration, and a series of concentration gradients, its 0 hole and IC are designed by equal difference₅₀Value is shown in Table 2.Selection OD values exist 2.0 or so, IC₅₀The relatively low corresponding antibody dilution of value is optimal primary antibody dilution factor.As a result show, selection 1:24000 is most Good antibody dilution.

The optimum antibody dilution factor of table 2 optimizes

Antibody extension rate (1:X)	0 hole OD values	IC50(μg/L)
			12000	2.500	4.16
16000	2.323	2.87
			20000	2.125	2.09
24000	1.925	1.82
			28000	1.803	1.78

The foundation of 3.4 standard curves

By cefalexin phosphate buffered saline into 7.2 μ g/L, 3.6 μ g/L, 1.8 μ g/L, 0.9 μ g/L, 0.45 μ g/ L, 0 μ g/L6 series concentrations, each concentration repeat 5 holes, are determined according to indirect competitive ELISA method, replication 5 times.With head The logarithm value of cefalexin solution concentration is abscissa, and B/B0 is that ordinate draws standard curve, obtains IC₅₀.This kit IC₅₀It is worth for 1.81 ± 0.16 μ g/L.

3.5 cross reactions are tested

By the cephalosporins medicine such as cefalexin phosphate buffered saline into debita spissitudo, with the ELISA method of foundation Determine the IC of each medicine₅₀Value, each 3 multiple holes of medicine, using monoclonal antibody to the cross reacting rate of cefalexin as 100%, Cross reacting rate of the monoclonal antibody to each medicine is calculated using formula 1.As a result (table 3) shows, it is indirect that the present invention is set up ELISA and kit are respectively 100%, 118% and to the cross reacting rate of cefalexin, cefadroxil and Cefradine 103%, and with the equal no cross reaction of other drugs.

Cross reacting rate of the kit of the present invention of table 3 to various cephalosporins medicines

Medicine name	IC50(μg/L)	Cross reacting rate (%)	The range of linearity (μ g/L)
				Cefalexin	1.83	100	0.45~7.2
Cefadroxil	1.55	118	0.45~7.2
				Cefradine	1.78	103	0.45~7.2
Penicillins	>1000	<0.1	--
				Other cephalosporinses	>1000	<0.1	--

The assembling of the ELISA kit of embodiment 4

4.1 ELISA kits of the present invention are made up of following part：

1) it is coated with coating antigen cefalexin-OVA solid phase carrier (ELISA Plate)；

2) 6 bottles of cefalexin standard liquid, concentration is respectively 7.2 μ g/L, 3.6 μ g/L, 1.8 μ g/L, 0.9 μ g/L, 0.45 μ g/L、0μg/L；

3) preserving number is CCTCC NO:The monoclonal antibody of C201340 hybridoma 3A6 secretions；

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

5) concentrated phosphoric acid salt buffer：NaCl 80.0g, KH₂PO₄2.0g, Na₂HPO₄·12H₂O 29.0g, KCl 2.0g, plus distilled water is to 1000mL；

6) concentrated cleaning solution：NaCl 80.0g, KH₂PO₄2.0g, Na₂HPO₄·12H₂O 29.0g, KCl 2.0g, Tween205mL, plus distilled water is to 1000mL；

7) substrate solution A：3,3', 5', 5- tetramethyl biphenyl diamines (TMB) 200mg, absolute ethyl alcohol 100mL, plus distilled water is extremely 1000mL；

8) substrate solution B：Na₂HPO₄14.6g, citric acid 9.3g, 0.75% hydrogen peroxide urea 6.4mL, plus distilled water is extremely 1000mL, adjusts pH to 5.0~5.4；

9) terminate liquid：2mol/L sulfuric acid solutions.

The preparation of 4.2 ELISA Plates

Cefalexin-OVA is diluted to 0.2mg/L with coating buffer, 100 μ L are added per hole, 4 DEG C are overnight, coating buffer of inclining, 250 μ L cleaning solutions are added per hole to wash 3 times, are patted dry, and confining liquid 250 μ L, 37 DEG C of incubation 120min, hole of inclining then are added per hole Interior liquid, cleaning solution is washed 3 times, is patted dry, and is preserved with masking foil vacuum sealing.

The mensuration program of the enzyme linked immunological kit of embodiment 5

The preparation of 5.1 reagents

1) sample diluting liquid：Used after the concentrated phosphoric acid salt buffer provided in kit is diluted into 10 times with tri-distilled water.

2) cleaning solution：Used after the cleaning solution provided in kit is diluted into 10 times with tri-distilled water.

3) Substrate cocktail：According to each institute's expense, the substrate solution A and substrate solution B of preparation are pressed into volume 1:1 mixes, It is now with the current.

5.2 sample pre-treatments

1st, milk：Measure milk 1mL, room temperature 4000rpm centrifugations 10min；Intermediate layer is taken, (volume ratio is with PBS/ methanol 9:1) direct sample detection after 40 times is diluted.

2nd, pig, chicken, the pre-treatment of ox muscle samples：Tissue sample 2.0 ± 0.02g of homogeneous thing is weighed in 50mL centrifuge tubes In, PBS 10mL are added, acutely after vibration 5min, room temperature 4000rpm centrifugations 10min；Supernatant intermediate layer is taken, 8 times are diluted with PBS Direct sample detection afterwards

5.3 determination step

1) it is loaded：Cefalexin series concentration standard liquid or the μ L of sample solution 50 are added into ELISA Plate micropore, then The μ L of monoclonal antibody working solution 50 are added, are placed in wet box, 37 DEG C of constant-temperature incubation 30min；

2) wash：The liquid in hole is poured out, adding the μ L of cleaning solution 250 in every hole washs 3 times and pat dry；

3) the sheep anti-mouse igg antibody working solution of horseradish peroxidase (HRP) mark is added：Horseradish peroxidating is added in per hole The μ L of sheep anti-mouse igg antibody working solution 100 of thing enzyme (HRP) mark, are placed in wet box, 37 DEG C of constant-temperature incubation 30min；

4) wash：The liquid in hole is poured out, the μ L of cleaning solution 250 are added in every hole, 3 times is washed and simultaneously pats dry；

5) substrate is added：The μ L of Substrate cocktail 100 are added in per hole, are placed in wet box, 37 DEG C of constant-temperature incubation 15min；

6) terminate liquid is added：The μ L of terminate liquid 50 are added in per hole；

7) determine：The OD value (OD values) in every hole is determined at 450nm with ELIASA.

5.4 results judge

Standard curve：

With the standard items OD values divided by " zero " hole OD values (B/B0) that are determined for ordinate, the logarithm value of cefalexin concentration Make standard curve for abscissa, and carry out linear regression, provide regression equation.

Cefalexin concentration is calculated in milk or muscle：

The inhibiting rate (the OD values divided by " zero " hole OD values of the sample obtained) of sample is calculated, the recurrence of standard curve is substituted into In equation, and the coefficient of dilution 40 is multiplied by, calculates cefalexin concentration in milk or muscle (μ g/kg), be converted to according to formula 2 Cefadroxil or Cefradine concentration (μ g/kg).

The sensitivity of the kit of embodiment 6, precision, the degree of accuracy, replica test

The sensitivity test of 6.1 kits

With the IC of standard curve₅₀Value and tissue minimum detection limit (LOD) refer to as the sensitivity of detection kit of the present invention Mark.The dilution of cefalexin standard items is turned into 7.2 μ g/L, 3.6 μ g/L, 1.8 μ g/L, 0.9 μ g/L, 0.45 μ g/L, 0 μ g/L 6 Concentration, each 5 multiple holes of concentration, according to indirect competitive ELISA method replication 5 times, take the IC of 5 measure₅₀Average value. LOD is determined by following steps, the OD values of 20 portions of blank milk or muscle samples is determined, according to the regression equation meter of standard curve Corresponding cefalexin concentration is calculated, the average value of cefalexin concentration is then calculatedWith standard deviation (SD), according to public affairs FormulaCalculate the minimum detection limit in milk or tissue.The IC of the present invention₅₀It is worth for 1.81 ± 0.16 μ g/L, Lowest detection line of the cefalexin in milk and animal muscle refers to table 4.

The minimum detection limit of three kinds of medicines in the milk of table 4 and muscle

The precision test of 6.2 kits

Cefalexin standard items are diluted to 7.2 μ g/L, 3.6 μ g/L, 1.8 μ g/L, 0.9 μ g/L, 0.45 μ g/L, 0 μ g/L 6 concentration, per concentration 5 multiple holes, according to indirect competitive ELISA method replication 5 times, using the regression equation of standard curve The measured value of each concentration cefalexin standard liquid is calculated, the interior coefficient of variation between plate of computing board the results are shown in Table 5.

Error in the plate of the standard curve of table 5 and between plate

The degree of accuracy of 6.3 kits, replica test

Three kinds of cephalosporins medicines of three concentration are respectively added in milk sample, its TIANZHU XINGNAO Capsul 56.7%~ Between 110%, with interassay coefficient of variation≤10.8% in batch；It is added in animal muscle tissue, its TIANZHU XINGNAO Capsul exists Between 66.5%~93.8%, with interassay coefficient of variation≤12.5% in batch.Specific measurement result is shown in Table 6-9.

TIANZHU XINGNAO Capsul in the milk of table 6

TIANZHU XINGNAO Capsul in the chicken muscle of table 7

TIANZHU XINGNAO Capsul in the pig muscle of table 8

TIANZHU XINGNAO Capsul in 9 Ns of muscle of table

Claims (8)

1. the monoclonal antibody of a kind of anti-cefalexin, cefadroxil and Cefradine, it by preserving number is CCTCC NO that it, which is,: Secreted by C201340 hybridomas 3A6.

2. the hybridoma 3A6 described in claim 1, is deposited in China typical culture collection center, preserving number is CCTCC NO:C201340。

3. the monoclonal antibody described in claim 1 is preparing the enzyme-linked of detection cefalexin, cefadroxil and Cefradine Application in immune reagent kit.

4. include the kit of monoclonal antibody described in claim 1.

5. kit according to claim 4, the kit is to be used to detect that cefalexin, cefadroxil and cephalo are drawn Fixed enzyme linked immunological kit.

6. the kit described in claim 4 or 5 is in the detection of cefalexin, cefadroxil and Cefradine non-diagnostic purpose Application.

7. a kind of enzyme-linked immunoassay method for being used to detect cefalexin in food, cefadroxil and Cefradine medicament residue, Comprise the following steps：

(1) cefalexin and oralbumin coupling are obtained into coating antigen；

(2) it is CCTCC NO with preserving number:C201340 hybridoma 3A6 prepares monoclonal antibody；

(3) with the coating primordial covering solid phase carrier of step (1)；

(4) enzyme linked immunosorbent detection is carried out after testing sample is extracted.

8. according to claim 7 be used to detect cefalexin in food, cefadroxil and Cefradine medicament residue Enzyme-linked immunoassay method, it is characterised in that：The extracting method of the testing sample is：

Milk：Milk 1mL, room temperature 4000rpm centrifugation 10min are measured, intermediate layer is taken, is PBS with volume ratio: methanol= Direct sample detection after 9: 1 40 times of diluted；

Meat：Weigh meat sample homogeneous thing 2g, add PBS 10mL, acutely after vibration 5min, room temperature 4000rpm from Heart 10min；Supernatant intermediate layer is taken, sample detection after 8 times is diluted with PBS.