Preparation Of Monoclonal Antibody Against Ofloxacin And Establishing Of ELISA

With the improving of the living level, the demand of the animal derived food is increasing, so the problem of the veterinary drug residues in animal derived food is concerned by public. On one hand, these residues can do harm to person's health directly or indirectly. On the other hand, in some trade countries such as Japanese, the European developed nations, the technique trade barrier of the food animal edible tissues imported from other countries is being increased, wherefore, as a member of the WTO, the loss that animal derived food of China can't be export because of the exorbitant standard of veterinary drug residues is very serious.The Ofloxacin(OFLX) belongs to the antibacterial agent that belongs to the fluoroquinolone (FQs) group. It is a kind of antibacterial agent with efficiently broad-spectrum and is very being widespread application in the clinic. But in recent years, the use of OFLX is being increased, and the reports about the adverse effect and resistance of OFLX are becoming more and more, so its latent serious adverse effect is concerned by public gradually. Furthermore, as the veterinary drugs and additives of animal feed, the OFLX is widely used for animals, as a result, the possibility of OFLX residues in animal edible tissue is becoming higher. Not only the side effect can endanger the human's health directly, but also a lower density of residues of OFLX may make the sensitive germ to OFLX bear the OFLX and this endanger the human's health indirectly.Currently, the high performance liquid chromatography is the mainly detection method of the OFLX, but it is only used for the corroborative analysis generally because of its complicated sample handles and expensive instruments. Therefore, to establish a kind of efficient method to detect the OFLX residues, not only may be applied to market monitor the OFLX residues in the food animal edible tissues, but also be used for elementary sieve of the large-scale samples for import and export detection, consequently, it will create a good society and economic benefits.According to the basic principle of immunology, the complete antigen of the OFLX were prepared and the monoclonal antibody (McAb) of the anti- OFLX (OFLX-McAb) were obtained, An indirect competitive inhibition enzyme-linked immunosorbent assay (ic-ELISA) was established to detect the OFLX residues in food animal edible tissues, and it will lay foundations for further studying of the fast test kits for detecting OFLX in animal derived food.The OFLX was conjugated directly to bovine serum albumin (BSA) and ovalbumin (OVA) by EDC method. The conjugates OFLX-BSA and OFLX-OVA were analyzed by nondenaturing gel electrophoresis and UV absorbance method, the molecule conjugate ratio of OFLX to BSA and OVA were 2:1 and 3:1 respectively. Splenocytes from mice immunized with BSA-OFLX were fused with SP2/0 myeloma cells. Three hybridomas of stable secreting OFLX-McAb were selected and were named 1E8,2A3,2F10,2H3,3D8,3G7and 4F4, after the OVA-OFLX as coating antigen was used to recognize the antibodies produced against OFLX. The OFLX-McAb of 4F4 was used to establish the ELISA method after the character of the three hybridomas being analyzed. The number of chromosome of the hybridoma cell line was from 88 to 95. The subclasses of McAb was IgG1; the molecular was 152000Da; the ELISA titers of ascites was 1:10000; the effective concentration of McAb was 402.88μg/mL; the affinity constant was 4.854×108M-1; the cross-reactivities with Fleroxacin , Enrofloxacin and Pefloxacin were 25% ,0.78% and 0.78%, respectively; the non-fluoroquinolones were tested and there was no cross-reaction between them.According to competitive principle, an ic-ELISA method was established for the quantitative detection of OFLX and its optimized reaction condition as follow:The OVA- OFLX was diluted to microtiter plates at a concentration of 0.5μg/mL and incubated 2h at 37℃. Plates were washed three times with PBST for 30 second per times, and 100μL 1% gelatin was added to each well to eliminate nonspecific binding by blocking the plastic surface where protein was not bound. After 1h of incubation at 37℃, Plates were washed three times with PBST, and 1:5000 OFLX-McAb and varying concentrations of standard OFLX (50μL/well) were added. After 1h of incubation at 37℃, Plates were washed three times with PBST, and 100μL/well goat anti-mouse IgG-HRP (1:10000) was added and incubated for 1h at 37℃. Plates were washed four times, and 100μL/well TMB substrata solution was added, followed by the addition of stopping solution(2M H2SO4) after 30 minutes of incubation in the dark at 37℃. Absorbance at 450nm was determined by an enzyme immunoassay reader. Percent inhibiting(b/b0×100%) was calculated from the absorbance obtained in the presence(b) and absence(b0) of OFLX in standard. A linear dose-response standard curve was prepared by plotting log [OFLX] versus percent inhibiting. The regression equation of standard curve was y= -3.1968x + 3.7028,The correlation coefficient R2=0.9958 ,the lower limit detection was 1.43ng/mL and a linear range was 1.43~4000ng/mL at a 50% inhibition of 31.25ng/mL. The average recovery rate of standard was 97.44%.With Shim-pack C18 column and RF-10AXL fluoroscope, A high performance liquid chromatography method has been developed, on the foundation of ameliorating the condition on literature[48]. The regression equation of standard curve was y=0.0003x-3.802,the y denote the concentration OFLX(ng/mL),and the x denote the peak area value. The correlation coefficient R2=0.9999,the lower limit detection was 0.048pg and a linear range was 0.048ng/mL~500ng/mL. The average recovery rate of standard was 93.7%. Collect the chicken organizational samples (muscle, liver) that were sold on the market, each sample was divided into three groups (2g/group), adding the standard OFLX 1, 0.5 and 0.25μg to each group respectively. The sample was drown from twice with the organizational homogenizer and the phosphate buffered solution(pH7.0), its final volume was to 25mL (stock solution). The concentration of standard OFLX were 10,20,40ng/mL in the stock solution. The stock solution was used to ic-ELISA directly, and the HPLC was carried out after the stock solution been purified with SPE column and filtered through the filter membrane (0.45μm). The results of two methods were as follow: The spiking concentration of standard OFLX was from 0.5μg/g to 0.125μg/g. The average recoveries of ic-ELISA and HPLC method were 87.0% and 107.85%, in the muscle sample, respectively.The spiking concentration of standard OFLX was from 0.5μg/g to 0.125μg/g.. The average recoveries of ic-ELISA and HPLC method were 107.87% and 93.44%, in the liver sample, respectively.Using the ic-ELISA method had been established, the recoveries in the sample of chicken organizes was 85% above, and matched the common regulate of ELISA method. The result of ic-ELISA method is corroborated by HPLC method.It was the first inland report that the OFLX-McAb was applied to the ELISA method for monitoring the OFLX residues in the food animal edible tissues. The success of this experiment will lay foundations for further studying of the fast test kits for detecting OFLX and the ELISA method for detecting the veterinary drugs residues in animal derived food.