Preparation Of Monoclonal Antibody Against Ciprofloxacin And Its Application In The ELISA

With the improving of the living level, the demand of the animalderived food is increasing, so the problem of the veterinary drug residues inanimal derived food is concerned by public. On one hand, these residuescan do harm to person's health directly or indirectly. On the other hand, insome trade countries such as Japanese, the European developed nations, thetechnique trade barrier of the food animal edible tissues imported fromother countries is being increased, wherefore, as a member of the WTO, theloss that animal derived food of China can't be export because of theexorbitant standard of veterinary drug residues is very serious.The ciprofloxacin (CPFX) belongs to the antibacterial agent thatbelongs to the fluoroquinolone (FQs) group. It is a kind of antibacterialagent with efficiently broad-spectrum and is very being widespreadapplication in the clinic. But in recent years, the use of CPFX is beingincreased, and the reports about the adverse effect and resistance of CPFXare becoming more and more, so its latent serious adverse effect isconcerned by public gradually. Furthermore, as the veterinary drugs andadditives of animal feed, the CPFX is widely used for animals, as a result,the possibility of CPFX residues in animal edible tissue is becoming higher.Not only the side effect can endanger the human's health directly, but also alower density of residues of CPFX may make the sensitive germ to CPFXbear the CPFX and this endanger the human's health indirectly.Currently, the high performance liquid chromatography is the mainlydetection method of the CPFX, but it is only used for the corroborativeanalysis generally because of its complicated sample handles andexpensive instruments. Therefore, to establish a kind of efficient methodto detect the CPFX residues, not only may be applied to market monitorthe CPFX residues in the food animal edible tissues, but also be used forelementary sieve of the large-scale samples for import and exportdetection, consequently, it will create a good society and economicbenefits. According to the basic principle of immunology, the complete antigenof the CPFX were prepared and the monoclonal antibody (McAb) of theanti-CPFX (CPFX-McAb) were obtained, An indirect competitiveinhibition enzyme-linked immunosorbent assay (ic-ELISA) was establishedto detect the CPFX residues in food animal edible tissues, and it will layfoundations for further studying of the fast test kits for detecting CPFX inanimal derived food. The CPFX was conjugated directly to bovine serum albumin (BSA)and ovalbumin (OVA) by EDC method. The conjugates CPFX-BSA andCPFX-OVA were analyzed by nondenaturing gel electrophoresis and UVabsorbance method, the molecule conjugate ratio of CPFX to BSA andOVA were 13:1 and 6:1 respectively. Splenocytes from mice immunizedwith BSA-CPFX were fused with SP2/0 myeloma cells. Three hybridomasof stable secreting CPFX-McAb were selected and were named 1D10, 3C6and 3G7, after the OVA-CPFX as coating antigen was used to recognize theantibodies produced against CPFX. The CPFX-McAb of 3C6 was used toestablish the ELISA method after the character of the three hybridomasbeing analyzed. The number of chromosome of the hybridoma cell line wasfrom 88 to 95. The subclasses of McAb was IgG1; the molecular was155.57KD; the ELISA titers of ascites was 1: 100×214; the effectiveconcentration of McAb was 3.64mg/ml; the affinity constant was 1.01×108M-1; the cross-reactivities with enrofloxacin and norfloxacin were125.85% and 19.50%, respectively; the non-fluoroquinolones were testedand there was no cross-reaction between them. According to competitive principle, an ic-ELISA method wasestablished for the quantitative detection of CPFX and its optimizedreaction condition as follow: The OVA-CPFX was diluted to microtiter plates at a concentrationof 1μg/ml and incubated overnight at 4℃. Plates were washed three timeswith PBST for 3 minutes per times, and 150μl 1% gelatin was added toeach well to eliminate nonspecific binding by blocking the plastic surfacewhere protein was not bound. After 1h of incubation at 37℃, Plates werewashed three times with PBST, and 1:18000 CPFX-McAb and varyingconcentrations of standard CPFX (50μl/well) were added. After 1h ofincubation at 37℃, Plates were washed three times with PBST, and100μl/well goat anti-mouse IgG-HRP (1:4000) was added and incubatedfor 1h at 37℃. Plates were washed four times, and 100μl/well OPDsubstrata solution was added, followed by the addition of stoppingsolution(2M H2SO4) after 20 minutes of incubation in the dark at 37℃.Absorbance at 492nm was determined by an enzyme immunoassay reader.Percent inhibiting(b/b0×100%) was calculated from the absorbanceobtained in the presence(b) and absence(b0) of CPFX in standard. Alinear dose-response standard curve was prepared by plotting log [CPFX]versus percent inhibiting. The regression equation of standard curve wasy= -37.24x+112.11,The correlation coefficient R2=0.9979,the lowerlimit detection was 4.10ng/ml and a linear range was 4.10～400ng/ml at a50% inhibition of 46.54ng/ml. The average recovery rate of standard was100.21% and the coefficients of variation was 6.91%. With Shim-pack C18 column and RF-10AXL fluoroscope, A highperformance liquid chromatography method has been developed, on thefoundation of ameliorating the condition on literature[36]. The regressionequation of standard curve was y = 6×10-8x,the y denote the concentrationCPFX(μg/ml),and the x denote the peak area value. The correlationcoefficient R2=0.9998,the lower limit detection was 0.00006μg/ml and alinear range was 0.00006～0.5μg/ml. The average recovery rate of standardwas 102.04% and the coefficients of variation was 3.35%. Collect the chicken organizational samples (muscle, kidney) thatwere sold on the market, each sample was divided into three groups(2g/group), adding the standard CPFX 0.625, 0.3125 and 0.15625μg toeach group respectively. The sample was drown from twice with theorganizational homogenizer and the phosphate buffered solution(pH7.0),its final volume was to 25ml (stock solution). The concentration ofstandard CPFX were 50,25,12.5ng/ml in the stock solution. The stocksolution was used to ic-ELISA directly, and the HPLC was carried outafter the stock solution been purified with SPE column and filteredthrough the filter membrane (0.45μm). The results of two methods wereas follow: The spiking concentration of standard CPFX was from 0.15625μg/gto 0.625μg/g. The average recoveries of ic-ELISA and HPLC methodwere 73.71% and 80.21%, and the coefficients of variation were 8.75%and 1.27% in the muscle sample, respectively. The spiking concentration of standard CPFX was from 0.15625μg/g...