Preparation And Analysis Of Monoclonal Antibodies Against Chlorothalonil And Procymidone

Because organochlorine pesticides are so stable in chemical properties and uneasy to be degraded in organic body,that could be accumulated in adipose tissue and potentially harmful to human health.Therefore,it is urgent to establish a economical,suitable and rapid method which could be practical for the qualitative and quantitative analysis of organochlorine pesticides.In this paper,chlorothalonil(CTN) and procymidone(PRM) of organochlorine pesticides were taken count into study because they were widely used.CTN and PRM were modificated and activated by glycol and then linked to carrier protein through N,N-carbonyldiimidazole(CDI).Then complete antigen of CTN and PRM were adopted to immunize mice cooperatively and respectively in succession to obtain antiserum.Afterwards,anti-CTN and PRM monoclonal antibodies were obtained by technology of hybridoma cell and the characteristics of cloned cells and monoclonal antibodies were analyzed.Detailed presentations are as followed.1.Preparation for artificial antigen of CTN and PRMDue to their low molecular weight of CTN(265.9) and PRM(284.1),they are both haptens of only reactinogenicity but of no immunogenicity.They could stimulate animal to produce antibodies only after they were changed into complete antigen by conjugated with carrier protein.According to the chemical properties of chlorine atoms on benzene ring of both CTN and PRM,chlorine atoms were firstly replaced by hydroxide radical of glycol and active intermediates of chlorothalonil hydroxy-substituted(CTNH) and procymidone hydroxy-substituted(PRMH) were made.Then intermediates were linked to bovine serum albumin(BSA) and ovalbumin (OVA) respectively through CDI as complete immunogens of CTNH-BSA, PRMH-BSA and coating antigens of CTNH-OVA,PRMH-OVA in ELISA.There were proved that CTNH and PRMH were successfully combined to BSA and OVA through the identification of ultraviolet spectrum and infrared spectrum.The conjugation mole ratios were respectively CTNH:BSA=11:1,CTNH:OVA=8:1, PRMH:BSA=9:1 and PRMH:OVA=8:1.2.Production of anti-CTN and PRM antibody and foundation of ELISAThe conjugate of CTN-BSA and PRM-BSA was injected into BALB/c small mice through multi-site and multi-pathway protocol cooperatively(with equal quantity of CTN and PRM mixture of 100μg/mouse and 200μg/mouse,as mixture group) and respectively(100μg/mouse,as control group).BALB/c small mice's bodies began to obviously produced antibodies after 25 days when they were firstly injected into antigens.The highest ELISA titer of the antiserum reached to 1:12800 reached to after 55 days.Working parameters of competitive ELISA technique for CTN and PRM detection was optimized,including selection of coating method,optimum dilution ratio of antiserum which were described as below:coating concentration of antiserum of mouse No.6 immunized by CTNH-OVA and PRMH-OVA(mixture group) were both 8μg/mL,which were deluted to 1:12800 by PBS(pH 7.4);coating concentration of antiserum of mouse No.7 immunized by CTNH-OVA and No.11 by PRMH-OVA (control group) were separately 4μg/mL(1:12800) and 2μg/mL(1:6400).Calibration graphs was formed by the logarithm of the CTN and PRM concentration against the inhibitory rate,the equation of the mixture group is as follow:y=-26.08x+98.933,(R²=0.973);y=-24.0x+100.4,(R²=0.982),respectively. The corresponding to sensitivity(IC₅₀) reached 79 ng/mL and 125ng/mL correspondently and the linearity range for detection were 10ng/mL 1000ng/mL.While the equation of the control group are:y=-23.58x+91.933,(R²= 0.979) and IC₅₀ to CTN is 59ng/mL for mouse No.7;y=-31.08x+119.27,(R²=0.976) and IC₅₀ to PRM is 93ng/mL for mouse No.11,with the same linearity range for detection as mouse No.6.3.Preparation of anti-CTN and PRM monocional antibodies and establishment of ELISAThe splenic cells from BALB/c mice of mixture group(after the fourth-time immunization) were fused with the murine Sp2/0 myeloma cells with a fusion ratio up to 50.3%.Each one strain of fusion cell(named as 1H4 and 5C2,respectively) were respectively selected from positive fusion cells which have the stable ability of generating anti-CTN and PRM monoclonal antibodies through limiting dilution method.Working parameters of indirectly competitive ELISA technique for CTN and PRM detection were optimized through square titration which were described as below:coating concentration of CTNH-OVA and PRMH-OVA were separately 2μg/mL and 4μg/mL,delution rate of monoclonal antibodies produced by strains of 1H4 and 5C2 were 1:64 and 1:32.Competitive inhibition curve for CTN and PRM are separately as follow:y=-25.08x+93.267,(R²=0.9782) and y=-23.64x+93.813, (R²=0.9603) The corresponding to sensitivity(IC₅₀) reached 54 ng/mL and 71ng/mL accordingly,with the same linearity range of 10ng/mL～1000ng/mL.