Preparation And Characterization Of Monoclonal Antibodies Against Citrinin(CTN) For Immunoassay

Mycotoxins are the toxic chemical compounds of secondary metabolites from fungi causing hazardous consequences on any living organisms.These harmful chemical compounds produced by different fungi molds contaminate human food and animal feeds.Citrinin(CTN)is a hepato-nephrotoxic mycotoxin produced by fungi genera of Aspergillus,Monauscus and Penicillium.CTN contaminates grains,fruits,juices and vegetables,and causes various toxic effects on humans and animals.In many toxicity studies,the kidney was identified as the principal target organ for citrinin,and significant differences of the susceptibility of different species to citrinin have been observed.CTN is commonly found with other mycotoxin particularly with ochratoxin A(OTA),and the combination toxic effect of CTN with other mycotoxin is heavy on human and animal health.In vitro and in vivo studies provided clear evidence of reproductive toxicity and embryo toxicity of citrinin to male and female.The commonly used analytical methods for CTN detection are thin layer chromatography(TLC),high-performance liquid chromatographic technique(HPLC),liquid chromatography tandem mass spectrometry(LC-MS/MS),ultra-high-performance liquid chromatography and fluorescence detection(UHPLC-FL),gas-chromatography-selected ion monitoring(SIM)mass spectrometry(GC-MS)and an enzyme-linked immunosorbent assay.However,these methods have some disadvantages,such as lacking automation,low sensitivity,low specificity,complex equipment's,incompatible with the real sample,energy and time consuming.For the above reasons,the present study produced and characterized a monoclonal antibody against CTN for immunoassays such as colloidal gold immunochromatograhic test strip and indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)methods,which is rapid,inexpensive,sensitive,and specific with low limit of detection and low half inhibition concentration.Citrinin is a small non-immunogenic toxin with a molecular weight of 250.25.Thus,it is necessary to conjugate it with carrier proteins for immune-response to generate antibodies.Different methods were applied to conjugate CTN with carrier proteins.In this study,CTN was conjugated to bovine serum albumin(BSA)and ovalbumin(OVA),respectively,by amide bonds through active ester method using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC)and N-hydroxysuccinimide(NHS).CTN-protein conjugates were synthesized and the success of CTN-protein conjugation was analyzed with non-denaturing agarose gel electrophoresis,SDS-PAGE and UV-spectroscopy methods.In this study,Female Balb/c mice were immunized with CTN-BSA conjugate.As the result of indirect non-competitive ELISA assay,the CTN-BSA immunized mice showed a high anti-serum titer(1:32000,v:v)when compared to the non-immunized control mouse.After the anti-serum titer reached a high level,the mice were chosen for cell fusion and spleen cells from the immunized mice were fused with Sp2/0 myeloma cells to obtain hybridoma cells.Positive clones were transferred from 96 to 48-well plates for growth and were subsequently sub-cloned several times until a single positive clone was obtained.Finally,a positive hybridoma cell of interest was screened out and named 21H27.The isotyping result showed that the 21H27 anti-CTN mcAb was the IgG2a subclass.The chromosome number result revealed that positive clone 21H27 was 102±4,indicating that the hybridoma cell was produced from the fusion of spleen cell and Sp2/0 myeloma cell.Pristane/incomplete adjuvant primed mice were prepared for ascites production.Ascitic fluid of hybridoma cells were produced in mice abdomen,and purified using caprylic/ammonium sulfate precipitation method.The average affinity of anti-CTN mcAb was 2.03×108 L/mol.Cross-reactivity test of anti-CTN mcAb was carried out with structurally related mycotoxins and molecules such as Zearalenone(ZEN),Trichothecene(T-2),Patulin(PT),Ochratoxin A(OTA),bovine serum albumin(BSA),ovalbumin(OVA)and Sterigmatocystin(STG).The results indicated that the anti-CTN mcAb was specific to CTN,with no cross reactivity to other molecules and toxins.ELISA assay result can be influenced by different factors.Therefore,selection of optimum parameters would prefer to maintain the stable results for ELISA assay.In the present study,the optimum coating antigen was 5?g/mL,and PBSM was selected for dilution which had the ability to block nonspecific bindings during antigen-antibody reaction.Furthermore,the best HRP antibody conjugate dilution was determined as 1:8000 in PBSM.Artificially contaminated samples with CTN were tested using competitive indirect ELISA(ic-ELISA),and the results showed that linear range of detection was 0.01-5.96 ng/mL and the IC50 was 0.28 ng/mL with a lower detection limit(LOD)of 0.01 ng/mL.The average recovery was(93.82±1.62)%with a coefficient variation of(1.03-4.31)%.Hence,anti-CTN mcAb secreted by 21H27 hybridoma cell was successfully produced,and this anti-CTN mcAb with low LOD,low IC50,high affinity and specificity would provide base information to assess the risk of contamination of citrinin mycotoxin in food and feed stuffs,and give insight for further research studies.Immunochromatographic assay based on colloidal god has shown significant advantage for detection of small molecules.Preparation of colloidal gold solution depends on different factors particularly the amount of sodium citrate.Concentration of coating antigen and the pH are the most significant factors to prepare the best color of colloidal gold solution.In this study,2 mL of sodium citrate and pH 8.2 adjusted with potassium chromate were applied for preparation of good color gold solution,and this pH was appropriate for the gold conjugate-anti-CTN mcAb.In addition to this,freshly prepared(within one-week shelf life)anti-CTN mcAb gold conjugation and optimum concentration of coating antigen were the appropriate parameters for the best achievement of the immunochromatographic strip assay.