Preparation Of Monoclonal Antibodies Against Ponceau 4R And Development Of An ELISA Method

Ponceau 4R (P4R, Carmines) is the most widely used and the largest amount of azo pigment. It is widely used in China’s food production and processing industries, because of its low cost, bright color and strong coloring power. But some recent studies have shown that P4R has potential toxicity. Since 2008, P4R have been banned to add to the food in Europe, North America and other countries. Although the use of P4R has been strictly limited in China, the abuse phenomenon still exists.Based on its characteristics, P4R has been used by utilized by some food processors to add to drinks, snacks, seafood, meat and other products excessly, which poses a threat to food safety. The main method to detect P4R residue is traditional instrumental analysis. Such as gas chromatography, mass spectrometry, spectrophotometry and electrochemical methods, and the most common method is HPLC. The instrument detection method not only require expensive instrumentation. But the instrument detection method not only needs the valuable instrument, the tedious sample processing also needs the technical personnel to operate. All these unfavorable factors cause traditional detection methods not to be suitable for the field sample inspection. A simple, effective and rapid method is required for the field detection. Based on the specificity of antigen antibody binding, immune detection method has a high selectivity and sensitivity. Furthermore, the method is simple, efficient and easy to carry. Thus, it is suitable for the rapid screening of large quantities of field samples.In this study, two kinds of P4R antigens were synthesized by using the method of halogen acid and glutaraldehyde. After dialysis, identification and comparison, we chose the more stable and the best immunogen, and the coating antigen which was more accurate and eliminate the spacer interference. Immunize BALB/c mice with synthetic antigen. The hybridoma cells we need were screened by cell fusion technique and ELISA. A monoclonal antibody with high specificity and high affinity to detect P4R was made, on basis of which a suitable ELISA detection method was established.1. Synthesis and identification of P4R complete antigensP4R is a haptens, and its molecular structure is not directly linked with the protein to the carboxyl or amino group. Therefore, In this study the hydroxyl and halogenated carboxylic acid sodium hypochlorite reaction on the molecular spacer containing carboxyl group increased. Then, the NHS method was used to connect the antigens A with the carrier protein (BSA, OVA) to get complete antigens A. At the same time, the P4R molecule was designed with nitric acid to add a nitro group, and the amino group was reduced to amino group by using palladium carbon and hydrogen, then it is connected with the carrier protein to obtain the complete antigens B by glutaraldehyde. Those two kinds of complete antigens were identified by UV spectrum scanning and SDS-PAGE, which proved that the connection was successful, and we found that the effect of complete antigen A is better than antigen B. So we finally chose the complete antigen A for immune animal. The protein concentrations of P4R-BSA and P4R-OVA were measured by BCA method, respectively, for 4.65mg/mL and 3.17mg/mL.2. Preparation of monocolonal antibody against P4RUsing P4R-BSA as immunogen to immunized BALB/c mice, P4R-OVA as coating antigen to coat ELISA plate. Seven days after fifth immunization, thetiter of blood serum reach more than 1:16000. By using cell fusion technique, ELISA screening method and the method of limited dilution, three strains obtained sustainable secretion of the P4R antibody hybridoma cell lines were prepared by injecting cells of hybridoma cell lines were obtained. Ascites if monoclonal antibodies were prepared by injecting cells of hybridomas into mice abdomen. Named them as 6A3,6B6 and 7C6. The protein concentration of ascites were 29.8mg/mL, 25.1mg/mL and 24.7mg/mL, respectively. Titers of the monoclonal antibodies were 32000, 32000 and 64000, respectively. Using HiTrap Protein G HP to purify ascites 7C6. Identify the effect of purification by SDS-PAGE. The result showed that complex proteins were significantly reduced. The cross test showed that the monoclonal antibody has 100% inhibition rate for P4R, and it has no cross reaction with Rhodamine B, Pararosaniline, Neutral red, Orange II, Tartrazine, Sunset yellow and Amaranth.3. The establishment of ELISA detection methodAn indirect competitive ELISA method for detection of P4R residues was established using purified monoclonal antibody. The best dilution concentration is 1:800 and the best working concentration of purified antibody is 1:16000. The standard curve of R to antibody-antigen reaction was prepared with the linear equation y=0.2207x+0.0003 (R2=0.9931), the linear range was 5-5000ng/mL. ICso= 172.97ng/mL, LOD under 2ng/mL, LOQ under 5ng/mL.4. The addition and reclamation test of P4R in the meatThe addition and reclamation test of P4R was carried on in the meat. The sample was determined after pre-processing. The intervention disappeared when the extraction of muscle sample was diluted 16 times. The standard curve equation was y=0.2731x-0.1457 (R2= 0.9564), the linear range was 5-5000ng/mL, IC50=256.94ng/mL, LOD under 5ng/mL, LOQ under 15ng/mL. When adding 50,200, 1000ng/mL P4R to the meat, the recovery ratio was between 85.2 to 94.4%.