Preparation Of Monoclonal Antibodies For The Residual Markers Of Trimethoprim Analogues And Establishment Of Enzyme-Linked Immunosorbent Assay

Trimethoprim analogues called antibacterial synergist can enhance the antibacterial effect of other antibiotics.Although the use of these drugs is more extensive,there are still many adverse reactions,such as trimethoprim（TMP）could affect the hematopoietic system in mammals,Diaveridine（DVD）has genetic toxicity on mammalian cells,and Baquiloprim（BQP）has liver toxicity.Besides,there are serious drug resistance problem due to the illegal use for trimethoprim analogues.As a result,many countries and organizations have established the residual markers and maximum residual limits（MRLs）for these drugs.The indirect competitive enzyme-linked immunosorbent assays（ic-ELISA）was the most common immunoassay for monitoring trimethoprim analogues residues because of their low-cost,simplicity,rapidity.The paper described the hapten design,the synthesis of artificial antigen,and preparation of antibody.Finally,we obtained a generic monoclonal antibody against six trimethoprim analogues,and developed the ic-ELISA method for monitoring these drugs in milk,honey,pig muscle,pig liver,chicken muscle,and chicken liver.The details include the following contents.1.The hapten DVD-HS was synthesized through the condensation reaction and identified successfully by mass spectrometry.The hapten TMPCOOH was also synthesized from TMP and identified successfully by mass spectrometry.Two haptens were coupled with bovine serum albumin（BSA）and ovalbumin（OVA）by DCC method respectively,then were identified successfully by UV spectrum.The hapten DVD was linked to BSA and OVA via glutaraldehyde method and identified successfully by UV spectrum.2.The artificial antigen mentioned above,such as DVD-HS-DCC-BSA,DVD-GA-BSA,TMPCOOH-DCC-BSA,was used for immunogens in female Balb/c mice.The homologous ELISA was used for monitoring the antisea titers and specificity of mice.These results demonstrated that the antisera from DVD-HS-DCC-BSA had no titers and specificity,and the antisera from DVD-GA-BSA had fairly high titers and specificity for only DVD but no any recognition capacity on other drugs,the antisera from TMPCOOH-DCC-BSA could recognize six trimethoprim analogues.3.The spleen cells from immunized mice with high titer and specificity were selected for the cell fusions.The positive hybridoma cells were screening by ic-ELISA and were subcloned.Finally,we achieved a hybridoms cell line named TMP/2G1.The average chromosome number of hybridoms cell TMP/2G1 was 103.4.We prepared monoclonal antibody via ascites tumor,and the m CAb was confirmed as IG1 subclass.The checkerboard assay was carried out by homologous and heterogenous coating,and the results showed that the homologous coating was optimum.The ic-ELISA was established by optimizing conditions.The results showed that the optimizing concentration of coating antigen TMPCOOH-DCC-BSA was 100mg/L,and the best dilution of monoclonal antibody TMP/2G1 was 1:16000.The standard curve,where the regression equation was y=（A-D）/[1+（1000x/C）^{^B}]+D,A=0.9244,B=6.5644,C=2.34793,D=0.09546,r²=1.00000,was developed for TMP and the IC₅₀value was 0.232±0.007mg/L（n=5）.Repeating the standard curve,the inter-and intra-cofficient of variation was below 15%.The m CAb had good specificity for 6 trimethoprim analogues and the cross-reactivity was trimethoprim（TMP）（100%）,diaveridine（DVD）（44%）,aditoprim（ADP）（15.7%）,ormetoprim（OMP）（24%）,baquiloprim（BQP）（5.3%）,brodimoprim（BDP）（195%）respectively.4.Six trimethoprim analogues at three different concentratons（1×LOQ,2×LOQ,4×LOQ）was spiked into six samples containing milk,honey,pig muscle,pig liver,chicken muscle,chicken liver.The acetonitrile was used for the extraction methods,the recoveries was 81.4%～107.7%.The inter-assay and intra-assay coefficient of variation were both below 20%which indicated that the ic-ELISA had good accuracy and reproducibility.Compared with HPLC-MS/MS,the ELISA results showed a good correlation,which proved that the developed ELISA was reliable.5.The stability assay was carried out for TMP standard solution,antibody,and antigen at 4℃,37℃and 37℃respectively.The results demonstrated that the substances can be preserved for one year.In this study,we prepared a broad spectrum antibody for six trimethoprim analogues by a desired hapten design which was better than similar products at home and abroad.Based on this m CAb,we established the ic-ELISA method for six trimethoprim analogues in six different samples,and it was significant for food security.