Construction, Selection Of Phage-Displayed Single-Chain Antibody Library Against Methamidophos And The Expression, Characterization Of The Single-Chain Antibody

Methamidophos (O,S-dimethyl phosphoramidothioate), an acutely toxic organophosphate, is still being used in violation of a ban because of its effective for the control of insect. Therefore, it is necessary to detect methamidophos residues for its threat. Immunoassay is being demonstrated as a sensitive, simple, and rapid alternative to traditional methods for pesticide analysis. There have been reported about polyclonal and monoclonal antibodies against methamidophos, but production of PAbs and Mabs is time-consuming and requires expensive instruments for culture cells, immunized-animals, and expensive costs. Recombinant antibodies, the third generation antibodies, can be produced cheaply and quickly through recombinant approaches. Here, we describe the construction, selection of phage-displayed single-chain antibody library against methamidophos and the expression, characterization of the single-chain antibodies.1 Immunization of Balb/c mice and analysis of characterization of antiserumThe hapten of HM3 (3-(methoxy(methylthio)phosphorylamino)propanoic acid), was conjugated with proteins (BSA and OVA) using the active ester method. Balb/c mice were immunized in three different dose of an immunogen HM3-BSA. Identification of antiserum shows that H3 Balb/c mouse whose serum possessed high titers and the ability of recognition free methamidophos were obtained in the high dose group after 8 times immunization in 6 months.2 Cloning and construction of phage-display scFv library against methamidophosmRNA was extracted from the splenocyte of H3 Balb/c mouse and used to amplified VH and VL genes by RT-PCR. The predominant PCR products were of the expected sizes for VH(340bp) and VL(320bp) fragments. The approximately mol VH and VL genes which have been purified were successfully fused with a linker DNA (Gly4Ser)3 by splicing overlap extension PCR (SOE-PCR). The ScFv genes about 750bp generated by PCR using the primers that contain the slice sites of restriction endonuclease. After digested with Sfi I and Not I, the ScFv genes were ligated into the phagemid vector pCANTAB5E and subsequently transformed into the competent E.coli TG1 cells. The phage-display library consisted of 9.8×105 clones. The scFv genes of randomly picked clones were checked by PCR and digestion with HindⅢand EcoRⅠ, and it was found that all contained inserts of the expected size (750 bp). The phage-display library exhibited high diversity as judged by the BstNⅠrestriction pattern.3 Selection and identification of specific phage-scFv antibodies against methamidophos from phage-display scFv libraryThe phage-displayed scFv antibody library against methamidophos containing 1.75×1013pfu/mL was constructed by infection with the helper phage M13K07. Then the library was biopanned 5 rounds through the stringency of selection by using the decreased concentration of coating conjugate HM3-OVA and the increased washing times. The phage recovery, the OD of polyclonal phage-ELISA, and the positive rate of clones were gradually increased from rounds 3 to 5. A great number of clones randomly picked from the 5th were further studied by monoclonal phage-ELISA and CI-ELISA, and 6 positive clones with the highest specificity for methamidophos were obtained. Sequence analysis of the 6 clones indicated that all VH and VL genes were homologous with the variable region of mouse antibody, and all sequences had different amino acid substitutions.4 Expression and characterization of soluble scFv antibodies expressed in E. coliRecombinant phages of the above 6 positive clones were used to infect E. coli HB2151 for expression of soluble scFv antibodies. The infected E. coli HB2151 were induced overnight by IPTG to express soluble scFv antibodies in small amounts. Both supernatants and pellets were collected. Periplasmic scFv was extracted by osmotic shock and culture supernatants were concentrated by Tangential flow filtration. Approximately 30KD scFv protein, which was consistent with expected molecular weight, could be detected both in the periplasmic extract sample and in the concentrated supernatants compared to negative control in SDS-PAGE analysis. Indirect ELISA also showed that soluble scFv expressed both in supernatant and in periplasmic had a good binding activity with the coating antigen. Data of CI-ELISA showed that soluble scFv of periplasmic extract of the 6 clones could compete with free methamidophos in various degrees, of which the Met-93 clone and the Met-28D4 clone possessed the highest I50 values (887.66μg/mL) and the lowest I50 values (146.74μg/mL) respectively.Under the optimum conditions, expression scale-up with the Met-28D4 clone was accomplished by increasing the culture volume (1L). The products were purified and the scFv yield was 0.98 mg/L. Based on the purified scFv, CI-ELISA were constructed for methamidophos. The linear range, the I50 values, and the limit of quantification (I20) of CI-ELISA were 1～500μg/mL,118.69μg/mL, and 3.36μg/mL, respectively. In the linear range of CI-ELISA, the intra-assay CV is 2.3%, the inter-assay CV is 4.8%, the recovery rate from rice and cabbage are 83.8% and 87.0% respectively. The Cross-reactivity with other organophosphate pesticides (dichlorvos, dimethoate, phorate, parathionmethyl, isocarbophos) was below 0.1% except that acephate had a low cross-reactivity of about 4.9%, which showed that the soluble scFv antibodies were highly specific to methamidophos. The stability test of scFv antibodies showed that the binding activity of purified scFv retained only 6 weeks when stored at 4℃in the presence of protein stabilizers. The short-term stability of scFv antibodies may meet the demands of study on the characterization of scFv antibodies, but they are often limited in the practical applications.5 Construction, production, and characterization of recombinant scFv antibodies against methamidophos expressed in Pichia pastorisPichia pastoris (x-33) was used to express soluble scFv antibodies of the Met-28D4-ScFv. The primers used for the PCR were synthesized according to antibody gene-specific primers and the cloning sites of the Pichia expression vector pPICZa C. The specific scFv gene was amplified from the Met-28D4-pCANTAB5E-ScFv and then subcloned into the expression vector pPICZa C. The resulting plasmid, pPICZa C-scFv, was linearized and transformed into Pichia pastoris (X-33). Transformants picked randomly were checked by PCR, and it was found that all had integrated target gene. Five clones, which were resistant to the high concentration of 2000μg/mL Zeocin and determined as Mut+ phenotype were obtained in the selection of transformants containing multiple copies of target genes. Then, the 5 transformants were cultured and induced with methanol. Approximately 30KD scFv protein, which was consistent with expected molecular weight, could be detected in the culture supernatants at 48 h in SDS-PAGE analysis. The amount of scFv protein produced by these 5 transformants was similar, and the expression of scFv was highest at 72 h after induction. The bioactivity of Pichia produced scFv was confirmed by indirect ELISA, which also suggested that the bioactivity of scFv in the culture supernatant reached the peak in 72h.The clone named X-33-Pp-Met-28D4-1, which showed steady and strong expression was selected for further optimization for high-level protein production. In our lab culture conditions (30℃,250 rpm), the optimum process parameters for the expression of scFv produced with X-33-Pp-Met-28D4-1 are (a) an induction time of 72 h, (b) a pH of 6.5, (c) an inoculum density of OD6oonm=1.5, and (d) a methanol concentration of 1.0%. The scFv produced with X-33-Pp-Met-28D4-1 under the optimal conditions was purified and the yield was 25 mg/L, which is higher than that from unoptimized conditions based on recommendations from Invitrogen (20 mg/L). The bioactivity of X-33-Pp-Met-28D4-derived scFv antibodies was confirmed by CI-ELISA. The I50 values for methamidophos in assays was 93.52μg/mL, which indicate that the quality of the P. pastoris-derived scFv is comparable to that of parental E. coli-scFv. The Cross-reactivity of purified X-33-Pp-Met-28D4-derived scFv with other organophosphate pesticides (dichlorvos, dimethoate, phorate, parathionmethyl, isocarbophos) was below 0.1% except that acephate had a low cross-reactivity of about 3.8%. It was found that the stability of P. pastoris-derived scFv was better than that of E. coli-scFv.The results generated in this study could be the foundation for large-scale production of specific antibodies against methamidophos, which would bring great significance in theorethics and practical for the development of recombinarit antibodies of other low-molecular-weight chemical pesticides.