Generation of Internal-Image Functional Aptamers of Okadaic Acid via Magnetic-Bead SELEX     

Chao Lin  Zeng-Shan Liu  Dong-Xu Wang  Lin Li  Pan Hu  Sheng Gong  Yan-Song Li  Cheng Cui  Zong-Cheng Wu  Yang Gao  Yu Zhou  Hong-Lin Ren  Shi-Ying Lu 《Marine drugs》2015,13(12):7433-7445

Okadaic acid (OA) is produced by Dinophysis and Prorocentrum dinoflagellates and primarily accumulates in bivalves, and this toxin has harmful effects on consumers and operators. In this work, we first report the use of aptamers as novel non-toxic probes capable of binding to a monoclonal antibody against OA (OA-mAb). Aptamers that mimic the OA toxin with high affinity and selectivity were generated by the magnetic bead-assisted systematic evolution of ligands by exponential enrichment (SELEX) strategy. After 12 selection rounds, cloning, sequencing and enzyme-linked immunosorbent assay (ELISA) analysis, four candidate aptamers (O24, O31, O39, O40) were selected that showed high affinity and specificity for OA-mAb. The affinity constants of O24, O31, O39 and O40 were 8.3 × 10⁸ M⁻¹, 1.47 × 10⁹ M⁻¹, 1.23 × 10⁹ M⁻¹ and 1.05 × 10⁹ M⁻¹, respectively. Indirect competitive ELISA was employed to determine the internal-image function of the aptamers. The results reveal that O31 has a similar competitive function as free OA toxin, whereas the other three aptamers did not bear the necessary internal-image function. Based on the derivation of the curvilinear equation for OA/O31, the equation that defined the relationship between the OA toxin content and O31 was Y = 2.185X − 1.78. The IC₅₀ of O31 was 3.39 ng·mL⁻¹, which was close to the value predicted by the OA ELISA (IC₅₀ = 4.4 ng·mL⁻¹); the IC₁₀ was 0.33 ng·mL⁻¹. The above data provides strong evidence that internal-image functional aptamers could be applicable as novel probes in a non-toxic assay.  相似文献   

Production and characterization of rabbit anti-idiotypic antibodies directed against a murine monoclonal anti-B. abortus antibody     

R. Adone  F. Ciuchini  R. Bianchi  G. Piccininno  C. Pistoia 《Veterinary research communications》1995,19(6):457-461

The protective anti-B. abortus monoclonal antibody ISS/32 (Ab1) was used as an immunogen to induce anti-idiotypic antibodies (Ab2) in rabbits. The purpose was to produce and characterize anti-idiotypic antibodies that share conformational similarity with the corresponding bacterial epitope recognized by Ab1. The rabbit anti-IdAb so induced was isolated and affinity-purified. Its specificity for the paratope of Ab1 was determined by evaluating its ability to compete withB. abortus for binding to Ab1 in a competitive ELISA assay. The anti-idiotypic ISS/32 antibodies were able to compete withB. abortus for binding to Ab1 in a dose-dependent manner. Hence, the data indicated that the rabbit anti-Id ISS/32 antibodies reacted with or near the antigen-binding site of Ab1.Abbreviations Ab antibody - anti-Id anti-idiotypic - ELISA enzyme linked immunosorbent assay - IgG immunoglobulin - i.p. intraperitoneal - mAb monoclonal antibody - PBS phosphate-buffered saline - s.c. subcutaneously - TD PBS + 0.05% Tween 20% + 1% yeast extract  相似文献