Development Of A Colloidal Gold Probe-based Silver Enhancement Immunochromatographic Assay For Detection Of Abrin-a

Two hundred grams of abrin kernal was homogenated after soaked in 5% cold acetic acid for 12h, then crude extract was obtained from 35% to 95% ammonium sulfate fractional precipitation. Farther purification was carried out by Sepharose 4B affinity chromatography and DEAE-Sepharose FF ion-exchange chromatography. Two fractions were collected and designated as P1 and P2, respectively. The molecular weight of intact P1 was 64.3KDa measured by SDS-PAGE. It was splited into two polypeptide chains of 29.1KDa and 35.4KDa when treated withβ-mercaptoethanol. P1 was identified as abrin by double-antibody sandwich ELISA.Recovery and amplification culture the frozen hybridoma cell(4G1) which secretes mouse anti-abrin-a monoclonal antibody to collecte ascites. Mice ascites was purified by ammonium sulfate- protein A chromatography, improved ammonium sulfate- protein A chromatography and caprylic- ammonium sulfate- protein A chromatography. The purity and immunological activity of the purified products were analyzed by SDS-PAGE and ELISA. The results showed that the activities of monoclonal antibody purified with different methods were normal. The purity of IgG2b from the method of caprylic- ammonium sulfate- protein A chromatography had reached electrophoretically pure. Rabbit blood serum was collected from the rabbits immunized by abrin-a and purified by caprylic-ammonium sulfate-protein A chromatography. The purity and immunological activity of the purified products were examined by SDS-PAGE and ELISA. The results showed that there was a high purity of polyclonal antibody and the activities of 1mg/ml rabbit anti-abrin-a polyclonal antibody were 1:819200.High-affinity anti-abrin-a monoclonal and polyclonal antibodies were used to develop a sandwich immunochromatographic assay and silver enhancement technology was used to increase the sensitivity sill further. Using a matrix of double distilled water or soybean milk added with abrin-a, the visual detection limit of abrin was found to be 10 ng/mL. The detection limit of abrin was 0.1 ng/mL, an increase in sensitivity of 100-fold when the silver enhancement technology was employed. The assay was portable and very simple to perform and the detection was completed within 20 min without complicated handling procedures. There was no significant cross-reactivity with several homologous toxins and associated agglutinin. The assay reagents could be stored for 12 weeks at 4°C without significant loss of activity. These characteristics make the strip assay to be an ideal candidate for the development of a rapid toxin detection kit.