The rabbit-ISAAC system. (

<p>A) A scheme depicting the rabbit-ISAAC procedure. Briefly, rabbits were immunized and later sacrificed 1 week after the final boost. Rabbit IgG⁺ cells were sorted, and single antigen-specific ASCs were detected using a microwell array chip. Next, rabbit immunoglobulin cDNAs were amplified from single cells, cloned into expression vectors and co-transduced into CHO-S cells. The antigen specificity of the produced antibodies can be determined 7 days after the detection of antigen-specific ASCs on the microwell array chip. (B) Detection of single HEL-specific ASCs in PBLs from HEL-immunized rabbits by the rabbit-ISAAC technique. Antibody secretion was detected with HEL (top left) or BSA (negative control, top right). The cells were then stained with Oregon Green (middle), and the antibody immunospots and cell signals were merged (bottom). Arrowheads indicate HEL-specific ASCs. (C) Amplification of rabbit V_H (top) and V_L (bottom) with a single-cell 5′-RACE procedure. The PCR products were analyzed by agarose gel electrophoresis. Sizes are shown at the left. Each lane shows the 5′-RACE products of individual cells. “N” indicates a cell-free RT-PCR reaction. (D) Competitive ELISA of HEL-specific RaMoAbs. The antigen specificity of antibodies derived from single ASCs using ISAAC was examined by competitive ELISA using varying amounts of HEL. The data represent means ± s.d. of 3 independent experiments.