Production and Characterization of IgA Monoclonal Antibody Against Ovalbumin (1)
SECRETORY IMMUNOGLOBULIN A (S-IgA) is the most prominent immunoglobulin on mucosal surfaces. The targets of IgA are not restricted to pathogens because it has also been suggested that IgA prevents food antigens from entering the blood circulation.
As an immunogen, 100 ug of OVA was mixed with 1 ug of Cholera toxin (CT) in 15 uL of PBS. The mice were intranasally immunized with 7.5 L of the antigen and CT mixture per nostril on days 0, 7, 14, and 21. On day 52, the mice were boosted intranasally with the same dose of antigen and CT. Concentration-dependent and saturable binding of A11F4, IgA, k chain, to the immobilized OVA was observed.
A11F4 gave bands corresponding to molecular masses of more than 250 kDa under non-reducing conditions. The major bands presumably represent a dimeric IgA form. The band corresponding to much higher molecular mass may represent a polymeric form of IgA. Under reducing conditions, a single band corresponding to the IgA heavy chain (65 kDa) was observed for the A11F4.
The association rate constant (kass) was calculated to be 164 (M^-1, s^-1) and the dissociation rate constant (kdiss) to be 7.59 x 10^-4 (s-^-1). The dissociation constant at steady state (KD) was calculated to be 4.63 x 10^-6 (M).
The collection of IgA-switched B cells is necessary to obtain IgA-producing hybridomas, which have been typically generated through fusion between myeloma cells and Peyer’s patch cells until now. B cells from the spleen, lung, and submandibular gland have also been examined as alternative sources.