For IgG1 mAbs, immobilized rabbit anti-mouse IgG (1:1,000) was used like a capture antibody and HRP-goat anti-mouse IgG (1:1,000) for detection.9IgG1 myeloma MOPC 21 was used as a standard. == Cell viability detection by MTT-like assay == Vero cells were plated at 2 104cells/100l of 10% FBS-M199 into Tacalcitol monohydrate the wells of a 96-well plate (Falcon 353072, Corning), and then cultured for 16h. IgG/IgA and parental IgG1 mAb, Stx1-induced apoptosis was examined using 2 different cell lines, Ramos and Vero cells. The cross IgG/IgA inhibited apoptosis more efficiently than the parental IgG1 mAb in both instances. The results indicated that the use of high affinity binding sites as variable regions of IgA would increase the power of IgA specific for virulence factors. Keywords:annexin V, apoptosis, caspase-3, dimer, immunoglobulin A, recombinant antibody, Shiga toxin == Abbreviations == Chinese hamster ovary becoming a member of chain secretory IgA Shiga toxin 1 B subunit of Stx1 == Intro == Immunoglobulin A (IgA) is the major class of antibodies present within the mucosal surface as secretory IgA (SIgA). SIgA is definitely believed to prevent pathogens and their virulence factors invading through the mucosal barrier.1,2 Like a virulence element of enterohaemorrhagicEscherichia coli(EHEC) strains such as O157:H7 andShigella dysenteriae,3,4we have been investigating Shiga toxin 1 (Stx1). Stx1 comprises one cytotoxic subunit and 5 binding subunits (Stx1B).5We produced recombinant Stx1B as Tacalcitol monohydrate an antigen to investigate the immune response leading to efficient IgA production.6The immunogenicity of Stx1B is not high enough to induce Stx1B-specific IgA responses efficiently in mice.7This may indicate a limitation of vaccination against Stx1. On the other hand, preformed IgA against Stx1B may be useful for passive immunity in the form of a restorative antibody. However, production of IgA monoclonal antibodies (mAb) turned out not to become straightforward, especially Spp1 in the case of antigens with poor immunogenicity like Stx1B. In our case, we succeeded in the production of an Stx1B-specific IgA mAb, G2G7.8However, this IgA mAb did not, but another IgG1 mAb, D11C6, did neutralize the toxicity of Stx1 holotoxin.9To obtain antibodies with useful variable areas and with the IgA constant region, we produced a recombinant cross IgG/IgA, in which variable areas were from IgG1 mAb, while the heavy chain constant region was from IgA mAb.10This hybrid IgG/IgA was shown to neutralize Stx1, of which the toxicity toward Vero cells was measured.11 Through manifestation of immunoglobulin heavy and light chains together with joining (J) chains in Chinese hamster ovary (CHO) cells, we were able to produce a dimeric form of the cross IgG/IgA. The dimerization of IgA is known to be required for the formation of SIgA.1,12In CHO cells capable of stably expressing the dimeric IgG/IgA, both dimeric and monomeric forms were present. After separation by means of size-exclusion chromatography, we shown the dimeric form was 10-collapse more effective than the monomeric Tacalcitol monohydrate one as to toxin neutralization.11However, comparison of the dimeric IgG/IgA and parental IgG1 mAb in terms of toxin neutralization was not performed. Stx1 is known to induce apoptosis of Burkitt’s lymphoma cells and kidney-derived Vero cells.13,14In this study, we focused on comparison of the dimeric form of IgG/IgA and the parental IgG1 mAb as to toxin neutralization. We utilized 2 types of cells, Ramos cells (Burkitt’s lymphoma) and Vero cells, using 2 different assays that reflect apoptosis induction by Stx1 holotoxin. == Results == == Preparation of dimeric cross IgG/IgA by size-exclusion chromatography == We previously founded a CHO-K1 cell clone stably expressing dimeric cross IgG/IgA antibodies specific Tacalcitol monohydrate for Stx1B.11This clone expresses heavy, light and J chains. Because the weighty chain consists of VH, C1, C2 and C3 domains, this antibody is able to dimerize through a J chain. A serum-free tradition supernatant was prepared, concentrated and subjected to size-exclusion chromatography on Sephacryl S-300 (Fig. 1). Each portion was examined by SDS-PAGE under non-reducing conditions, followed by immunoblotting with anti-IgA antibodies like a probe. The dimeric cross IgG/IgA (molecular mass higher than 220 kDa) was primarily separated in fractions 46 to 52. Some IgA molecules remain Tacalcitol monohydrate monomers with this clone. The monomeric cross IgG/IgA (molecular mass between 120 and 220 kDa) was found between fractions 52 to 57. To keep the incorporation of monomers as low as possible, we pooled fractions 47 to 50 in the present study. For biological assays, the antibody concentration in the pooled portion was determined by sandwich ELISA. == Number 1. == Separation of dimeric and monomeric forms of a recombinant hybrid-IgG/IgA specific for Stx1B. A serum-free tradition supernatant (60 ml) of CHO-K1 cells triple transfected with vector constructs for H, L and J chains of the hybrid-IgG/IgA was concentrated and separated on a column of Sephacryl S-300. Elution.