Journal of Immunological Methods, 127 (1990) 147-148 147 Elsevier JIM 05513 Letter to the editors Immobilization of immunoglobulins on ethanol-treated ABS beads for radioimmunoassays Sotiris E. Kakabakos 1, Gregory P. Evangelatos 1 and Dionyssis S. Ithakissios 2 / National Centre for Scientific Research "Demokritos" Athens 15310, Greece, and 2 Department of Pharmacy, University of Patras, Patras 26110, Greece (Received 24 August 1989, revised received 1 December 1989, accepted 4 December 1989) Dear Editors, The loss of coating reagents that has been observed when adsorptive techniques are used for the immobilization of antibodies or antigens onto plastic supports is one of the main disadvantages that limits the widespread application of these methods in immunoassays (Parsons, 1981; Tijssen, 1985). As part of a program to gain insight informa- tion on the parameters affecting coating condi- tions several factors were studied that might in- crease the adsorption of anti-triiodothyronine rab- bit IgG (anti-T 3 IgG) on acrylonitrile-butadiene- styrene (ABS) beads (Sekisui Chemical Co., Kita- Ku, Osaka, Japan), which has been a very little studied material. In initial studies, the adsorption of anti-T 3 IgG onto ABS beads, under conditions retaining their immunoreactivity, was found to depend on such experimental variables as pH, ionic strength, con- centration of protein, temperature and incubation time of the coating procedure. Native anti-T 3 rab- bit IgG was adsorbed to a greater extent at acidic pH, whereas immobilized immunoreactivity fol- lowed a different pattern, being high when adsorp- tion was done at pH < 4 and > 9 and minimum around neutrality. In addition, when adsorbing acidic pH pretreated anti-T 3 IgG at pH 7 (Ishikawa Correspondence to: D.S. Ithakissios, Department of Pharmacy, University of Patras, Patras 26 110, Greece. et al., 1981), it was possible to obtain high im- mobilized immunoreactivity, in comparison with those obtained using untreated ones, under opti- mum coating conditions (Fig. 1). On the other hand, when ABS beads that had been incubated in absolute ethanol for 15 rain prior to coating were used, the immunoreactivity of adsorbed anti-T 3 80- S < ~ 70- -e q = 50- ~_~ = < 40- == o _ __= 30- ~ Q z = ~- o 2o- -- .~ lo- --= ~ - I I:: IF: m= 0 a b c d Fig. 1. Immunoreactivity of native anti-T 3 IgG adsorbed at: pH 3.5, 0.4 M using acetate buffer (a); pH 7.0, 0.1 M using phosphate buffer (b); pH 9.6, 0.05 M using carbonate buffer (c), as well as of acidic pH pretreated anti-T 3 IgG adsorbed at pH 7.0, 0.1 M using phosphate buffer (d) on untreated (open bar) and ethanol treated (striped bar) ABS beads. The con- centration of anti-T3 IgG in the coating solutions was 1 rag/1 and the coating volume was 0.5 ml per bead in all cases. Solid bar: non-specific binding (NSB). The NSB beads were coated respectively with native or acidic pH pretreated normal rabbit IgG. 0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)