Journal of Immunological Method~, 140 (1991) 159-165 ,!5 1991 ElsevierScience PublishersB.V.0022-1759/91/$03.50 ADONIS 002217599100206Z Simple colorimetric cell-cell adhesion assay using biotinylated lymphocytes Rachael Pearce-Pratt, David M. Phillips and Aldar S. Bourinbaiar The Population Council. Center for Biomedical Research. 1230 York A i'cnue. New Y,,rk. N Y 1(~12 I. I.,. S.A. (Received 13 December1990, revisedreceived4 March 1991. accepted 12 March 1991) A new approach for quantitating lymphocyte adhesion based on labeling the lymphocyte plasma membrane with water-soluble biotin was developed. Adherent biotinylated lymphocytes were quantitated by measuring OD values of a colored substrate representing the amount of bound avidin-peroxidase. The lymphocyte adhesion assay based on the high affinity of avidin to biotin was considerably more sensitive when compared to rose bengal or [3H]thymidine labeling methods. The end-point of sensitivity is approximately 1000 lymphocytes which is clearly an improvement over the rose bengal or radiolabeling techniques with a detection limit of respectively 15 x 10 3 and 7.5 x 10 3 lymphocytcs added to wells at the beginning of the assay. The method has the advantage of being rapid and simple and offers an alternative to adhesion assays based on cell ELISAs using cell-specific monoclonal antibodies. Key words: Avidin-peroxidase; Biotin: Cell adhesionmolecule: ELISA: Epithelium: Lymphocyte Introduction The binding of lymphocytes to target cells is the earliest step in the immune recognition process and is mediated by cell adhesion molecules (CAM). The cell-cell interaction process resulting in cell adhesion has recently become a subject of inten- sive investigation (for review see Springer, 1990). The nature and mechanism of cell adhesion can be approached by quantitating the dependence of lymphocyte attachment on CAM which control the cell-cell conjugate formation. In addition to tedious visual estimation requir- Correspomlence to: R. Pearce-Pratt,The Population Coun- cil, Center for Biomedical Research, 1230 York Avenue. New York. NY 10021. U.S.A. Abbret,iation: 1407, Intestine41)7. ing the counting of bound cells under a micro- scope (Lackie and De Bono, 1977; Beesley et al.. 1978) ahernative methods based on automated counting of adherent cells using a particle counter (McFaul and Bowman, 1990) have been proposed. However, the usual strategy consists of radiolabel- ing lymphocytes with either [3Hlthymidine (Roszkowski et al., 1989) or StCr (Haskard et al.. 1989) and measuring radio-activity or staining with a dye such as rose bengal (Chong and Parish, 1985) and measuring the optical density. Although the approach based on isotope incorporation is sensitive, it has the disadvantage of being time consuming and requires special disposal and safety precautions. Staining with rose bengal dye is more rapid and simple but suffers from low sensitivity. Therefore. there is still a demand for a better alternative method for quantification of adherent lymphocytes.