Volume 142, number 2 FEBS LETTERS June 1982 IDENTIFICATION OF THE SPECIES-SPECIFIC ANTIGENIC DETERMINANT(S) OF HUMAN PLASMA FIBRONECTIN BY MONOCLONAL ANTIBODIES V. E. KOTELIANSKY, E. L. ARSENYEVA, G. T. BOGACHEVA, M. A. CHERNOUSOV, M. A. GLUKHOVA, A. R. IBRAGHIMOV, M. L. METSIS, M. N. PETROSYAN and O. V. ROKHLIN USSR Cardiology Research Center, Academy o f Medical Sciences, Petroverigsky Lane 10, Moscow 101837, USSR Received 7 April 1982 1. Introduction Fibronectin is a high-M r glycoprotein which is found in the blood plasma and other body fluids, in connective tissues and in basement membrane [ 1-7]. Fibronectin is involved in a variety of cell functions - adhesion on different surfaces, cell motility, phago- cytosis [ 1-7]. Fibronectin is a multidomain disulfide- bonded dimer, the dimensions of the molecule are 15 X 8 nm;/3-form is the main element of secondary structure [8,9]. Fibronectin has an affinity to the cell surface, collagen, heparin, fibrinogen, DNA, actin and some other macromolecules [1-7]. Similar chemical properties and functions are characteristic for fibro- nectins isolated from different sources. Moreover, conventional antibodies generated against one of the types of this protein usually exhibit significant cross reactivity with fibronectins of the other origin [10,11 ]. The immunologic similarity of fibronectins can be illustrated by the fact that antibodies against human plasma fibronectin cross-react with the homologous protein from sponges [12]. However, monoclonal anti- bodies have been obtained which distinguish between different forms of this protein [13-15], thus demon- strating the difference between plasma and cellular fibronectin [ 13]. In [ 14,15] monoclonal antibodies specifically recognizing fibronectin from amniotic fluid were described. Monoclonal antibody recognizing a single antigenic determinant can provide a sharp experimental tool for the conformational and func- tional analysis of large multidomain proteins. For example, monoclonal antibodies were very useful in the localization and isolation of the ceil adhesion frag- ment and the site of structural difference between cellular and plasma forms of fibronectin [ 13,14]. We describe here species-specific monoclonal anti- bodies sensitive to the conformational transitions of the fibronectin molecule. We have also localized the antibody-binding site, i.e., the antigenic determinant specific for human and rhesus macaque plasma fibro- nectin. 2. Materials and methods Fibronectin was isolated from human plasma by affinity chromatography on gelatin-Sepharose fol- lowed by ion-exchange chromatography on Whatman DE-52 cellulose [ 16]. The characteristics of our human plasma fibronectin preparation have been pub- lished [9]. Fibronectins from pig, rat, calf, chicken and rhesus macaque plasma were isolated by affinity chromatography on gelatin-Sepharose and concen- trated by ammonium sulfate precipitation (40% satu- ration). Hybridomas secreting antibodies specific for fibro- nectin were prepared by the general method in [17]. BALB/c mice were immunized with intravenous injections of fibronectin (100/ag), followed by booster injections of the same amount of protein after 45 days. After 4 days the spleens were excised, and the spleen cells were fused with mouse myeloma cells P3/NSI-1-Ag 4-1. After growth on a selective medium containing hypoxanthine, aminopterin and thymidine the hybrid cells were seeded into 24-weU plates (Lindbro, Flow Labs). Positive clones secreting antifibronectin antibodies were detected by a solid- phase radioimmunoassay; these were then subcloned by limiting dilution and expanded as ascites tumors in BALB/c mice. The antibodies were precipitated from Published by Elsevier Biomedical Press 00145793/82/0000-0000/$02.75 © 1982 Federation of European BiochemicalSocieties 199