Generation And Evaluation Of Monoclonal Antibodies And Single-chain Antibody Against ICAM-1

ICAM-1 is cell surface adhesive molecules with MW of 76000-114000,andbelongs to the superfamily of immunoglubulins. The structure of ICAM-1 protein iscomposed of external, transmembrane and a short internal parts. The external partincludes 5 immunoglobulin-like functional regions, connecting head-to-tail with eachother, region 1 to region 4 poseess 88, 97, 99, 101amino acid residuals,respectively.Region 1 and region 2 are adheresive regions, to which the LFA-1 binds.Region 3 mainly binds to Mac-1.The interaction of ICAM-1and LFA-1 intermediate adhesion of epithelium tolymphocytes, some monocytes and neutrophils. It can also promote the inactivationand endosmosis of T cells by binding to LFA-1 and Mac-1(CD11b/CD18). This is ofimportance to maintain the normal physiology and balance the diseaseprogress.Abnormal expression of ICAM-1 might enhance or aggravate the incidenceand development of many kinds of diseases. For example, autoimmune disease mayresult in the increase of ICAM-1 expression. Inhibiting the expression of ICAM-1 willcease certain pathological injuries. Monoclonal antibodies against ICAM-1 mightblock the adhesion. This study firstly developed monoclonal antibodies againstICAM-1.The VH and VL genes of the McAb were cloned from hybridoma secretingthe McAbs, and constituted into a ScFv gene and then subcloned to pET28a andpET20b. The ScFv was correctly expressed. After denaturing, renatureing andpurification, the ScFv was proved to be effective in inhibiting inflammation.Therefore,a new type of inflammation method has been established.Two monoclonal antibodies (2A3, 3H7 ) of IgG1 subclass were raised againstICAM-1. Their titers in cell culture supernatant were 1:360, 1:680 respectively, titersin ascites were 1:2×104,1:8×105 respectively, the best coating concentrations of...