Useing AFM To Observelo Monoclonal Antibodies And The Cytomembrance Surface Morphology Of Human Lymphocytes Combined With Membrance Antibodies

ObjectiveUsing Atomic Force Microscope(AFM) to observe10monoclonal antibodiescommon used and cytomembrane surface morphology of human normallymphocyte, and lymphocyte combined with different membraneantibodies.Providing morphology figure of monoclonal antibodies andantibodies binding to the surface of cells on nanometer level,which gives a newmethod for immunohistochemistry in nanometer level research, antigenaccurate location and understanding of exception expressed phenomenonfurther.Methods1.Monoclonal antibodiesAdhesion precipitation and10%formalin fixed method of steam wereapplied to the10kind of monoclonal antibodies (Cytoke, tratin-pan, Vimentin,Leukocyte common antigen, S100, Dog-1, Cytokeratin7, Thyroglobulin, CD3protein, CD20protein, CD30protein),then scanning by AFM, and observe themorphology of different monoclonal antibodies. 2.LymphocyteThe freshed lymph nodes or tonsil from the patient was pressed onto theadhesion microscope slides.There were five management for five groups. Thefirst group was not mixed any antibody. The second group was added inEMA,the third was CD3protein,CD20protein was forth, and CD3and CD20protein were added into the last one. Using the method of cells smear andchoose the AFM tapping mode to scan cytomembrane surface structure of fivegroup of human lymphocyte. Applicating the AFM image analysis software tomeasure and analysis the partial cell membrane parameters of MeanRoughness (Ra) and The Maximum Height (Rmax).Then All data were analyzedby SPSS13.0statistical software.Results1pathological cytology resultsThe results of each group of human lymphocyte with immunohistochemicalstains in light-microscope have been observed by two pathological diagnosisdoctors.Squamous cells in EMA negative control group showed positivereactions on cytomembrane while negative reactions on cytoplasm andcyteblast, but negative in lymphocyte.Lymphocytes in CD3protein experimentalgroup, CD20protein experimental group,CD3protein CD20proteinexperimental group were positive reactions on cytomembrane, but negative insquamous cells. However, we could not distinguish wether the lymphocytesexpressed CD3and CD20simultaneously.2. Scanning cells by AFMApplicating AFM scaning10kinds of monoclonal antibodies, we got different morphology.The2D shape Figures was divided into four types: round,the irregular long rod-shaped, triangular and diamond.The cells contour of eachgroup have no significant differences. The most lymphocyte is round oroval,and a narrow halo around. The surface of the control group cells is up anddown.The upheavals arranged irregularly.Compared with the control group,thecytomembrane surface form of the three experimental group （CD3proteingroup,CD20protein group,CD3protein and CD20protein group） shows thatthere are some granular materials adhere to the cytomembrane surface.Thegranular materials are similar size, regular shape, any number, unevendistribution.They forms pits between upheavals with different width and depth.In partial view of the experimental group of CD3protein and CD20protein(thelast group),we found both antibodies CD3protein and CD20protein wereshowed in lymphocytes，There are no obvious differences in the morphology ofcytomembrane surface between EMA negative control groups and blank controlgroup.3Statistical analysis resultsThe mean of the width of10kinds of monoclonal antibodies is from560nmto1.90μ m.The width and the maximum height of CD3protein,CD20protein,CD30protein are less than the other groups,but the mean roughnessare greater. There are significant difference between experimental groups andcontrol groups in Ra and Rmax values.But no difference among the threeexperimental groups.conclusionApplicating AFM scaning10kinds of monoclonal antibodies, we gotdifferent morphology.The2D shape Figures was divided into four types: round, the irregular long rod-shaped, triangular and diamond. It confirms that thepolymorphism of different antibodies and each antibody is relatively specific invisually form. We observed membrane antibody binding lymphocyte cell surfacestructure, and saw a clear, intuitive image in application on AFM. What is moreimportant, it illustrated CD3protein and CD20protein on the same lymphocyticsurface. It not only is the morphological basis of study on normal lymphocytesand lymphoma tumor cells in the abnormal expression of the antigen andprovide of expression, but also provide clues for the further application ofimmunohistochemical diagnosis.