B. Van Camp

Pea lectin as a universal cell binding agent for ELISA (screening) tests

Abstract A cell binding assay (CBA) was developed in which plant lectins are used as binding agents between microtiter plates and human cells. After binding, the cells were fixed by mild glutaraldehyde treatment. Their antigenic activity was investigated by enzyme-linked immunosorbent assay (ELISA) with several monoclonal antibodies. The binding method resulted in cell layers that remained firmly attached to the plates during the washing and incubation procedures of the ELISA. A comparative phenotype analysis, performed by indirect membrane fluorescence, showed that cells bound by this method, do not lose their antigenic activity. This binding assay can be used as a rapid, large scale screening test for monoclonal antibodies to membrane antigens of malignant and normal cells.

Differential gene expression of the neural cell adhesion molecule (N-CAM) in a panel of multiple myeloma cell lines.

A striking feature of myeloma plasma cells concerns their expression of the neural cell adhesion molecule (N-CAM). The regulation of this particular expression is, however, not known. In this study, the N-CAM (CD56) gene regulation was examined in a panel of multiple myeloma (MM) cell lines. In this panel, both N-CAM-positive and -negative cells were analysed, reflecting the in vivo situation where a minority of MM patients have CD56-negative plasma cells at diagnosis or where in cases of extramedullary involvement CD56 expression decreases. At least two N-CAM mRNAs were found in the cell lines expressing the 140u2009kDa isoform. With one exception, no N-CAM transcripts could be detected in the N-CAM-negative cell lines. No structural differences could be found in the genomic organization of the N-CAM gene, or in the regulatory promoter region when CD56-positive and -negative cell lines were compared. In transfection studies, however, transcriptional activity of the N-CAM promoter was observed in N-CAM- negative cells, leading us to conclude that the up-regulation of N-CAM in MM cannot be explained by a simple transcriptional gene activation.

Enumeration of T Lymphocytes and their Subclasses in Peripheral Blood by Immunogold Staining

Abstract Immunogold staining was used to enumerate T lymphocytes and their subclasses, as defined by monoclonal antibodies in the peripheral blood of a healthy population. Leukocytes were first incubated with the monoclonal antibodies and then with colloidal gold-labeled secondary antibodies. The cells were fixed and cytocentrifuge preparations were made. Granulocytes an monocytes in the preparations were labeled by cytochemical staining of their endogenous peroxidase activity. The preparations were examined in light microscopy and the percentage of positive lymphocytes was determined. The results were compared to those obtained by immunofluorescence microscopy. On stable preparations, lymphocytes could be recognized by their morphological characteristics. Cells reacting with the monoclonal antibody had numerous red-blue granules all around their surface membrane. The red-blue color of these granules is the color of the gold marker. This immunogold staining method forms a reliable tool for the enumeration of T lymphocytes and their subclasses. Applied on small quantities of whole blood, after lysis of the red blood cells, it could form an excellent routine procedure for this purpose, especially in laboratories lacking immunofluorescence equipment.

A Lectin Cell Binding Assay

Abstract A simple and sensitive procedure is described using plant lectins to perform a cell binding assay. This cell binding assay can be used in the screening procedure, to detect monoclonal antibodies to membrane antigens of malignant and normal cells.

Strategy to Obtain Monoclonal Anti-Idiotype Antibodies

Abstract Monoclonal anti-idiotype antibodies can be obtained after immunizing mice with human-mouse hybrids secreting the surface immunoglobulin of malignant B-cells. This allows the preparation of monoclonal anti-idiotype antibodies for therapeutical use in non secreting B-cell malignancy.

Specificity Screening of Monoclonal Antibodies Obtained after Immunization with a Human Myeloma Cell Line

Abstract Mice were immunized with a human myeloma cell line (ARH-77), in order to obtain monoclonal antibodies specific for the plasma cell surface membrane. Screening in different systems demonstrated that most monoclonal antibodies were HLA-Dr related. One antibody (6A10) precipitated a surface antigen of + 35.000 MW present on ARH-77 and a few lare bone marrow cells, negative for intracellular Ig.