Lena B. Lundin

Methods for derivation and detection of anti-parasite monoclonal antibodies

We describe detailed methods for derivation and cloning of myeloma hybrids which secrete antibodies specific for antigens of protozoan parasites. The methods were designed to enable the derivation of large numbers of specific monoclonal antibodies and to give high cloning efficiencies of desired hybrids. Although special attention is paid to derivation and detection of anti-parasite antibodies, the methods can be applied to many different antibody-antigen systems. Using the described methods we have isolated more than 90 myeloma hybrids which secrete antibodies specific for antigens of African trypanosomes and Theileria parasites, thus illustrating their effectiveness.

Differences in the ConA-induced redistribution and agglutination patterns of EBV genome-free and EBV-carrying human lymphoma lines.

Summary EBV-carrying human lymphoid lines showed a reduced redistribution of concanavalin A (ConA) receptors after ligand contact, compared with EBV negative lines. ConA agglutinability showed the opposite pattern, high in EBV positive, low in EBV negative lines. The same differences were also found in a comparison between two EBV negative lines and their in vitro EBV-converted, viral genome-carrying sublines, suggesting that the viral genome is directly responsible.

Relationship between amount of Epstein-Barr virus-determined nuclear antigen per cell and number of EBV-DNA copies per cell.

EPSTEIN–BARR virus-determined nuclear antigen (EBNA) can be detected in all EBV–DNA-carrying cells1,2. This includes Burkitts lymphoma (BL) and nasopharyngeal carcinoma (NPC) tumour biopsy cells and EBV-carrying lymphoid lines1–5. Of the known EBV-associated antigens only EBNA is regularly associated with the presence of viral genetic material, no matter whether the cell line produces virus or not6,7. The composition and function of EBNA are largely unknown. Some recent reports suggest that EBNA is a protein that binds to DNA in vitro8,9 and is associated with chromosomes in vivo at least in part1. EBNA might be a virally determined or virally altered chromosomal protein of the non-histone type9. It is tempting to speculate that EBNA has a role in EBV-induced cell transformation (“immortalisation”10) and/or in the control of viral gene expression. There is a wide variation in the average number of EBV genome equivalents between different EBV–DNA-carrying cell lines. In virus non-producer cell lines this number is a better approximation of the true EBV genome number for individual cells than in virus producer lines, and is relatively stable over long periods of serial passage11. The EBNA content of EBV-containing cell lines also seems to vary between different cell lines, as judged visually in the fluorescence microscope. We have measured the amount of EBNA per cell, by quantitative immunofluorimetry in different cell lines, in parallel with determining the average number of EBV–DNA copies, and established a correlation between these two parameters.

SUPPRESSOR CELLS IN TRYPANOSOMA CONGOLENSE-INFECTED MICE

Spleen cells from mice infected with T. congolense strongly suppressed lymphocyte stimulation induced in normal spleen cells by incubation with mitogens or allogeneic cells. Cell dilution studies showed that suppressor activity was extremely strong. Suppressor cell activity was markedly reduced by treatment of spleen cell populations with mitomycin-C and was unaffected by treatment with anti-Thy.1 sera and complement. Removal of cells which bound carbonyl iron or which bound to nylon columns, decreased but did not abolish suppressor activity.

Immune Depression in Trypanosoma Congolense-Infected Mice

The capacity of spleen cells from Trypanosoma congolense-infected mice to respond to the mitogens concanavalin A and bacterial lipopolysaccharide and to allogeneic lymphocytes is severely depressed or abolished. Moreover these cells cannot serve as stimulators of DNA synthesis in mixed lymphocyte reactions. The lack of responsiveness or of stimulation cannot be attributed to the dilution of appropriate B or T lymphocytes by the large number of “null” cells found in the spleen of infected mice. These “null” cells bear approximately ten times more H-2 antigen than normal lymphocytes but are devoid of Ia antigen.

Expression of theta antigen on mouse thymocytes during the cell cycle

SummaryCells from normal mouse thymus were subdivided according to size and analysed for surface antigen expression, protein content and DNA expression and content. It could be shown, that the expression of theta-antigen followed protein accumulation in individual cells. Protein content on the other hand, was correlated with DNA synthesis even if it varied over a greater range.Only one subgroup of small non-DNA-synthesizing cells was calculated to have a higher density indicative of a more condensed state together with an increased surface antigen content. These cells might represent the G0 state of the remnant proliferating population and most resembled the small cortical cells.