N.R. Ling

Use of antibody-coated red cells for the sensitive detection of antigen and in rosette tests for cells bearing surface immunoglobulins

Conditions for an improved chromic chloride method for the attachment of antibody to red cells are described. The method, which is applicable to whole Ig fractions as well as affinity-purified antibodies, is reproducible and has a sensitivity for detection of antigen in the nanogram range. The coated cells may be used in a rosette assay for the detection of cell surface-bound antigens.

ELISA measurement of IgG subclass production in culture supernatants using monoclonal antibodies

Specific and sensitive ELISA to quantitate the human IgG subclasses in cell culture supernatants are described. These assays detect a minimum of 5 ng/ml IgG1, 90 ng/ml IgG2, 8 ng/ml IgG3 and 8 ng/ml IgG4 and can generally measure IgG subclasses in lymphocyte cultures containing a minimum of 200 ng/ml of total IgG. The isotype specificity of these ELISA is demonstrated and each individual ELISA shown to react with a number of paraproteins of the relevant subclass independently of their light chain type or their (major Caucasian) allotype. These assays have been used to determine the IgG subclass response of normal human lymphocytes to pokeweed mitogen in vitro.

Soluble forms of CD21 and CD23 antigens in the serum in B cell chronic lymphocytic leukaemia

By using pairs of monoclonal antibodies (MAbs) to different epitopes on CD21 and CD23 antigens, it has been shown that both antigens are readily detectable in cell-free supernates of cultures of B cells expressing these antigens on the cell surface. The antigens remained in the soluble fraction after high speed centrifugation. Sera from normal individuals contained significant amounts of CD21 antigen, whereas little CD23 antigen was detectable. By contrast CD23 but not CD21 antigen was present in urine. Sera from patients with B cell chronic lymphocytic leukaemia (B-CLL) contained increased amounts of both antigens. The levels were related to the surface expression of antigen on the leukaemic cells and the number of cells in the blood. The possible functional role of soluble forms of B cell antigens and the diagnostic potential of their detection in body fluids are discussed.

The separation of human serum IgG into subclass fractions by immunoaffinity chromatography and assessment of specific antibody activity

Murine monoclonal antibodies ( McAbs ) with specificity for subclass-specific or subclass-restricted determinants on human IgG have been coupled to Sepharose to generate affinity columns. The judicial use of positive and negative chromatography and the exploitation of the special properties of individual McAb affinity columns has allowed the preparation of individual IgG subclasses from polyclonal IgG containing less than 1% contamination by any other IgG subclass. The specificity of the antibodies present in each polyclonal IgG subclass preparation has been assayed against a bacterial toxoid (tetanus), 2 bacterial cell wall antigens (E. coli and pneumococcal) and coat antigen(s) of a DNA virus (CMV). Antibodies were predominantly IgG1 to tetanus toxoid, IgG2 to pneumovax and E. coli cell walls, and IgG1, 2 and 3 to CMV coat antigens.

Cellular origins of serum complement receptor type 2 in normal individuals and in hypogammaglobulinaemia.

A soluble form of complement receptor 2 (sCR2) is found in normal human serum. Amounts present arc about 30 90 ng/ml, which is of the same order as reported tor soluble CR1. Although B cells express surface CR2 and are the main peripheral blood source of sCR2 they do not appear to be the major tissue source of serum sCR2. Scrum levels of sCR2 of patients with hypogammaglobulinaemia were not significantly different from those of normal individuals even in the case of two brothers with Brutons X‐linked agammaglobulinaemia (XLA) lacking (CD 19+) B cells. On gel filtration through Sephacryl S‐300 the sCR2 from XLA serum behaved exactly like sCR2 from normal serum or sCR2 affinity purified from cell supernates of a B lymphoblastoid line or from the T‐ALL line MOLT‐4. In all cases a single peak appeared at the same point in the chromatogram. Possible alternative sources of serum sCR2 are follicular dendritic cells (FDC) which are known to express CR2 strongly and T6+ lymphocytes within the thymus. Peripheral T cells from adults have not been reported to express CR2. However, investigation showed that cells from the Brutons XLA cases produced small amounts of sCR2 in culture and although no CD21 was detected on the surface of the mononuclear cells by flow cytometry, the more sensitive direct antibody rosette test readily detected CD21. Further studies showed that non‐B cells from control samples of cord blood or blood of young children also weakly expressed CD21.

The development of difference turbidimetric analysis for monoclonal antibodies to human IgG.

Abstract Individual monoclonal antibodies to human IgG have been shown to form immune complexes of defined stoichimetry. These complexes are non-precipitating and do not exhibit turbidity. Combinations of two monoclonal antibodies directed against spatially distinct antigenic determinants produce complexes exhibiting marked turbidity. Such combined monoclonal antibody ‘cocktails’ may be applied to quantitative techniques.

Properties of Soluble CR2 in Human Serum

A soluble form of complement receptor number 2 (sCR2) found in human serum closely resembles that produced in culture by B lymphoblastoid cells. Epitope analysis with a panel of CD21 monoclonal antibodies revealed only minor differences between antigen from the two sources. Purified sCR2 from both sources bound to C3dg prepared from human or mouse serum and to u.v.-inactivated Epstein-Barr virus. SDS-PAGE analysis of culture supernates of B-lymphoid cells labelled by growth in medium containing 35S-methionine revealed a major component of molecular weight approximately 130 kDa and another band at 30 kDa. Incubation with endoglycosidase F reduced the size of the high molecular weight component. Gel filtration of untreated serum or culture supernate revealed that, in its native state, sCR2 behaved as a molecule or complex of apparent molecular weight 320 kDa. Possible explanations are discussed.

Ingestion of dyed-opsonised yeasts as a simple way of detecting phagocytes in lymphocyte preparations. cytophilic binding of immunoglobulins by ingesting cells

Procion-dyed yeasts which have been incubated in fresh serum and washed are readily ingested by human blood monocytes and tumour macrophages during a 30 min incubation period. Uptake is enhanced by centrifugation. Intracellular yeasts can be readily distinquished from extracellular by their much slower uptake of toluidine blue. Yeast ingestion is a much more reliable test for blood monocytes than the latex bead test and it is easier to read. The ingestion test may be combined with a rosette test for surface immunoglobulins (SmIg). Since the yeasts take up immunoglobulins from human serum during the complement-coating stage it is necessary, in a combined ingestion-SmIg test, to use fresh serum from another species (sheep) for opsonisation of the yeasts. A technique is described for reducing the number of immunoglobulin-bearing monocytes to a low level with a combined ingestion-SmIg rosette technique to detect residual immunoglobulin-bearing phagocytes.

Origin and properties of soluble CD21 (CR2) in human blood

By analysis with a panel of CD21 MoAbs it is shown that a large part of the soluble CD21 in human blood plasma is of the long isoform (CD21L), as judged by comparison with antigen produced by mouse L cells transfected with CD21L‐cDNA and reactivity with the restricted CD21 MoAb R4/23. This is compatible with the hypothesis that soluble CD21 in the blood is mainly derived from follicular dendritic cells (FDC). Cells from a human keratinocyte cell line transfected with cDNA from the Burkitt lymphoma cell line Raji also produced soluble CD21L (sCD21L), whereas the short form of sCD21 (sCD21S) was the major component of sCD21 produced by the B lymphoblastoid cell line LICR‐LON‐HMy and the T cell line Jurkat. Confocal studies of FDC isolated from human tonsil revealed that CD21 was present in the cytoplasm. On gel filtration sCD21 from untreated serum has an apparent size considerably greater than the 130 kD found by SDS–PAGE analysis. This may be partly accounted for by the non‐globular shape of the molecule, but may also indicate, as reported by others, that in its native state sCD21 is complexed with other proteins. However, no evidence of complexing with sCD23 or C3d could be found.

Immunoglobulin production by cultured human lymphocytes

The synthesis and secretion of immunoglobulin induced in cultured B-lineage cells is of interest for several reasons: (i) analysing the B-cell repertoire, (ii) recall of immunological activity retained in the circulating lymphocyte population, and (iii) study of factors needed for clonal expansion, immunoglobulin class switching, IgV-region mutation and maturation of cells to Ig secretion. Methods available are outlined and alternative procedures for cell separation and purification, helper cell provision and Ab/Ig assay systems are discussed. The aim is to provide practical guidance for those who intend to begin work in what is a vitally important, but experimentally difficult, area. There are a bewildering number of methods described in innumerable publications, old and new. The review provides a personal assessment of the present state of knowledge and prospects for improvements when all the new observations relating to cell-cell interactions and cytokines are integrated into existing technologies. The survey is chiefly concerned with physiologically based procedures, but artificial auxiliary methods are also briefly mentioned.