Monoclonal antibody against a cross-reactive idiotypic determinant found on human autoantibodies with anti-I and -i specificities.

A monoclonal antibody has been raised against a cross-reactive idiotypic determinant expressed on human autoantibodies with anti-I and -i specificities. The McAb is directed against a conformational epitope requiring interaction of H- and L-chains for maximal expression. This epitope was strongly expressed on the monoclonal protein of one out of 100 patients with paraproteinaemia and the peripheral blood lymphocytes of one out of 50 cases of B-cell leukaemia. A small amount of the epitope is detectable among immunoglobulins in normal serum.

Immunogenic and antigenic epitopes of immunoglobulins--V. Reactivity of a panel of monoclonal antibodies with sub-fragments of human Fc gamma and abnormal paraproteins having deletions.

The specificity of a panel of 27 monoclonal antibodies reactive with human Fc gamma has been further defined through reactivity profiles with sub-fragments of Fc gamma and IgG paraproteins having deletions within C gamma 2 or C gamma 3. Antibodies are identified that are directed to discontinuous epitopes requiring native Fc gamma for expression. Other antibodies are reactive with isolated C gamma 2 or C gamma 3 domains. Sub-class specific and sub-class restricted antibodies allow correlation of epitope expression with primary structure.

Immunogenic and antigenic epitopes of immunoglobulins--VII. The topographical distribution of Fc gamma epitopes and the relationship of an iso-allotypic specificity to the presence of histidine 435.

Epitopes recognised by a panel of 23 anti-Fc gamma monoclonal antibodies (McAbs) have been subdivided into three groups each having a distinct topographical distribution. One group of mutually inhibitory McAbs are reactive with epitopes expressed on the fy "surface" of the C gamma 2 domain. A second group recognises epitopes in the region of arginine 355 of the C gamma 3 domain whilst the third group recognises epitopes expressed in the inter C gamma 2/C gamma 3 domain region--as evidenced by inhibition of Staphylococcus aureus protein A binding. An antibody of the latter group reactive with IgG1, 2, 4 and IgG3m(15,16) proteins but not IgG3m(5) or IgG3m(21) proteins allows histidine 435 to be identified as a critical residue for expression of the epitope recognised by this antibody.

Autoantibodies to acetylcholine receptor in myasthenia gravis: light chains.

We studied the light chain type of autoantibodies to acetylcholine receptor (AChR) by affinity chromatography with monoclonal anti-kappa and anti-lambda antibodies. The autoantibodies in four of eight myasthenic patients were of a single light chain type; the others comprised both types. In Graves' disease and cold-reactive hemolytic anemia, the pathogenic autoantibodies are confined to a single light chain type in individual patients, and in other diseases, doubtfully pathogenic autoantibodies are invariably mixtures of both light chain types. AChR antibodies may comprise both pathogenic and nonpathogenic types of autoantibody.

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.

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.

Synergy test for recognition of epitopes on soluble proteins; its application in the study of CD21 and CD23 antigens and their respective antibodies.

Antigens such as CD21 and CD23, which express only one copy of an epitope require two monoclonal antibodies (mAbs) for their detection and estimation. This requirement is exploited in two ways in a technique based on the chromic chloride haemagglutination test. For simple titration of antigen two portions of red cells each coated with one of a pair of synergising mAbs are used in a 1:1 combination. For testing the antigenic specificity of a mAb and assessing its region of epitope binding, the mAb under test is serially diluted in fluid containing a standard amount of antigen and red cells are added to which have been attached a different mAb. If the red cell-bound mAb recognises a determinant topographically distinct from that of the soluble mAb, red cell agglutination to high titre occurs. In titrations of ascitic fluid containing approximately 1 mg/ml mAb, titres of log2(9) to log2(16) were recorded from a starting dilution of 1 in 200. Hence the test is very sensitive and only minute amounts of a mAb are required for testing. The same test system can be used for assessing the relative display of epitopes on antigen obtained from different sources, e.g., culture supernates and body fluids. The method is of general applicability to monomeric antigens and its use is illustrated by analysis of CD21 and CD23 antigens and antibodies.

Quantitation of human total IgG, kappa IgG and lambda IgG in serum using monoclonal antibodies.

Monoclonal antibodies to human IgG have been applied, in parallel with polyclonal antisera, to the routine quantitation of human IgG. Two monoclonal antibodies directed against spatially distinct epitopes have been used in combination to form insoluble complexes exhibiting turbidity. Quantitation was performed using a centrifugal fast analyser and a correlation coefficient of 0.979 was obtained for the two estimations. The technique has been further developed to allow the separate quantitation of kappa IgG and lambda IgG in whole serum.

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.

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.

Variants of lymphoid lines produced with ricin A-chain monoclonal antibody conjugates.

Conjugates of ricin A-chain with monoclonal anti-light chain antibodies specifically killed cells hearing kappa or lambda immunoglobulin (Ig) light chains. Exposure of cells from B-lymphoblastoid cell lines (B-LCL) to conjugate for less than 30 h had only a slight effect on cell growth, but on 48 h exposure a marked killing effect was achieved. After recovery of growth, cells were re-exposed to conjugate for 9-14 days. Treatment of cells from the EB4 line (sIgG lambda) in this way yielded 4 variants which showed a marked reduction in levels of surface Ig lambda and secreted Ig lambda with slight, or no, reduction in MHC class II expression and similar growth rates to the parent line. Variant lines retained their phenotype over long periods of culture.

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.

Lymphocyte stimulation with skin cells. A function of maturation.

Cell monolayers grown from trypsin dispersions of whole rat embryos were tested for their capacity to stimulate allogeneic rat thymus cells in mixed cell cultures. Cells from whole embryos of only 15-days gestation were virtually ineffective at stimulating allogeneic thymus cells. Cells from 19-day-old whole embryos produced definite stimulation and stronger stimulation was obtained with skin cell monolayers prepared from these embryos. The average amount of allogeneic stimulation obtained with rat skin cells increased with the age of the donor animals. The greatest degree of stimulation occurred when the skin cells were obtained from rats about 1 week postpartum. Thereafter, the stimulating potential of allogeneic skin cells declined and was found to stabilize at the levels observed when adult skin cells were used as the stimulator cells. Neither rat brain cells nor rat kidney cells (taken from embryos, neonates, and adults) were capable of inducing allogeneic rat thymocytes to undergo significant transformation in culture. We conclude that the in vitro lymphocyte response to skin cell stimulation is a function of skin cell maturation, and that the inability of kidney cells or brain cells to provoke the same sort of stimulation is not attributable to antigen loss during or after foetal development, but exists as a feature of these cells at the earliest stages of differentiation.

Studies on the reappearance of MHC class II antigens on cells of a variant human lymphoblastoid line.

EB4 lymphoblastoid cell line cells were cultured for 32 days with a ricin A chain-conjugated monoclonal antibody (McAb) to a beta chain determinant present on DP and DR antigens. A single colony of cells which survived this treatment was grown up and surface expression of MHC class II and B cell antigens measured. All class II antigens (DQ as well as DP and DR, alpha as well as beta chains) were initially greatly diminished, but substantial recovery of expression occurred within 20-100 days of culture (approx. 21-105 generations), although recovery was still incomplete. The CD19 (p95) B cell antigen was present in greater amount and the CD22 B cell antigen and surface immunoglobulin in lower amount on the variant line cells. It was confirmed that unconjugated anti-class II McAb binds to surface antigens but is unable to induce modulation even over an 8-day culture period. Evidence is presented that the gradual re-expression of class II antigens on the variant line cells is not due to the appearance of a mutant or to recovery from modulation. It is suggested that the variant line cells produce an excess of a regulatory molecule when grown in the conjugate.

Use of monoclonal antibodies reactive with secretory epithelial cells for the immunocytochemical identification of plasma cells.

Six monoclonal antibodies (McAbs) were identified as plasma cell-reactive when screened on sections of human tonsil. They were all produced following immunisation of mice with cells of a human plasmacytoid line. Three of the antibodies also stained the cytoplasm (but not the surface) of blood B cells and were unreactive with other leucocytes; one McAb showed broad lymphocyte reactivity and two were completely unreactive with blood leucocytes; on testing with a panel of cell lines specificity for the plasmacytoid line was demonstrated by three of the McAbs. In spite of the marked restriction shown by the reactivity of these antibodies in tests on cells of haemopoietic origin, tests on other human tissues - including thyroid and pancreas - showed that a related antigen was present in the cytoplasm of secretory epithelial cells. The overall patterns of reactivity of the individual McAbs on various tissues and blood lymphocytes were different. Comparisons were made with the established McAb OKT10, which binds to plasma cells, early stem cells and activated lymphocytes; its binding to plasma cells was confirmed and it was shown that it did not stain secretory epithelia. The potent reactions obtained with the new McAbs suggest that antibodies to antigens associated with epithelial cell secretory apparatus provide potentially useful reagents for studying plasma cells.

Evaluation of monoclonal antibodies having specificity for human IgG sub-classes: results of an IUIS/WHO collaborative study.

Seventy-four monoclonal antibodies (McAb) of putative specificity for human IgG (11), the IgG sub-classes (59) or Gm allotypes (4) have been evaluated for reactivity and specificity in eight laboratories employing different assay techniques or protocols. For the IgG, IgG3, IgG4, G1m(f) and G3m(u) specificities McAb have been produced that can be satisfactorily applied in most methodologies employed and have potential as reference reagents. The IgG1 and particularly IgG2 specificities proved problematical with all McAb evaluated demonstrating apparent assay restriction and whilst performing well in some assays proved to be poor or inactive reagents in others. However, the study identifies McAb individually suited to application within most commonly employed methodologies. Epitope display is the probable variability rather than capricious behaviour by the McAb. IgG1 and IgG2 were the least immunogenic of the sub-class proteins and there is evidence that epitope display is influenced by the physical and chemical procedures used to immobilize or fix antigen - a common requirement in the assay systems studied.

Efficiency of induction of immunoglobulin synthesis by autologous human T cells and T cell clones: relation to surface isotype of the B cell.

Efficient immunoglobulin (Ig) production was induced when human B cells were cultured with autologous T cells activated by immobilized CD3 mAb in cultures supplemented with IL-2. Negatively purified B cells or B cells positively selected with mAb to CD19, CD21 or CD72 surface antigens produced IgM, IgG and IgA, whereas B cells selected for surface IgD or IgM produced predominantly IgM indicating that little or no isotype switching was occurring. Results are compared with reports describing high levels of mu to gamma and mu to alpha switching in single B cell systems. The limited proliferation of B cells in our culture system may account for the difference. When untreated T and B cells were cultured together in the presence of immobilized CD3 mAb, B cell numbers peaked at 6-10 days whereas T cells continued to proliferate maximally. All 52 T cell clones tested induced the production of IgM, IgG and IgA from unselected or CD19 selected B cells, but efficiency of production of Ig overall and of the different isotypes varied with different T clones. All T clones which induced high IgM, IgG and IgA production induced IgE production too, but some less active T clones also induced IgE production under non-switching conditions indicating that direct contact with activated T clone cells efficiently induces IgE as well as IgG and IgA production from B cells already expressing these isotypes. Less Ig was produced with optimal numbers of untreated T clone cells than with X-irradiated cells, confirming that proliferating T cells can inhibit as well as activate Ig production from B cells.

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.