Search

Lymphocytotoxic (LCT) and platelet reactive (PR) antibody (Ab) responses were serially determined in 49 patients with acute leukaemia. LCTAb were found in 20 patients and occurred in 13 patients with acute myeloid leukaemia and 7 patients with acute lymphoblastic leukaemia. Four differing patterns of LCTAb responses could be defined. Thirteen of 22 subjects showed marked reduction or loss of LCTAb. Indirect platelet immunofluorescence, measured by flow cytometry, provided the most convenient means of detecting PRAb which were found in 11 subjects and generally showed moderate or weak reactivity.

Eight monoclonal IgG antibodies to the Rh antigen D, produced by Epstein-Barr virus transformed B-lymphoblastoid cell lines from two individuals, have been assessed for their suitability as blood grouping reagents. All showed similar specificity and agglutinated all red cells with partial D antigens tested except category DVI cells. They gave strong reactions with Du red cells in indirect antiglobulin tests, and they all gave good reactions with R1R1, R2R2 and R0r cells in manual tests using antiglobulin, enzyme or albumin methods. Initial studies showed that some of the monoclonal antibodies worked well at high dilutions on a Technicon Autogrouper 16C when used for routine D-typing of blood donors. Antibody production by the cell lines was stable for many months in continuous culture. These monoclonal antibodies may be useful diagnostic reagents.

Pig lymphocytes typed for products of the MHC were primed in vitro against allogeneic lymphocytes. When rechallenged with a panel of different cells, specific 'typing' responses were distinguishable which were not obscured by high non-specific responses or by a restimulator gene dose effect. A gene additive effect was observed in both primary MLR and in the primed lymphocyte test (PLT).

Both monoclonal human antibodies to HLA-DR antigens and supernatants from oligoclonal B-cell lines can be conveniently screened for activity by microELISA assays which use 1/10 of the volume of reagents used in conventional ELISA assays. Target cells are fixed to the bases of wells in Terasaki plates, 5 microliters volumes of supernatants incubated in these wells, and target bound antibody detected by peroxidase-conjugated anti-immunoglobulin followed by the substrate ABTS(2,2'-azino-di-(3-ethylbenzthiazoline sulphonate). The plates are read on a micro EIA reader. Supernatants can also be assayed for immunoglobulin content and isotype in Terasaki plates by coating the wells with isotype-specific anti-immunoglobulin, adding test supernatant and developing with appropriate peroxidase-conjugated anti-immunoglobulin sera and ABTS. When assaying for immunoglobulin content, the plates can be read either with a reader or by eye. Advantages and modifications of these procedures are discussed. There are no apparent practically important disadvantages to these procedures as compared with more conventional ELISA assays.

The matchability of a patient awaiting kidney transplantation is his propensity for being 'well-matched'. We present two methods for estimating matchability. It is commonly supposed that phenotype frequency is a good predictor of matchability. We show a correlation of only 0.3 between matchability and phenotype frequency using the definition of 'well-matched' used by the UK Transplant Service. We also show that ABO type and HLA homozygocity can affect matchability.

A polyclonal human lymphoblastoid cell line transformed in vitro with Epstein-Barr virus produced specific anti-Rhesus D antibody. It was repeatedly enriched by rosetting procedures and subsequently cloned. The cloning conditions employed a combination of mouse macrophage feeder layers, antimycoplasma agents, and low density passage. Formal evidence of monoclonality was obtained in one case which was of human IgG1 isotype and was secreted at the level of 15-20 micrograms/ml. All clones showed long-term stability in culture after 10 months of continuous passage. Both polyclonal and monoclonal cell lines possessed antigens characteristic of highly differentiated B cells, yet they also expressed Epstein-Barr virus nuclear antigen (EBNA). This study exemplifies a simple method for obtaining monoclonal antibody secreting plasmacytomas of human origin.

HLA-DR4 frequency was raised in patients with persistent acute arthritis following human parvovirus infection, suggesting that DR4 positive individuals are more susceptible to develop joint complications following human parvovirus infection.

Human B-cell lines secreting antibodies which react preferentially with the HLA Class II antigen DR5 have been produced. Supernatants from these cell lines reacted with lymphocytes from all DR5 positive donors and a minority of DR6 positive donors but were negative on lymphocytes of other phenotypes. The cytotoxic activities of the supernatants apparently depend on IgM antibody. These results demonstrate the potential of B-cell lines for the in vitro production of antibodies to HLA-DR antigens.

An automated HLA typing method has been developed and standardized. The procedure involves simultaneous excitation and reading of two fluorochromes, carboxy-fluorescein diacetate and propidium iodide, by a purpose-built Terasaki tray scanner known as the Astroscan 2100. Data obtained from 95 donors typed by this method and by the classical NIH dye exclusion method prove this system to be suitable for routine HLA-A,-B typing (similarity coefficient 0.9861). This comparison has highlighted significant observer bias, whereby the observer tends to downgrade very weakly positive reactions to negative and upgrade moderately positive reactions to strong positive. Elimination of this bias reduces the chances of any clinically disastrous consequence.

HLA class I phenotyping was performed using T-lymphocyte populations isolated by immunomagnetic beads (IMBs) coated with monoclonal antibodies with specificity for CD2, CD4 or CD8. The results were compared to those obtained using density gradient-separated lymphocytes (PBL). The typing trays were read by the automated simultaneous double-fluorescence (SDF) technique previously established in our laboratory using an Astroscan 2100 system. The aims of the present study were to establish whether the advantages of IMB lymphocyte separation and automated plate reading by SDF were complementary and whether the results obtained by IMB-SDF and PBL-SDF were concordant. Similarity coefficients for paired results obtained by IMB-SDF and PBL-SDF varied between 0.825 using anti-CD8-coated IMBs and 0.914 using anti-CD4-coated IMBs with a consistent excess of stronger results observed with the PBL-SDF technique. The variations observed did not result in incorrect phenotype assignment but would significantly influence a cross-matching test. These results illustrate the feasibility of using IMB-separated lymphocytes for HLA phenotyping by SDF.

Several mouse monoclonal antibodies recognising different epitopes on human erythrocyte sialoglycoproteins alpha and beta (syn: glycophorins A and C, respectively) react with human renal endothelium. Those monoclonal anti-blood group M and anti-blood group N antibodies which recognised the M/N epitopes on sialidase-treated human erythrocytes also reacted with human renal endothelium whilst those which recognise the M/N epitopes on native, but not on sialidase-treated human erythrocytes were not reactive with human renal endothelium. These results provide evidence for the occurrence of alpha and beta molecules in renal endothelium and suggest that alpha in renal endothelium is incompletely sialylated.

A rapid, reproducible and sensitive assay was developed to investigate the ability of human lymphocytes to form rosettes with erythrocytes sensitised with human monoclonal anti-D. Erythrocytes sensitised with a known number of anti-D molecules per cell were incubated with lymphocytes immobilised on plastic by poly(L-lysine), the resulting rosettes fixed, unbound erythrocytes removed by washing and the cell preparation stained. IgG1 and IgG3 anti-D-coated erythrocytes gave similar rosette formation at sensitisation levels in the range of 5000-15,000 molecules per cell, although at lower sensitisation levels IgG3 gave greater rosette formation than IgG1. A minimum of 500 IgG3 and 1000 IgG1 anti-D molecules per erythrocyte were required for rosetting.

HLA DQ alpha and DQ beta cDNA probes were used to study TaqI generated restriction fragment length polymorphisms (RFLPs) in DR4-positive patients with Felty's syndrome (FS), seropositive rheumatoid arthritis (RA), and in HLA-DR4 positive controls. The results of this analysis revealed two DQ beta RFLP patterns (DQ beta 3a and DQ beta 3b) associated with DR4, of which DQ beta 3b was found at significantly higher frequency in patients with FS (73%) or with RA (52%) than in DR4 controls (29%). Hind III generated RFLPs provide evidence that DQ beta 3b is in strong linkage disequilibrium with the gene encoding the serologically recognized epitope TA10. Results obtained using a DQ alpha chain probe revealed polymorphic differences between DQ alpha chain genes associated with different DR types, thereby providing a possible explanation for the lack of association between RA and other DR haplotypes in linkage disequilibrium with TA10. We conclude that both DQ alpha and DQ beta genes may be important in determining HLA-linked susceptibility to severe forms of RA.