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Antibodies to the D antigen of the Rh system use a restricted set of immunoglobulin V and J gene segments, especially VH DP50 and DP63, JH6, Vlambda DPL16 and Jlambda 2/3. These gene segments may confer a natural affinity on the antibodies for the D antigen and this hypothesis has been tested by constructing two single-chain Fv phage-antibody libraries based on the germline gene segments DP50 and DP63; structural variability was obtained by insertion of 11 amino acids in random sequence in the VHCDR3. 10 anti-D antibodies were selected from these libraries, each with a unique VHCDR3. In contrast, selections with the CcEe antigens produced antibodies reacting with the Rh polypeptide molecules but without strict blood group specificity. One of these latter DP50-based antibodies was converted into 12 different antibodies with specificity for E by replacing the original germline light chain with chains from a rearranged L chain library. The CDR1 and CDR2 sequences of the DP50-based antibodies were common to both anti-D and anti-E molecules; differentiation between D and E specificity was dependent on VHCDR3 sequences and their correct pairing with an appropriate L chain.

Sera containing anti-D, taken from 44 RhD-negative women with RhD-positive infants, were tested in antibody-dependent cellular cytotoxicity (ADCC) and monocyte monolayer assays (MMA) which used similar target and effector cell populations. In addition, the anti-D concentration was measured in the Auto Analyzer and the number of IgG1 and IgG3 anti-D molecules bound to the target red cells was measured by flow cytometry. The results of the functional assays and Auto Analyzer quantitation were examined for correlation with IgG subclass quantitation and all results were compared for their ability to predict the severity of haemolytic disease of the newborn (HDN). ADCC correctly predicted HDN in 39/44 (88.6%) cases, Auto Analyzer quantitation in 35/44 (79.5%) and the MMA in 32/44 (72.7%). For all three assays, the number of correct predictions was highest when the maternal serum contained both IgG1 and IgG3 anti-D. ADCC activity and HDN were correlated with the number of cell-bound IgG1 molecules (r > or = 0.58), but MMA activity was most closely correlated with the number of cell-bound IgG3 molecules (r = 0.68). Hence the superior predictive value of ADCC is due to its ability to reflect the IgG1 component of maternal anti-D, which has a better correlation than IgG3 anti-D with the severity of HDN.

We previously reported the initial characterization of five human single-chain Fv (scFv) antibody fragments specific for the blood group antigens B, D(Rh), E(Rh), Kpb and HI. The scFvs were isolated from a phage-antibody library constructed from the variable region genes of two non-immunized donors. In this paper we report the specificity, affinity and kinetics of antigen binding of these scFv fragments. All five scFvs agglutinated the appropriate red cell phenotype following the addition of a monoclonal antibody which recognizes a peptide tag incorporated into the scFv. The anti-B and anti-HI scFv molecules, which recognize high density carbohydrate antigens, spontaneously polymerized and agglutinated red cells directly. None of the antibody fragments showed cross-reactivity with other red cell antigens, with the exception of the anti-E which reacted weakly with E-negative cells. Specific scFv binding was confirmed by ELISA, flow cytometry and radioactive labelling. The anti-D scFv recognized 17,600 sites on cDE/cDE red cells with an association constant (Ka), of 5.2 x 10(7) M-1 and a rate constant for dissociation (koff) of 1.9 x 10(-2) s-1. The anti-E scFv recognized 29,800 and 39,800 sites on cDE/cDE red cells in two experiments with Kas of 8.4 x 10(6) and 4.4 x 10(7) M-1. The koff for this antibody was 2.7 x 10(-2) s-1. The results demonstrate that scFv antibody fragments specific for cell surface antigens and possessing affinities typical of the primary immune response can be obtained from a phage-display library.

Two human monoclonal antibodies, one IgG3 and one IgG1, with anti-Rh D specificity, were tested for their ability to clear red cells. Samples of red cells from 12 D-positive subjects were sensitised in vitro with various amounts of antibody, the number of antibody molecules bound to the cells was estimated, and the cells were reinjected into the donor's circulation. Both antibodies mediated clearance but substantially fewer IgG3 than IgG1 antibody molecules were required to produce a given rate of clearance. The IgG3 antibody was slightly more effective than polyclonal anti-D, as judged by comparison with previously published results. Since there is believed to be an association between the rate of red cell clearance by anti-D and the ability of the antibody to suppress D immunisation, the IgG3 monoclonal antibody may prove suitable for the immunoprophylaxis of Rh D haemolytic disease.

The exploitation of Epstein-Barr virus transformation to generate human lymphoblastoid clones (LCL) producing antibody of predefined specificity has proved highly inefficient. Observations reported here on a cloned LCL producing anti-Rhesus(D) may provide an explanation for the low success rate. Over a few months this clone manifested a progressive loss of capacity to maintain growth in culture. Evidence consistent with terminal differentiation to a cell with the properties of a nonproliferating plasma cell was obtained. These late cells differed from those in the earlier actively cycling phase in that they were no longer able to respond to autostimulatory growth factors although they continued to produce them. This irreversible senescence leading to the death of the clone may be a common feature of virally transformed B cells and this would explain many of the difficulties encountered on this route to the production of human monoclonal antibodies.

The prophylactic effect of anti-D in the prevention of maternal immunization to the D antigen of the Rh system depends in the first instance on its ability to bind to fetal Rh-positive red cells. The functional activity of the injected anti-D in this respect is dependent on both the plasma concentration and functional affinity constant of the antibody, and both factors should be taken into account in assessing IgG anti-D preparations for clinical use. A radio-immunoassay utilizing 125-I-labelled human monoclonal anti-D has been developed which measures the amount of antibody bound to red cells after the addition of an aliquot of the test anti-D preparation. The assay is thus a functional test in that it measures the ability of the anti-D to react with the antigen. The functional activity of the test anti-D is compared to that of the international standard, and the concentration is then expressed in terms of microgram-equivalents, that is, its equivalence in functional terms to the stated amount of international standard. The radio-immunoassay was compared to the currently used agglutination assay, and the results were found to give good agreement in 18 out of the 21 samples assayed.

An international collaborative study of anti-D assays has been carried out by 21 laboratories in 11 countries. Samples of anti-D immunoglobulin assayed in this study included two dilutions of a preparation used in clinical trials to determine a dose-protection relation, a national standard, commercial clinical preparations and the proposed international reference preparation in coded ampoules. Manual, automated haemagglutination and isotope labelling methods all gave similar relative potencies. Several of these estimates were significantly (P=0.95) heterogeneous and some modifications to improve assay design and procedure are suggested. The coded preparation was shown to be stable and suitable for comparative assays. It was estimated to contain 60 microgram of of anti-D IgG immunoglobulin per ampoule, and 150 i.u./ampoule when assayed against the International Standard for Incomplete Anti-D Blood Typing Serum. Thus for this preparation 1 microgram of IgG anti-D immunoglobulin identical to 2.5 i.u. anti-D antibody. At its 28th meeting the Expert Committee on Biological Standardization of WHO established the preparation 68/419 as the International Reference Preparation of Anti-D Immunoglobulin and assigned to it a potency of 150 i.u. per ampoule. The Preparation has been widely used (with a nominal content of 60 microgram of IgG anti-D immunoglobulin) for control of clinical preparations.