Idiotypic and anti-idiotypic determinants on lymphocytes during anti-Rh immunization

Evolution of idiotypic determinants on lymphocytes membrane and presence of other lymphocytes carrying anti-idiotypic determinants, were studied in Rh negative human volunteer blood donors during immunization towards Rh factor. For this purpose E-Rh Rosettes, direct immunofluorescence, inhibition of E-Rh Rosettes by anti-idiotypic sera as well as EA-Rh Rosettes--induced with Fab'2 fragments from anti-Rh antibodies and lymphocytes from the same subject--were examined and compared to the evolution of circulating antibodies. E-Rh Rosettes preceded or accompanied production of anti-Rh antibodies; their frequency decreased after the fifth month following immunization, meanwhile EA-Rh Rosettes increased in parallel with antibody decrease. The direct immunofluorescence and the inhibition of E-Rh Rosettes, by anti-idiotypic sera, show the presence of idiotypic determinants on lymphocyte membranes; the presence of EA-Rh Rosettes, coinciding with the decrease in antibodies demonstrate the existence of lymphocytes bearing auto-anti-idiotypic determinants.     

Incidence of Maternal Rh Immunization by ABO Compatible and Incompatible Pregnancies

The incidence of maternal Rh immunization in Rh-negative women following a single ABO compatible Rh-positive pregnancy is about 17%. This incidence was determined by following Rh-negative women through two Rh-incompatible pregnancies and analysing their sera for anti-Rh at the time of delivery of their second observed pregnancy. Maternal Rh immunization occurs almost exclusively after delivery; however, antibodies may not be detectable in the absence of further antigenic stimulation.The incidence of maternal Rh immunization when maternal-foetal ABO incompatibility is also present is 9–13% and 17% for group O and non-group O women respectively. This study emphasizes the need to offer Rh-immune prophylaxis to Rh-negative women having Rh-positive infants whether or not ABO incompatibility exists between the mother and infant.     

Automatic pre-transfusion serology]

This paper describes an automated apparatus combining Rosenfield's and Lalezari's antibody screening and identification basic technics. PVP bromelin and low ionic strength acid polybren channels are used; agglutinates are decanded; the remaining cells are hemolyzed and the optical density is then measured through a colorimeter and recorded on a chart; speed is of 40 samples an hour. This machine was also used for irregular antibody screening and identification. Sensitivity is shown to be equal to that of manual technics for ABO, Lewis, Lutheran as well as K, S, M, Kpb, Xga, U and Vel antibodies detection. Nevertheless, a much greater sensitivity is achieved (titers 3 to 10 times higher) than by manual technics for Rh, -k, S, Fya antibodies detection. Polybren channel is suitable for anti-Rh, Duffy, I and M (human detection; bromelin channel however, has a greater sensitivity for other specificities. Anti-M and anti-N sera from rabbits were shown to be non specific when using this machine. Over almost 15 000 sera tested, no antibody (detected by manual techniques) escaped the automated screening. This antibody detection machine was applied to compatibility tests prior to transfusion. (21 480 units were tested. aimed to be transfused to 5 611 patients). A third, PVP without bromelin, was set in parallel in order not to let escape any anti-M, even a weak one. The sera distributor was slaved to the cells distributor so that the whole procedure was automated. Furthermore, each serum was tested against red cells to be transfused, but also against the patient's own red cells to be transfused, but also against the patient's own red cells and against two selected red cells panels, so as to ensure irregular antibody detection at the same time. Using this machine, 3 to 4% of the cell samples were rejected, i.e. more than with usual techniques. All manually detected antibodies were identified, but also some others, which showed only weak reactions by classical techniques. Total results can be obtained within 20 to 30 minutes, which is quite rapid, compared to techniques using for example antiglobulin tests.     

An investigation of the immune response of homozygotes for the Rh haplotype --D-- and related haplotypes. Using cells of rare Rh phenotypes

Immune sera from people homozygous for the "deleted" gene complexes --D--, cD--, CwD--, . D . and (C)Div-- as well as heterozygous --D--/--- --- --- were studied by their reactions, and in some cases by absorption-elution tests, with cells of the following rare Rh types: two unrelated examples of homozygous . D .; three unrelated examples of Rhmod; homozygous R33; cDe with a very weak e; apparent CwD(e)/--D--. The results showed that these immune sera are clearly heterogeneous: this heterogeneity was not entirely due to the strength of the antibody (as determined by titration against CDe/cDE cells) or to the phenotype of the antibody maker. Absorption and elution tests using two of the immune sera and . D ./. D . cells demonstrated a "new" antibody, anti-Dav, for a high frequency Rh antigen in addition to anti-Rh17.     

Fluorescein and tetramethyl rhodamine as haptens in enzyme immunohistochemistry

Fluorescein (Fl) and tetramethyl rhodamine (Rh) were evaluated as possible candidates for a double hapten sandwich system in enzyme immunohistology. Monoclonal antibodies were raised against Fl and Rh. Their fine-specificity was tested with a competition-like assay. A pair of Mab's was selected for immunohistology in which they functioned as a bridge between Fl/Rh conjugated antibodies and Fl/Rh labeled peroxidase and alkaline phosphatase, respectively. The binding of fluorescein labeled antibodies could be successfully demonstrated in histological slides. A large variability in the efficacy of staining was observed in the case of rhodamine labeled antibodies. The phenomenon is explained by assuming that tetramethyl rhodamine isothiocyanate reacts preferentially with lysine residues near to, or embedded in, hydrophobic regions in a protein. This condition may reduce the accessibility of the Rh moiety for anti-Rh antibodies.     

Fluorescein and tetramethyl rhodamine as haptens in enzyme immunohistochemistry

Summary Fluorescein (Fl) and tetramethyl rhodamine (Rh) were evaluated as possible candidates for a double hapten sandwich system in enzyme immunohistology. Monoclonal antibodies were raised against Fl and Rh. Their fine-specificity was tested with a competition-like assay. A pair of Mab's was selected for immunohistology in which they functioned as a bridge between Fl/Rh conjugated antibodies and Fl/Rh labeled peroxidase and alkaline phosphatase, respectively. The binding of fluorescein labeled antibodies could be successfully demonstrated in histological slides. A large variability in the efficacy of staining was observed in the case of rhodamine labeled antibodies. The phenomenon is explained by assuming that tetramethyl rhodamine isothiocyanate reacts preferentially with lysine residues near to, or embedded in, hydrophobic regions in a protein. This condition may reduce the accessibility of the Rh moiety for anti-Rh antibodies.Abbreviations AEC 3-amino-9-ethylcarbazole - AP Alkaline phosphatase - BSA Bovine serum albumin - CGG Chicken gamma globulin - c-IF Cytoplasmic immunofluorescence - DAB 3,3-Diamino benzidine HCl - ELISA Enzyme linked immunosorbent assay - Fl Fluorescein - F/P ratio Number of fluorochrome molecules per molecule of protein - Ig Immunoglobulin - Mab Monoclonal antibody - OPD Ortho-phenylenediamine-dihydrochloride - PBS Phosphate buffered saline - PBS-T PBS with 0.2% Tween-20 - PO Peroxidase - RAM/PO Peroxidase labeled rabbit antiserum directed against mouse immunoglobulins - Rh Tetramethyl rhodamine - RT Room temperature In honour of Prof. P. van Duijn     

Preparation and performance of bispecific F(ab' gamma)2 antibody containing thioether-linked Fab' gamma fragments

A simple and efficient method is described for the production of pure bispecific F(ab' gamma)2 heterodimers, in which the individual antibody Fab' gamma fragments are joined via a stable thioether linkage. Hybrid molecules were constructed from both mouse monoclonal and rabbit polyclonal antibodies with equal efficiency, in the combinations mouse-rabbit and mouse-mouse. Peptic F(ab' gamma)2 fragments from the two chosen antibodies were first reduced to provide Fab' gamma SH. The SH groups on one of the Fab' gamma SH partners were then fully alkylated with o-phenylenedi-maleimide to provide free maleimide groups. Finally the two preparations, Fab' gamma mal and Fab' gamma SH, combined under conditions which allowed cross-linking of the maleimide and SH groups and avoided reoxidation of SH groups. The major product isolated from the reaction mixture after chromatography was always the F(ab' gamma)2 heterodimer (50 to 70%), other products being unreacted Fab' gamma and trace amounts of putative F(ab' gamma)3. Immunochemical analysis revealed that the thioether-linked F(ab' gamma)2 molecules were essentially all heterodimers, most of which had been joined via their Fd chains. The dual specificity of F(ab' gamma)2 heterodimers was tested functionally in three systems: 1) the combination (anti-idiotype + anti-phycoerythrin) linked L2C cells to the fluorochrome phycoerythrin, allowing fluorescence analysis; 2) the combination (anti-idiotype + anti-saporin) linked L2C cells to the ribosome-inactivating protein saporin, and transformed a subtoxic dose of saporin into a highly toxic mixture which prevented further protein synthesis by L2C cells; and 3) the combination of anti-idiotype with 3G8 (antibody to the Fc gamma receptor CD16) subjected L2C cells to cytotoxic attack by human mononuclear effectors.     

Generation of monoclonal antibodies to Macaca mulatta (rhesus) IgA

One IgG1 and five IgM murine monoclonal antibodies (mAb) specific for rhesus (Rh) IgA were generated. These mAbs bound to Rh IgA but not IgG or IgM when tested by enzyme-linked immunosorbent assay. Immunoblotting revealed that the mAbs reacted with the alpha heavy chain of Rh but not human IgA. The IgG1 anti-Rh IgA mAb detected IgA-producing cells in sections of monkey gut examined by immunofluorescent staining. These mAbs should be useful for characterizing IgA responses in the Rh monkey.     

Molecular characterization of a human anti-Rh(D) antibody with a DH segment encoded by a germ-line sequence.

The lambda-light-chain and lambda-heavy-chain variable-region genes of an anti-Rh(D) (Rh, Rhesus; D, heavy-chain diversity region) human monoclonal antibody secreted by lymphocytes transformed by the Epstein-Barr virus have been cloned and sequenced. Sequence comparison of the anti-Rh(D)mAb lambda-chain variable region with those of the other available human lambda chains revealed that it belonged to the human V lambda I (V lambda, variable region of lambda chain) subgroup. The greatest sequence similarity (80%) was observed with that of another anti-Rh antibody lambda-chain directed against the Rh(c) antigen. For the VH (VH, variable region of heavy chain) sequence, the highest similarity (86%) was observed with the germline VHG3 gene which belongs to the VHI subgroup. The expressed DH sequence of the anti-Rh(D) antibody is also of germline origin and complementarity-determining region 3 is thus produced by VH-DH and DH-JH (J, joining region) joining without recombination of multiple DH gene segments.     

The kinetics of antibody binding to membrane antigens in solution and at the cell surface.

The reaction kinetics of 125I-labelled mouse monoclonal antibodies binding to three cell-surface antigens of rat thymocytes (Thy-1.1, W3/25) were studied. The differences between bivalent and univalent interactions were determined by using antibody in the F(ab')2 or Fab' form and by using antigen in polymeric or monomeric forms. Association rate constants (k+1), dissociation rate constants (k-1) and equilibrium constants were determined. Also, the dissociation kinetics of rabbit antibodies against rat Thy-1 antigen were studied. The major findings were as follows. (i) With F(ab')2 antibody there was no simple relationship between antigen density at the cell surface and extent of bivalent binding. Extensive univalent binding was observed unless the antibody had a high k-1 for the univalent interaction, in which case all binding was bivalent. (ii) k+1 values were similar for F(ab')2 or Fab' antibody, and for the different antibodies were in the range 0.8 x 10(5)--1.1 x 10(6) M-1.s-1. These differences were sufficient to affect the interpretation of serological assays with the different antibodies. (iii) Antibody bound bivalently dissociated much more slowly than that bound univalently. However, the k-1 values for the univalently bound antibody were sufficiently low in most cases that the lifetime of the univalent complex was similar to or greater than the time needed for the assay. Thus the results could be interpreted on the basis of irreversible reactions. The overall conclusion from the study is that for an understanding of the binding of antibody to cell-surface antigens the kinetics of the interaction are of major importance and theories based on equilibrium binding are inappropriate.     

Shape and volume of fragments Fab' and (Fab')2 of anti-poly(D-alanyl) antibodies in the presence and absence of tetra-D-alanine as determined by small-angle x-ray scattering.

The conformation of two fragments derived from anti-poly(D-alanyl) antibodies, the divalent fragment (Fab')2 and the monovalent fragment Fab', was studied by small-angle X-ray scattering before and after interaction with the tetra-D-alanine amide hapten. More than 90% of the combining sites were occupied by the hapten. No significant changes were observed in the volume or in the radius of gyration, with either of the fragments. This contrasts with the significant decrease in these two parameters found upon reacting the hapten with intact anti-poly(D-alanyl) antibodies (I. Pilz, O. Kratky, A. Licht, and M. Sela (1973), Biochemistry 12, 4998). For Fab', the radius of the whole particle was found to be 3.48 nm in the absence of the hapten and 3.46 nm in its presence, the radius of gyration of the cross-section was 1.37 nm without hapten and 1.38 nm in its presence, and the volume of the particle was 98 nm3 in the absence of the hapten and 91 nm3 in its presence. For (Fab')2 the respective values were 5.06 and 5.05, 1.38 and 1.37, and 182 and 182. These results suggest that a conformational change occurs within the antibody molecule, but not within its Fab fragment, upon reaction with the tetraalanine hapten.     

Response and specificity of antibodies for Shigella flexneri: demonstration of type-specific factors in immunoglobulin G fraction of antiserum

Considerable amounts of immunoglobulin G (IgG) antibody appeared in hyperimmune rabbit serum at a late period during a course of immunization with several injections of Shigella flexneri O antigens. High yields of IgG antibody possessing homogenous specificity could be fractionated from crude gamma-globulin solution on a diethylaminoethyl-Sephadex column with 0.02 m phosphate buffer (pH 6.6) containing 0.1 m NaCl. Specificities of IgG antibodies for six serotypes of S. flexneri were demonstrated to be high as compared with those of whole sera and their IgM antibodies. Type-specific factors for antigens I to VI were shown in each IgG fraction according to serotype employed. Further, in most sera, subtype-specific factors could be detected in the IgG fraction. These results suggest that it would be desirable to use IgG antibodies for the typing of S. flexneri.     

Characterization of two monoclonal antibodies reactive with the external domain of the platelet-derived growth factor receptor

Two monoclonal antibodies against the receptor for platelet-derived growth factor (PDGF) were obtained by immunizing mice with pure PDGF receptor preparations derived from porcine uterus. The antibodies, denoted PDGFR-B1 and PDGFR-B2, both bound to the external domain of the receptor, as demonstrated by indirect immunofluorescence and binding of 125I-labeled antibodies to intact human fibroblasts. Both antibodies precipitated pure 175-kDa 32P-labeled autophosphorylated porcine PDGF receptor as well as a Mr 175,000 glycoprotein from metabolically labeled cells. The monoclonal antibodies did not inhibit binding of 125I-PDGF to human fibroblasts and did not stimulate these cells to undergo mitosis. Both antibodies induced clustering and down-regulation of their antigen. However, this resulted in only a partial loss of cell surface binding sites for PDGF itself, consistent with the conclusion that the monoclonals recognized only one of two or several receptors for PDGF. Clustering and down-regulation were not seen when the cells were incubated with monovalent Fab' fragments of the PDGFR-B2 antibody. The antibodies also stimulated autophosphorylation of pure PDGF receptor, and PDGFR-B2 was shown to stimulate phosphorylation of phosphofructokinase, an exogenous substrate for the PDGF receptor kinase. High concentrations of PDGFR-B2 antibody, or Fab' fragments thereof, failed to enhance the PDGF receptor kinase activity, compatible with the possibility that dimerization was of importance in the antibody-stimulated kinase activity of purified PDGF receptors.     

Binding of antibromelain monomeric Fab' improves the stability of stem bromelain against inactivation

Antienzyme polyclonal antibodies against stem bromelain were raised in male albino rabbits and the Fab' monomers isolated from the IgG of the immune sera. Incubation of bromelain with the Fab' resulted in binding and gel filtration of the resulting complex suggested a 1:1 stoichiometry. Complexing with the Fab' resulted in significant stabilization of bromelain against thermal inactivation and alkaline pH.     

Analysis of the interaction of antibodies with immunoglobulin idiotypes on neoplastic B lymphocytes: implications for immunotherapy

We have examined the reactions of a panel of nine monoclonal anti-idiotype antibodies with the surface immunoglobulin in situ on guinea pig L2C leukemic lymphocytes. Equilibrium binding constants were shown to range between 10(7) and 10(8) M-1 for univalent Fab' gamma fragments and between 10(8) and 10(9) M-1 for intact IgG. Saturation of the cell surface binding sites was achieved with 2.9 X 10(5) Fab' gamma molecules/cell and 1.2 X 10(5) IgG molecules/cell for each antibody, a result that is consistent with a bivalent mode of interaction for the IgG. Despite these overall similarities in binding characteristics antibodies showed striking differences in their ability to clear Ig from the cell surface by antigenic modulation in vitro. This suggested differences in the readiness with which the antibodies cross-linked neighboring surface Ig molecules. Such an interpretation was supported by differences in the times required to achieve bivalent binding at 0 degree C, and in the rates at which labeled antibody dissociated from the cell surface in the presence or absence of an excess of unlabeled antibody. The data are consistent with there being two functionally distinct types of anti-idiotype antibody: those that form predominantly intra-Ig bridges, with each antibody Fab being linked to an Fab on one target molecule ("monogamous" binding) and not favoring modulation; and those that form predominantly inter-Ig bridges ("bigamous" binding) and favor modulation. The nature of interaction is presumably dictated by the orientation of the particular idiotope concerned. This distinction could be of great importance in the therapeutic use of anti-idiotype to ablate B cell neoplasms.     

The presence of anti-sheep red blood cell heterophile antibodies and their characteristics in murine schistosomiasis japonica

Sensitive methods of an enzyme-linked immunosorbent assay (ELISA) using red blood cells (RBC) have been developed and were applied to the detection of anti-sheep red blood cell (SRBC) heterophile antibodies (Ab) present in sera of Schistosoma japonicum (SJ)-infected mice. The indirect hemagglutination test (IHA) was used for the purpose as well. By these methods a significant increase in the heterophile Ab levels was demonstrated in the mice particularly after 6-10 weeks of infection. The heterophile Ab in SJ-infected mice were predominantly immunoglobulins resistant to 2-mercaptoethanol treatment and had temperature-independent reactivity. In an attempt to investigate the immunological specificity of the heterophile Ab, various absorption tests were performed; Davidsohn's differential absorption test revealed that the heterophile Ab were distinct from Forssman antibody, Paul-Bunnell antibody and heterophile agglutinins known to appear in serum sickness. The heterophile Ab were absorbed only with SRBC and goat red blood cells, not with other species of RBC such as human O Rh-, O Rh+, A Rh+, B Rh+, mouse, ox, chicken, horse, rabbit, guinea pig, and rat RBC, or syngeneic tissue homogenates including brain, lung, liver, spleen, kidney, muscle, and thymus. This heterophile antibody response is not a consequence of a specific immune response directed to the antigens of SJ parasites, since absorption of the heterophile Ab with SJ adult worms or an egg preparation did not reduce the heterophile Ab level.     

Internalization and recycling of CD4 transfected into HeLa and NIH3T3 cells.

The internalization of CD4, a T cell differentiation antigen and the receptor for the human immunodeficiency viruses (HIV-1 and -2), has been examined in HeLa and murine 3T3 cells transfected with CD4 cDNA. Fab' fragments of the anti-CD4 monoclonal antibody Leu3a were generated by pepsin digestion and used as a specific monovalent, non-crosslinking ligand for CD4. These Fab' fragments were shown to bind to CD4 on the transfected cells with an affinity similar to that of HIV gp120, and inhibited HIV infection of lymphocytic cells. The Fab' fragments were radioiodinated and used in an acid-stripping endocytosis assay to demonstrate that the CD4 expressed on transfected HeLa and NIH3T3 cells was internalized. Approximately 1.5-2% of the total cell-bound [125I]Fab' fragments were internalized per minute. Furthermore, the internalized [125I]Fab' fragments could be shown to recycle to the cell surface. After 30-60 min a steady state was reached between internalization and recycling, with approximately 30-40% of the total cellular CD4 pool residing inside the cell. Similar results were obtained in studies with the intact divalent radiolabelled Leu3a antibody. These data demonstrate that CD4 expressed on transfected non-lymphoid cells is constitutively endocytosed and recycled.     

Characterization of primate antibody responses to administered murine monoclonal immunoglobulin

Murine monoclonal antibodies (MAb) are currently being assessed for their utility as tools in cancer management. Anti-murine immunoglobulin responses have been observed in many patients receiving monoclonal antibody treatment. In this study, we evaluated the response of primates to the administration of a monoclonal antibody. MAb B6.2, an antibody generated against a human breast tumor metastasis, was used as a prototype MAb. Baboons were inoculated with MAb B6.2 whole IgG, Fab', or F(ab')2 fragments. Blood samples were drawn at periodic intervals post-inoculation and the sera collected. Anti-murine immunoglobulin responses were detected using a solid-phase radioimmunoassay. The specificity of the antibody response was analyzed to determine if the response was directed against the species of origin of the MAb (species specificity), against the class of the MAb (isotype specificity), or against the hypervariable region of the MAb (idiotype specificity). We found that primates develop a humoral immune response against all three forms of the monoclonal antibody [IgG, Fab', and F(ab')2]. Furthermore, this antibody response demonstrated a high degree of specificity for the antigen binding site suggesting an idiotypic specificity. Using a competitive radioimmunoassay, the antibody response was found to interfere with antigen binding of MAb B6.2. These studies suggest that monoclonal antibody treatment can generate an anti-idiotypic response which may alter the efficacy of this mode of treatment.     

An immunoblotting as a method for the diagnosis of typhoid fever

The patients' sera had been referred to the National Salmonella Centre for routine Widal serology. Sera were predominately from patients suspected of having been infected with Salmonella Typhi, but also included one serum from patient with typhoid fever who was culture positive for Salmonella Typhi. The immunoblotting procedure using Salmonella Typhi somatic (O=9,12 LPS) and flagellar (H=d) antigens was used for preliminary testing of selected patients sera previously evaluated by Widal agglutination assay as containing different levels of antibodies against O and/or H antigens of Salmonella Typhi. Following Chart et al., immunoblotting reactions were graded between 0 and 3, with 0 indicating an absence of antibody binding, and 3 where antibody binding was readily observed. Sera giving reaction of 2 or 3 were considered to be antibody positive for this study. Positive immunoblotting reaction to O=9,12 LPS antigen was obtained only with the serum of patient with typhoid fever. Presence of specific anti-LPS antibodies was also observed in two other patients' sera diluted 1:50, and in case of one of them also in dilution 1:200, but intensity of antigen-antibody reaction was under positive result criterion. The most other sera positive to O=9,12 antigen in law dilutions (1:50, 1:100) by Widal assay, showed the traces of non-specific reaction by immunoblotting. Presence of positive antigen-antibody reaction was indicated for five sera in dilution 1:50 when tested with the >55 kDa H=d flagellar protein subunit, including the serum of patient with typhoid fever. Only in this serum the high level of specific antibodies was detected also in dilution 1:200, what was not observed in case of the other four, which appeared negative. All the other sera were shown not to contain antibodies to flagella antigen. Although the presented results are preliminary and additional study of more sera of people infected with Salmonella Typhi is needed, it can be concluded after Chart et al., that an immunoblotting procedure incorporating O=9,12 LPS and flagellar H=d antigens is a useful method for providing serological evidence of infection with Salmonella Typhi. In our opinion it can serve as a rapid test for the diagnosis of typhoid fever.