Comparative properties of monoclonal antibodies comprising a high-affinity anti-fluorescyl idiotype family

The T-dependent BALB/c murine immune response to fluorescein (F1) is characterized by structural heterogeneity at the protein level exemplified in part by a significantly wide range of affinities (Ka), and apparent lack of dominant idiotypes. In order to generate an idiotype family, xenogenic anti-idiotype (anti-ID) antibodies raised against anti-F1 monoclonal antibody (MCA) 4-4 (Ka = 1.7 X 10(10) M-1) were used in a solid-phase radioimmunoassay (SPRIA) to screen 68 anti-F1 hybridomas generated from multiple cell fusions for idiotypically related immunoglobulins. Four affinity-purified MCAs (designated 9-40, 10-25, 5-14 and 5-27) bearing 4-4 idiotypic determinants (ID 4-4) exhibited discrete isoelectric focusing spectrotypes (pI range = 6.8-7.7), significantly different fluorescence quenching values (38-95%) of bound ligand, binding affinities ranging from 3.3 X 10(7) to 5.3 X 10(8) M-1, similar active site inaccessibility to iodide, and closely related fine-specificity patterns for fluorescyl analogues. Idiotypic relatedness of each MCA to prototype 4-4 was quantitated by SPRIA, the results demonstrating that: each 125I-labeled MCA bound significantly to solid-phase anti-ID 4-4, and the concns of heterologous MCAs 9-40, 10-25 and 5-14 required for 50% inhibition of 125I-4-4/anti-ID 4-4 binding were comparable to homologous Ig protein. The finding that ID 4-4 bearing anti-F1 MCAs exhibit various binding properties and affinities is consistent with variable-region somatic diversification in anti-F1 affinity maturation.     

A tumor-associated lactic dehydrogenase virus suppresses the host resistance to infection with listeria monocytogenes

Infection of mice with lactic dehydrogenase virus (LDV) causes a lifelong chronic infection which is followed by alterations in immune responses during the acute phase of the infection. LDV was found to impair many functions of the reticuloendothelial system and to suppress macrophage-dependent immune responses. We tested the effect of acute infection with LDV in mice on the macrophage-mediated resistance to infection with a virulent bacterium. We found that LDV reduces the host's capacity to resist infection with Listeria monocytogenes.Many tumor lines which are transferred in mice are infected with LDV, and their growth rate is affected by the presence of the virus. It is therefore important to distinguish between immune alterations in tumor-bearing mice which are caused by the progressive growth of the tumor and those which are secondary to the viral infection.We tested whether LDV and a circulatory factor from tumor-bearing mice with similar suppressive effects on anti-Listeria immunity are two different entities or whether they are similar. We found that the factor is associated with LDV-infected tumor cells and is absent in LDV-free tumor cells. Other biological and physical characteristics supported the assumption that the tumor-associated factor is the LDV.     

Binding and structural diversity among high-affinity monoclonal anti-digoxin antibodies

High-affinity monoclonal antibodies specific for the cardiac glycoside digoxin provide a useful system for the study of structure-function relationships between antibody combining site and specific antigenic determinants. Fifteen high-affinity monoclonal anti-digoxin antibodies were produced when spleen cells from A/J mice immunized with digoxin coupled to human serum albumin (Dig-HSA) were fused with the non-secreting murine myeloma Sp2/0 cell line. Each subcloned hybridoma antibody was analyzed for affinity and specificity for structurally related cardiac glycosides by a radioimmunoassay based on the adsorption of free [3H]digoxin to dextran-coated charcoal. All of the anti-digoxin hybridoma proteins demonstrated high affinity constants ranging from 10(9) to 10(12) M-1. Using seven different analogs of digoxin, binding specificities of the monoclonal antibodies were assessed by inhibition radioimmunoassay. The 15 hybridomas produced from fusions involving five mice could be divided into eight sets on the basis of these binding specificities. Certain antibodies exhibit a preference for the aglycone portion of digoxin, while others are more specific for the tridigitoxose sugar moiety of digoxin. Monoclonal antibody H- and L-chains were subjected to N-terminal amino acid sequence analysis. The antibodies may be divided into several sequence homology sets for both H- and L-chains. In most instances, homologous heavy chains are associated with a set of homologous light chains. Homologous partial sequences, however, do not correlate with similar antigenic specificities and affinities for digoxin. Thus the fine specificity for antigen is not dependent on VH- and VL-encoded sequences alone. These data illustrate the broad diversity of the elicited response to a single hapten, even in inbred mice.     

Effector functions of a monoclonal aglycosylated mouse IgG2a: binding and activation of complement component C1 and interaction with human monocyte Fc receptor

Aglycosylated monoclonal anti-DNP mouse IgG2a produced in the presence of tunicamycin was compared with the native monoclonal IgG2a with respect to its ability to interact with the first component of complement, C1, and to compete with human IgG for binding to human monocyte Fc receptors. The aglycosylated IgG2a was found to bind subcomponent C1q with an equivalent capacity to the native IgG2a, but the dissociation constant was found to be increased three-fold. When activation of C1 by the glycosylated and aglycosylated IgG2a was compared, the rate of C1 activation by the aglycosylated IgG2a was reduced approximately three-fold. In contrast aglycosylation was accompanied by a large decrease (greater than or equal to 50-fold) in the apparent binding constant of monomeric IgG2a to human monocytes. The data suggest that the aglycosylated IgG2a has a structure which differs in the CH2 domain from the native IgG2a, and that the heterogeneous N-linked oligosaccharides of this monoclonal IgG2a which occur at a conserved position in the CH2 domain play a role in maintaining the integrity of its monocyte-binding site. This lack of monocyte binding may result either from a localized conformational change occurring in a single CH2 domain or from an alteration in the CH2-CH2 cross-domain architecture which is normally structured by a pair of opposing and interacting oligosaccharides. The minimal changes in C1q binding and C1 activation suggest that the oligosaccharides are, at most, indirectly involved in these events.     

Identification of HTLV p19 specific natural human antibodies by competition with monoclonal antibody

A competitive binding assay using a monoclonal antibody to the human T-cell lymphoma/leukemia virus (HTLV) p19 was developed for use in detecting natural antibodies to the protein in human sera. The specificity of the assay for HTLV p19 was demonstrated using a variety of antisera. While sera known to contain antibodies to HTLV p19 competed in the assay, antisera prepared against purified HTLV p24, the major core protein of the virus, or against other disrupted type-C retroviruses did not. Sera of Japanese patients with adult T-cell leukemia and similar T-cell malignant lymphomas were examined by this technique for the presence of antibodies to HTLV p19. The results were compared with those obtained by a solid-phase radioimmunoassay (RIA) against disrupted HTLV. The majority of Japanese ATL patients possess natural antibodies to HTLV as shown by solid-phase RIA (88%) and also specifically to HTLV p19 (77%). Similarly, 50% of Japanese patients with similar T-cell malignant lymphomas possess HTLV antibodies by solid-phase RIA and nearly as many (42%) possess anti-p19 reactivity. Twelve and eight percent, respectively, of normal Japanese donors from the ATL endemic region possessed HTLV-specific antibody by the solid-phase RIA or competitive binding assay. Normal donors from nonendemic areas lacked antibodies to HTLV. These results extend our previous findings of natural antibodies to HTLV in Japanese patients with ATL. The finding of p19-specific antibodies in these Japanese sera, together with previous reports of natural antibodies to HTLV p24 in sera from this same geographic cluster, strengthens the association of HTLV with Japanese ATL.     

Topographic and functional assay of antigenic determinants of human prolactin with monoclonal antibodies

Six distinct antigenic determinants were identified on human prolactin (hPRL) by competition assays with murine monoclonal antibodies (MABs). The affinity of binding and the cross-reactivity of the antibodies with two non-primate prolactins was also determined. Binding of 125I-hPRL to MAB-coated microtitre plates in the presence of a second MAB resulted in either inhibition or enhancement of antigen binding to the plate. These results were interpreted in terms of conformational changes to epitopes, induced allosterically by the binding of a second MAB to the antigen. The topographic relationship of epitopes to the biologically active regions on the hormone was examined on the basis of the neutralizing potency of MABs in the proliferation of the prolactin-dependent cell line NB2. The NB2 growth-inhibitory activity was restricted to three distinct epitopes (NE02/6, 1208 and NE03) but absent from three other MABs tested (QB01, WC01/3 and 1200). On the basis of the competition and functional studies, an elemental scheme of the topographic localization of epitopes is presented. The experimental approach employed may contribute to studies on the allocation of biologically active sites on protein hormones.     

A novel H-2s class I molecule expressed on a B-cell leukemia from SJL/J mice

Anti-H-2.33 [(B10.D2 X A)F1 anti-B10.A(5R)], which predominantly contains antibodies recognizing H-2Kb and IAb molecules, was found to be cytotoxic against DMLM 1678, a B-cell leukemia of SJL/J (H-2s) origin. The antiserum precipitated a typical class I (H-2-like) molecule from labeled tumor cell preparations as judged by molecular mass, papain susceptibility and association with beta 2-microglobulin. Sequential immunoprecipitation studies revealed that it was distinct from either H-2Ks or H-2Ds, the 2 molecules expressing the private antigens of the H-2s haplotype. Absorption analysis using congenic mice mapped the gene controlling the expression of the novel molecule telomeric to the S-region within the major histocompatibility complex.     

Idiotypic studies of monoclonal anti-tobacco mosaic virus antibodies from one mouse

Monoclonal antibodies have been prepared from one BALB/c mouse immunized with tobacco mosaic virus. The monoclonal antibodies are distributed into three subgroups recognizing different epitopes on tobacco mosaic virus subunits. The idiotypic specificities of these monoclonal antibodies have been studied using syngeneic antiidiotypic sera. A sharing of idiotypic specificities has been observed between members of each subset. These idiotypes are not recurrent in BALB/c mice immunized with tobacco mosaic virus.     

A simple bactericidal antibody test for sero-diagnosis of typhoid fever

Serum samples were collected from 24 confirmed cases of typhoid fever, 15 clinically suspected cases and 23 normal healthy controls. The convalescent sera were obtained in 13 of the 24 confirmed typhoid cases. In all, 13 paired sera, 11 acute phase only, 15 clinically suspected and 23 normal serum samples were tested for eliciting bactericidal antibodies to Salmonella typhi. In addition, the Widal test was also performed for comparison. All the 24 acute phase sera as well as 13 convalescent sera were found to be positive by bactericidal antibody test (titre 1:80 or above). Of 15 clinically suspected cases, 5 were positive whereas one of the 23 normal controls sera gave a false positive reaction. In contrast, the Widal test could detect only one of the 24 cases in the acute stage, but all 13 cases showed antibodies at a diagnostic titre level during the convalescent stage. None of the 15 clinically suspected cases or 23 normal controls were positive by the Widal test. The feasibility of using a bactericidal antibody test in sero-diagnosis of typhoid fever is discussed.     

Anti-gD monoclonal antibodies inhibit cell fusion induced by herpes simplex virus type 1

Monoclonal antibodies directed against glycoprotein D of herpes simplex virus completely inhibited fusion of Vero cells infected with type 1 virus. In contrast, several monoclonal antibodies directed against other viral glycoproteins, including B, were ineffective or were only minimally inhibitory at the highest concentrations tested.     

Identification of a second protein (M2) encoded by RNA segment 7 of influenza virus

Recent analyses of the nucleotide sequences of RNA segment 7 of influenza virus from two strains of infuenza A virus (PR/8/34 and Udorn/72) have shown that in addition to the reading frame coding for the membrane protein (M₁) there is a second overlapping open reading frame that could code for 97 amino acids [Winter and Fields (1980). Nucleic Acids Res.8, 1965–1974;Allen et al. (1980). Virology107, 548–551;Lamb and Lai (1981). Virology112, 746–751]. We have identified such a protein (M₂) in infected cells and shown that it is synthesized from a separate mRNA. Hybrid-arrested translation experiments and the use of recombinant viruses of defined genome composition have demonstrated by both biochemical and genetic approaches that protein M₂ is coded for by RNA segment 7. Peptide mapping studies have shown that M₁ and M₂ are distinct proteins, and the peptide maps can be correlated with the nucleotide sequences coding for M₁ and M₂.     

A rapid solid-phase enzyme-linked binding assay for screening monoclonal antibodies to cell surface antigens

A solid-phase enzyme-linked binding assay is described for screening monoclonal antibodies to cell surface antigens. E. coli β-galactosidase was coupled to rabbit anti-rat Ig and used to detect the binding of rat monoclonal antibodies to cells which had been fixed to the wells of microtitre plates using a combination of poly-l-lysine and glutaraldehyde. This method was found to be advantageous for the large scale screening of monoclonal antibodies with a panel of cell types, and has been useful in the selection of antibodies which would be candidates for differentiation markers within the human and mouse haemopoietic systems.     

Sequence of the neuraminidase gene of influenza virus A/Tokyo/3/67 and previously uncharacterized monoclonal variants

A full-length cDNA copy of the neuraminidase (NA) gene of influenza strain A/Tokyo/3/67 was cloned into the plasmid pBR322, and the nucleotide sequence of the gene was determined. In addition, the sequence changes in six variants of A/Tokyo/3/67 selected with various monoclonal antibodies (Ab) to the NA were determined by dideoxy sequencing of the vRNA. In five of the monoclonal variants, a single change occurred, resulting in an amino acid substitution at residue 344. Arginine in the parent virus changed to every amino acid possible with a single nucleotide change. In another variant, arginine at position 253 changed to serine, a change that also occurred in field strains. All variants so far sequenced that were selected by monoclonal Ab to A/Tokyo/3/67 virus changed at position 344, except one which changed at residue 368. Both of these positions are in clusters of residues that vary considerably in field strains, the clusters being 344-347 and 368-370. Analysis of the three-dimensional crystal structure of the NA of A/Tokyo/3/67 shows that these clusters are directly adjacent on the protein, and likely comprise a single antigenic site. A total of three or four antigenic sites have been proposed for the NA protein, based on antigenic mapping with monoclonal Ab [R. G. Webster, V. S. Hinshaw , and W. G. Laver (1982) Virology 117, 93-104]. Variants selected by Ab to Tokyo/67 NA all change in this single antigenic site, whereas variants selected by Ab to other strains change in other regions. It is possible that, although there may be three or four antigenic sites on the NA molecule, there may be a single, dominant antigenic site for each strain.     

Sequence of the hemagglutinin gene of influenza virus A/Memphis/1/71 and previously uncharacterized monoclonal antibody-derived variants

A cDNA copy of the hemagglutinin (HA) gene of Mem/1/71HA Bel/42NA was cloned into the plasmid pBR322. The cloned gene contained the coding information for all of the HA except for the hydrophobic membrane insertion sequence of the C-terminus of HA2. The cloned HA DNA was used to make HA-specific primers from restriction fragments for internal priming on the viral RNA. These primers were used to sequence the complete HA gene of Mem/1/71, and to determine nucleotide sequence changes in four previously uncharacterized monoclonal antibody generated variants of Mem/1/71 (Webster, Laver, Air, Ward, Van Wyke, and Gerhard, Ann. N.Y. Acad. Sci. 354, 142-161, 1980).     

Partial elucidation of an anti-hapten repertoire in BALB/c mice: Comparative characterization of several monoclonal anti-fluorescyl antibodies

The anti-fluorescyl repertoire in BALB/c mice was examined by producing nine hybridomas secreting antibodies with fluorescein specificity. All nine purified monoclonal anti-fluorescyl antibodies contained kappa light chains and gamma heavy chains (IgG1 and IgG2). Isoelectric focusing profiles of reduced and alkylated Ig preparations demonstrated restricted, yet relatively different spectrotypes. Collectively, the hybridomas provided a diverse range of antibody affinities as determined by several methods, including dissociation rate and ligand inhibition studies. Homogeneity of purified preparations was confirmed by dissociation rate experiments which showed that each monoclonal anti-fluorescyl preparation exhibited a single first-order off-rate. Competitive inhibition experiments with structurally related ligands indicated a high degree of fine specificity. Absorption profiles of bound fluorescein revealed distinct relative differences within the active sites of the molecules studied, and suggested the lack of any apparent correlation between affinity and wavelength maximum. Characteristics of the clones are discussed in terms of the range and prevalence of anti-fluorescyl antibody affinities and the diversity of possible binding mechanisms.     

Mouse monoclonal anti-Ia antibodies recognize cross-reacting determinants expressed on distinct subsets of human Ia-like cell-surface molecules

Human Ia-like cell-surface molecules from a homozygous HLA-DR (6/6) B lymphoblastoid cell line have been analyzed using five mouse anti-Ia m.Ab cross-reacting with HLA-DR antigens. The surface-iodinated molecules immunoprecipitated by these m.Ab were analyzed by SDS-PAGE under reducing conditions and by SDS-PAGE followed by isoelectrofocusing. As read from the different migration patterns, three distinct combinations of human Ia-like molecules were identified by these m.Ab. Three anti-I-E-reactive m.Ab immunoprecipitated two-chain molecules whose apparent mol. wt (32K, 29K) corresponded to those of the classical HLA-DR antigens. One m.Ab which on mouse cells recognized a determinant shared by the I-A and I-E molecules precipitated not only the 32-29K bands, but also a 26K band from human cell extracts. Finally, an I-A reactive m.Ab precipitated a complex set of polypeptides including in addition to the 32-29K bands, three additional chains of 30, 28 and 26K. Sequential immunoprecipitation demonstrated that removal of the classical 29-32K HLA-DR chains by an anti-I-E m.Ab did not affect the subsequent immunoprecipitation of the additional chains by the anti-I-A or the anti-I-A + I-E m.Abs. These patterns and those obtained by 2D-gels analysis which demonstrated the complexity of the 26K band are compatible with the coexpression of at least three different subsets of molecules: (1) Ia-like molecules of 29-32K, recognized by all the m.Ab used; (b) molecules of 28-30K recognized by the anti-I-A m.Ab and (c) molecules apparently constituted by 26K chains, precipitated by the anti-I-A m.Ab and by the anti-I-A + I-E m.Ab.     

Simultaneous detection of Salmonella typhi antigen and antibody in serum by counter-immunoelectrophoresis for an early and rapid diagnosis of typhoid fever

Serum samples were collected from 26 proved cases and 15 clinically suspected cases of typhoid fever and from 23 normal controls. The convalescent phase serum samples were obtained in 18 of the 26 proved cases of typhoid fever. Salmonella typhi antigen and antibody were detected simultaneously in the serum samples by counter-immunoelectrophoresis (CIE). The conventional Widal test was also performed to elicit S. typhi H and O agglutinins. Out of the 26 cases proved by culture examination, 25 were detected during early stages of the disease (24 positive for S. typhi antigen and 1 for antibody) by CIE. Out of 15 clinically suspected cases of typhoid fever, 4 were positive for S. typhi antigen and none positive for antibody. All the 18 convalescent phase serum samples were positive for antibody, but were negative for S. typhi antigen. None of the 23 normal controls gave any false positive reaction for either antigen or antibody. In contrast, the Widal test could detect only 1 out of 26 cases in the acute stage, and all the 18 cases where convalescent serum samples were obtained were positive for antibodies at diagnostic titres. None of the 15 clinically suspected cases and 23 normal controls were positive by the Widal test. The feasibility of using simultaneous S. typhi antigen and antibody detection by CIE in diagnosis of typhoid fever is discussed.     

A simple, accurate method for reproducing details of histological sections using a microfiche printer

A rapid, inexpensive method is described for achieving accurate reproduction of histological sections. The method uses a microfiche reader-printer (for library use) which produces A4 size prints. Interchangeable lenses on the microfiche printer permit the magnification of the section to be varied over the range 6.6-72 times. Where large numbers of sections are involved the speed and low cost of the method offers considerable advantages over traditional hand tracing or photomicrography.     

Expression of defined idiotypes throughout the BALB/c anti-fluorescyl antibody response: Affinity and idiotype analyses of heterogeneous antibodies

Heterogeneous BALB/c anti-fluorescyl antibodies were shown to display increases (> 50-fold) in binding affinity from the primary through the tertiary responses. The structural basis of such affinity maturation and the diversity exhibited by anti-fluorescyl antibodies was examined by idiotypic analysis using a panel of anti-idiotype reagents specific for seven different monoclonal antifluorescyl antibodies. Because these clones exhibited binding affinities characteristic of a secondary or hyperimmune response, it was possible to examine the mechanism of affinity maturation by determining the prevalence of the seven idiotypes (Id-4-4-20, Id-20-19-1, Id-20-20-3, Id-6-10-6, Id-20-4-4, Id-4-6-10 and Id-6-19-1) in specifically purified heterogeneous preparations with low (i.e. primary response) or high (i.e. secondary and tertiary responses) binding affinities. Four of the idiotypes were not detected in heterogeneous preparations and thus each represented less than 0.1 % of the total anti-fluorescein repertoire. Although results indicated that each of three other clones expressed unique or private idiotypic determinants not present in the heterogeneous population, these idiotypes (Id-4-4-20, Id-6-10-6, Id-6-19-1) were detected and ranged from 0.2 to 2.0% of the repertoire. However, results indicated that each clone expressed unique or private idiotypic determinants not present in the heterogeneous population. Determinants expressed by such high-affinity monoclonal antibodies were expressed equally in all heterogeneous preparations examined. Because those determinants which were expressed were found in either low- or high-affinity heterogeneous antibodies, it is likely that the higher affinities exhibited by monoclonal antibodies derived from a secondary response are associated with unique idiotypic determinants which were not detected in polyclonal preparations. Hence, the process of affinity maturation may find as its structural correlate a mechanism such as somatic mutation which generates individual or unique idiotypes.