Specificity and variable region cDNA sequence of an isogeneic monoclonal antiidiotype to an anti-alpha(1----6)dextran.

We have characterized a monoclonal isogeneic antiidiotype, IdB5.7, from a BALB/c mouse immunized with the anti-alpha(1----6)dextran C57BL/6 45.21.1. It defined a hapten-inhibitable idiotope expressed on four of the 2 myeloma and 37 hybridoma anti-alpha(1----6)dextrans tested. Sequence comparison of Id+ and Id- anti-alpha(1----6)dextrans suggested that two extra amino acids at VH 100A and 100B and different residues at VH 101 abolish the expression of the idiotope in the Id- anti-alpha(1----6)dextrans. Sequence analysis of the VH of IdB5.7 showed a CDR1 longer than usual and a D segment in CDR3 formed by the fusion of two D minigenes. The IdB5.7 V kappa uses the V kappa 1 germline gene K5.1 with a few substitutions. The D-D fusion in VH CDR3 is a feature which has been reported in several other antiidiotypic antibodies.     

Variable region cDNA sequences of three mouse monoclonal anti-idiotypic antibodies specific for anti-alpha(1----6)dextrans with groove- or cavity-type combining sites.

The variables regions of three syngeneic anti-idiotypic antibodies (Ab2s) were cloned and sequenced. They are encoded by different VL genes, two are from different members of V kappa-Ox1 superfamily. The H chains are encoded by VH genes belonging to three different VH families, J558, Q52 and 7183. Together with a previous report from this laboratory, the nucleotide sequences of four Ab2s to anti-alpha(1----6)dextrans have been presented. They are derived from a number of unrelated germline genes, and differ from similar studies in anti-NP, anti-GAT and anti-Ars systems. Three of four Ab2s in the anti-alpha(1----6)dextran system appear to have D-D fusions, which has also been reported in several other Ab2s.     

Immunochemical analyses of C57BL/6J monoclonal anti-alpha (1----3) dextran antibodies

Twelve C57BL/6J anti-B-1355S monoclonal antibodies (five IgM lambda and seven IgM kappa) were characterized immunochemically by binding and inhibition ELISA. All 12 were negative for the expression of the cross-reactive idiotype (IdX) of BALB/c mice, as expected from previous work; no kappa IdX+ antibodies have been reported and IdX- lambda class antibodies were observed in B-1355-Con A induced immune sera [Geckeler W., Blomberg B., dePreval D. and Cohn M. (1977) Cold Spring Harb. Symp. quant. Biol. 41, 743-748]. The antibodies studied bind to B-1355 coated plates and this binding is inhibited by B-1355 but not by dextrans B-512 (F) or B-742S; the latter two have no linear alpha (1----3; 1) linkages. The nomenclature of Jeanes is used [Jeanes A. (1986) Molec. Immun. 23, 999-1028]; alpha (1----3; 1) refers to glucosyl diose residues linked alpha (1----3) linearly. In the case of B-1355 these linear stretches alternate with alpha (1, 6) linkages and are non-contiguous; alpha (1----3; b) refers to the linkage at a branching residue, e.g., a 1,3,6 linked moiety. The IgM kappa class antibodies are not inhibited by nigerose or nigerantetraose, suggesting that they have binding site sizes which are unusually large for B-1355 specific antibodies. The five IgM lambda antibodies are inhibited identically by equimolar amounts of nigerose and nigerantetraose, suggesting that their binding sites accommodate a disaccharide epitope. These antibodies are also inhibited by the alpha (1----6), alpha (1----4) triose, panose. The kappa class antibodies do not bind to alpha (1----3)-diglucosyl-(nigerosyl; N)-BSA. Four of the five lambda class antibodies show weak binding to N-BSA, while the fifth binds N-BSA better but less well than MOPC 104E (the BALB/c myeloma protein). All 12 antibodies are unique when compared to BALB/c antibodies derived from B-1355 immunization. The primary response of 15 C57BL/6J mice to B-1355 was re-assessed for kappa and lambda class antibody contribution. A patchy lambda class response was observed suggesting that previous lambda class responses may have been overlooked.     

The Hb-2d cross-reactive idiotype. A common idiotype expressed by monoclonal and polyclonal antibodies to human haemoglobin

Hb-2d is a monoclonal antibody of B10.D2 origin that is specific for the beta chain of human adult haemoglobin (HuHb-beta). Polyclonal anti-idiotypic sera to Hb-2d were produced in B10.D2, SJL/J and BALB/c mice. Using anti-idiotypic sera from SJL/J it was observed that Hb-2d expresses a cross-reactive idiotype (CRI) also found on both polyclonal and monoclonal antibodies to HuHb. Polyclonal antisera against HuHb-beta from H-2 congenic mice on the C57BL/10 (B10) background contained antibodies expressing the Hb-2d CRI; these antisera, however, contained little if any antibody to the antigenic determinant on HuHb-beta recognized by Hb-2d. Polyclonal antisera to HuHb-beta from the strains A.CA, A.SW, C3H.OL, BALB/cByJ, DBA/2 and SJL/J contained lower, but nevertheless detectable, amounts of antibody expressing the Hb-2d CRI. Unlike B10-H-2 congenic mice, antisera from the strains A.CA, A.SW and BALB/c bound to the same or a closely associated determinant as that recognized by Hb-2d. Two anti-HuHb monoclonal antibodies, Hb-48a and Hb-53a, both derived from B10-H-2 congenic mice, were shown to possess at least part of the Hb-2d idiotype. These antibodies are specific for epitopes on the human haemoglobin alpha chain (HuHb-alpha). It would appear, therefore, that Hb-2d possesses a CRI that is carried by antibodies to various antigenic determinants on HuHb. The linkage of these different antibodies by the CRI may allow for a common regulatory pathway.     

The complete amino acid sequence of the light chain variable region of two monoclonal anti-p-azobenzene-arsonate antibodies bearing the cross-reactive idiotype

Two monoclonal anti-p-azobenzene-arsonate antibodies produced by cell fusion of A/J spleen lymphocytes were selected. It had previously been shown that they both expressed the cross-reactive idiotype (CRI) and that the amino terminal sequence of their heavy chain variable region differed by only one amino acid substitution within the first 55 residues, at residue 41 in the second framework region. A novel, sensitive peptide-mapping method had indicated that the light chain of these two antibodies differed by at least three amino acid substitutions. Here, the complete amino acid sequence of the light chain variable region is presented. There are only 3 amino acid substitutions between the two antibodies, located in the first and second complementarity determining regions and the third framework region respectively. Although the variable region of the light chains of these two monoclonal antibodies show such a high degree of homology they differ by 26 and 27 substitutions from the reference sequence of the light chain of CRI⁺ anti-ABA serum antibodies. In addition, they are homologous to 4 other such CRI⁺ monoclonal antibody light chain sequences published so far, in which only 2 of the above 3 substitutions are not represented. The contribution of the light chain to the CRI is discussed.     

Isogeneic monoclonal antibodies against anti-alpha(1----3)dextran idiotypes. II. Neonatally induced idiotope-specific suppression: a comparative analysis

From a panel of isogeneic monoclonal anti-idiotope antibodies several were used as agents in neonatal idiotope suppression. They differed from one another in isotype, and in idiotope specificity, as described in the preceding report (Eur. J. Immunol. 1987. 17: 255). In their effects they were compared with respect to the following variables: (a) minimum dose required for suppression; (b) duration of suppression, and its relationship to the dose applied neonatally; (c) half-life of anti-idiotope in the immune system of the young mice; (d) specificity of suppression as achieved by a given anti-idiotope: in how far does it affect idiotopes defined by alternate anti-idiotopes? The following results were obtained: (a) the minimum effective dose varied widely between anti-idiotopes. One, belonging to the IgM class, was completely ineffective; others varied from ≈ 10 μg/mouse, required for complete suppression, to ≈ 100 μg/mouse. (b) The dose-response characteristic was independent of whether the state of suppression was tested (by immunization against α(1 → 3)dextran) 26 days or 70 days after neonatal anti-idiotope treatment. We take this as an indication that the anti-idiotope effect occurs during an early postnatal period. (c) There appeared to be a relationship between the rate of decay of anti-idiotope in the system and the dose required for complete suppression: the faster the decay, the more is needed initially. The persistence of effective molecules in the animals appears to depend on their isotype (as has been noted by others before): IgM decays fastest, and was ineffective in our experiments; IgG₁ stays longest, and the smallest dose was required for suppression. IgG_2b was intermediate. (d) The specificity of neonatal suppression was clearly correlated with the serological specificity of the anti-idiotope monoclonal antibodies, as well as with the representation of the corresponding idiotopes in physiological anti-dextran sera, as described in the preceding report: private anti-idiotopes suppressed their counterpart idiotopes only, while the public anti-idiotope suppressed all other idiotopes in concert.     

Rabbit antisera to the variable region domains of an anti-alpha(1----6) dextran using E. coli-produced VL and VH fusion proteins as immunogens.

Bacteria were engineered for the expression of mouse immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) fusion proteins. cDNAs encoding the VL and VH of anti-alpha(1----6)dextran hybridoma protein 19.22.1 were inserted into the pATH 10 prokaryotic expression vector downstream of trp operon sequences. V domains joined to approximately 330 amino acids of the trp E gene product encoded by the expression plasmids accumulated at high levels in E. coli. In addition, the VL domain was expressed with a 15 amino acid extension at low levels in lon mutant bacteria. The trp E-VL and trp E-VH proteins were used to raise antisera in rabbits and the V specificity of the sera demonstrated.     

Serum prostatic acid phosphatase (PAP): monoclonal enzyme-linked immunoassay compared to polyclonal radioimmunoassay

A new enzyme linked immunosorbent assay (ELISA) kit which utilizes a monoclonal antibody against prostatic acid phosphatase (PAP) was compared with current radioimmunoassay (RIA) methodology which uses a polyclonal antibody. Both assays are double antibody immunoassays with the major difference being the method of antibody preparation. A study of intrarun precision using control material showed an average within run coefficient of variation (CV) percent as 7.0 percent and 6.9 percent for ELISA and RIA, respectively, while between run CV percent averaged 11.1 percent and 11.5 percent, respectively. Thus, precision results compare similarly between the two assays. The specificity showed significantly different results. Patterns of correlation between the two methods indicate differing specificities of the primary antibodies. The values for ELISA were greater than RIA for control sera and patient samples when values fell outside the reference range; however, RIA values exceeded ELISA values with patient samples which fell within the reference ranges as provided by each manufacturer. Therefore, there exists a question of specificity of antibody employed in each of the two assays. The PAP antigen is prepared from two different sources for each kit. The ELISA manufacturer prepares antigen from seminal fluid and RIA manufacturer prepares antigen from normal human prostate. The question of specificity may be influenced by: (1) source of antigen used in immunizing animals and (2) monoclonal versus polyclonal means of producing antibody.     

Isogeneic monoclonal antibodies against anti-alpha(1----3)dextran idiotypes. I. Isotypes, idiotope specificity and representation of idiotopes in antisera from mice of various genetic constitutions

Eight isogeneic anti-idiotypic hybridomas were raised against BALB/c myeloma protein MOPC 104E and one against J558. Both myelomas react specifically with the alpha(1----3) glucosidic linkage of dextran B1355 fraction S (Dex). Six anti-MOPC 104E proteins were IgG1, one was IgG2b and one IgM. The anti-J558 protein was IgG1. Competitive interactions of the anti-idiotopes and antigen with anti-Dex proteins were measured. Dex itself was effective, but also an alpha(1----3) glucosidic heptasaccharide (N7-CHO). In order to assess the anti-idiotope specificity of hybridoma proteins, three anti-Dex molecules were used: MOPC 104E, J558 and hybridoma protein Hdex14. These differed from each other in VH amino acid positions 54-55, or 100-101, respectively. By their serological reaction pattern our anti-idiotope proteins could be divided into 3 groups: cross-reactive, partially cross-reactive and strictly specific for the immunogen. The latter ones were in the majority, and were called "private", in contrast to the cross-reactive "public" anti-idiotopes. The serological pattern was followed, in general, by the mouse-to-mouse distribution of idiotopes in physiological anti-Dex sera. Public idiotopes were closely correlated in their expression with anti-Dex activity. "Private" idiotopes showed no correlation, and displayed a characteristically high degree of fluctuation from mouse to mouse. Among the different mouse strains that were compared with respect to idiotope expression in anti-Dex sera, two stand out: C57BL-Igha, which carries chromosome 12 of BALB/c, (as selected through allotype) on the C57BL/6 genome, and BALB-Ighb, dex+, a recombinant in chromosome 12 linking the dex+ trait from BALB/c to the CH allotype from C57BL/6. The latter strain expressed significantly more of the private idiotopes than the former. This observation is discussed in terms of the position effect of classical genetics and network concepts.     

Conserved V(D)J junctional sequence of cross-reactive cytotoxic T cell receptor idiotype and the effect of a single amino acid substitution.

A dominant T cell population bearing the cross-reactive idiotype of T cell antigen receptor (TcR) has been obtained using an anti-TcR monoclonal antibody (mAb) developed in syngeneic mice. Forty-four cytotoxic T cell (CTL) clones with reactivity to a mAb (N9-127) were selected out of 396 H-2Db-restricted CTL clones specific for FBL-3 tumor antigen from C57BL/6 mice. These CTL clones were divided into two groups according to the blocking pattern of cytotoxic activities with mAb N9-127. All eight CTL clones chosen from both groups expressed TcR with a specific combination of alpha and beta chains (V alpha 1J alpha 112-2/V beta 10D beta 2.1J beta 2.7), and the difference in the blocking susceptibility resided in a single amino acid substitution (Gly to Asp) in the D-J joint of beta chain. This provides direct evidence for the molecular basis of cross-reactive idiotypes of TcR recognized by mAb.     

Specificity and prevalence of natural bovine anti-alpha galactosyl (Galalpha1-6Glc or Galalpha1-6Gal) antibodies

Immunity against the carbohydrate components of microorganisms mediated by antibodies is an important part of host defenses. Humans and closely related primates, but not other mammals, possess natural anti-Galalpha1-3Gal antibodies which also, although less avidly, react with melibiose (Galalpha1-6Glc). Using an enzyme-linked immunosorbent assay (ELISA) with melibiose-bovine serum albumin as an antigen, we analyzed bovine anti-alpha galactosyl antibodies with respect to specificity and distribution in individual animals. Inhibition assays showed that melibiose was the strongest inhibitor, followed equally by stachyose (Galalpha1-6Galalpha1-6Glcbeta1-2Fru) and raffinose (Galalpha1-6Glcbeta1-2Fru) and then by Galbeta1-6Gal, Gal, and Galalpha1-2Gal. Others, including Galalpha1-3Gal and Galalpha1-4Gal, only exhibited minor inhibition. Thus, these bovine anti-alpha galactosyl antibodies appeared to preferentially react with Galalpha1-6Glc or Galalpha1-6Gal. The distinction of this specificity from that (Galalpha1-3Gal) of human antibodies was further demonstrated by the poor reaction of bovine serum to the Galalpha1-3Gal antigen in comparison to human serum. All 27 healthy bovine serum samples of the three age groups (newborn, calf, and adult) tested contained such antibodies with titers increasing with age. The antibodies purified by affinity chromatography using a melibiose-agarose column were mainly of the immunoglobulin G (IgG) isotype with a concentration of >23 microg/ml in most samples. IgG1 was found to be the primary antimelibiose IgG isotype in all age groups by isotype-specific ELISA, but a significant increase in IgG2, an isotype more related to innate immunity, was observed in calves and adults, compared to newborns. The purified antibodies reacted with the type II bovine strain of Streptococcus agalactiae, a common pathogen of bovine mastitis. Thus, these anti-Galalpha1-6Glc or Galalpha1-6Gal antibodies in cattle might be involved in defense against microbes bearing this or the related epitopes.     

A dot-blot method for screening polyclonal and monoclonal antisera to poly(ADP-ribose)

ADP-ribosylation reactions play a key role at several points in cellular regulation and repair of DNA damage. The use of polyclonal or monoclonal antisera to poly(ADP-ribose) as probes to localize the site(s) of action of the polymer offers a promising tool for these studies. We report here a simple, sensitive method for detection and titration of these antisera to poly(ADP-ribose) using nitrocellulose membrane (NC) as a support for a dot-blot analysis. We take advantage of the fact that a highly labeled poly(ADP-ribose) preparation can be obtained by incubation of a 0.3 M KCl extract prepared from calf thymus nuclei with 32P-NAD. Such a preparation of labeled antigen is used as a reagent to detect the positive antibody spots on the NC with negligible background. Subsequent titration of the antisera and their semi-quantitative evaluation are also feasible using the dot-blot method. The sensitivity of the assay is only limited by the specific activity that can be achieved for the labeled polymer prepared as the antigen probe. The advantage of this method is that it eliminates the need to prepare pure, highly radiolabeled polymer as well as the fact that several samples can be handled on the membrane simultaneously. We demonstrate application of this technique for screening sera from patients with systemic lupus erythematosus (SLE) for anti-poly(ADP-ribose) antibodies. Further, we also extend the use of these sera for immunoquantitation of ADP-ribosylated proteins in six human tumor cells in tissue culture.     

Simultaneous binding of two monoclonal antibodies to epitopes separated in sequence by only three amino acid residues

Two monoclonal antibodies recognizing distinct epitopes the outer boundaries of which are separated by only three amino acid residues, a maximum of 10A, were demonstrated to bind simultaneously to a short synthetic peptide. The affinity of binding of the two monoclonal antibodies and of Fab' fragments derived from them was determined. The stoichiometry of the interaction was analysed by velocity sedimentation and by gel permeation chromatography experiments. The results indicate that the immune complexes formed are composed of two antibody molecules in association with one or two peptide molecules.     

The amino acid sequence of a monoclonal gamma 3-heavy chain from a patient with articular gamma-heavy chain deposition disease

Abnormal deposition of proteins, including monoclonal immunoglobulin gamma-heavy chains, may cause tissue damage and organ dysfunction. We here report the amino acid sequence of the free gamma-heavy chains present in serum and urine of the first reported case (patient G. L.) of synovial heavy chain deposition disease. The protein was heavily deleted and consisted of the hinge, in addition to the CH2 and CH3 domains, in a dimeric form, thus lacking its variable domain as well as the CH1 domain. The sequence was consistent with the gamma 3 subclass (gamma 3GL). Gm typing revealed the gamma 3 allotypes G3m(b0) and G3m(b1) in accordance with the residues Pro123, Phe128, Thr171 and Phe268 in gamma 3GL. Furthermore, the gamma 3GL molecule was glycosylated at Asn in position 129. Finally, the gamma 3GL protein was shown to contain a typical binding site for the first complement component, C1q, namely the residues Glu150, Lys152 and Lys154, with the potential of binding and activating complement, causing tissue damage following deposition.     

Identification of the amino acid residues contributing to monoclonal antibody-defined DQw1 epitopes.

The reactivity of monoclonal antibodies (mAbs) R1, S1, and S5, shown previously to recognize polymorphic epitopes on HLA-DQ molecules, have been found to correlate with the presence of certain DQB1 alleles. mAb S5 reacts with cells expressing DQB1*0503, 0601, 0602, 0603, or 0604 alleles while R1 and S1 react with all DQB1 alleles except *0201 and 0301. In the case of R1 and S1, sequence comparison of these chains suggests the involvement of residues 45-47 (GVY) in formation of the epitopes. This prediction has been confirmed by showing that a G----E mutation in position 45 of the DQB1*0302 gene eliminates binding of both mAbs.     

Preparation and functional properties of polyclonal and monoclonal antibodies to murine MD-1

Rabbits, rats and hamsters were immunized with KLH-coupled synthetic peptide sequences of the murine MD-1 molecule. Serum from immunized animals bound in Western gels to a 25 KDa protein extracted from LPS stimulated mouse spleen cells, as did a rat hybridoma (SH1.2.47) prepared from peptide-immunized rats. CHO cells transfected with a plasmid cDNA construct encoding murine MD-1, the target antigen for the antibodies in question, were also stained (in FACS) by the same antibodies. Patching and capping of the antigen(s) detected by any one of these sera abolished binding of all antibodies in subsequent FACS analysis, consistent with the hypothesis that they all detected the same antigen. In a final study to assess the possible involvement of MD-1 in regulation of cell activation for cytokine production following allostimulation, we found that all of the antibodies inhibited IL-2 and IFNgamma production, while enhancing IL-4 and IL-10 production, in mixed leukocyte reactions (MLR) in vitro.     

Comparative amino acid sequence analysis of the major outer capsid protein (VP7) of porcine rotaviruses with G3 and G5 serotype specificities isolated in Venezuela and Argentina

Summary Seven porcine group A rotavirus strains isolated in Venezuela were shown to be antigenically related to serotype G3 (five strains) or to serotype G5 (two strains), whereas two strains isolated in Argentina were classified as serotype G5. The serological classification of eight of these strains was confirmed by sequence analysis of the gene encoding the VP7 glycoprotein. A high degree of homology was observed among strains belonging to the same G serotype, although some variations in the serotype-specific regions were detected among different strains. Comparison with the published VP7 amino acid sequences of serotype G3 indicated that most porcine rotavirus strains are more closely related to each other and to human rotavirus strains than to rotavirus strains isolated from other species. Amino acid sequence comparison among serotype G5 porcine strains revealed that Venezuelan porcine isolates were more closely related to the American strain OSU, while the Argentinian strains had a higher similarity to the Australian strain TRF-41. This report confirms the worldwide distribution of these G serotypes among the porcine population.     

Reactivity of Leu 1 and T101 monoclonal antibodies with B cell lymphomas (correlations with other immunological markers)

The reactivity of Leu 1/T101 monoclonal antibodies (MoAb) was studied in a series of 69 lymphomas with B cell differentiation and was correlated with other cell markers. A three step immunoperoxidase technique on frozen sections was used to test a panel of 20 MoAb: anti-human Ig (heavy and light chains), To 15 (Pan B cells), Leu 1, T101, Leu 4, Leu 3a, Leu 5, OKT 8, OKT 6, Leu 7, anti-CALLA (IOT 5), Leu 10, anti-HLA-DR, OKM 1 and anti-dendritic reticulum cells (R 4/23). T101/Leu 1 antigen was detected in 24 cases: CLL (11 of 11), diffuse centrocytic lymphomas (four of 11), follicular lymphomas (none of 12), follicular and diffuse lymphomas (seven of 10) and one unclassified low grade lymphoma. This antigen was observed in only one high grade malignant lymphoma. In follicular lymphomas, two results deserve attention: (1) T101+ lymphomas showed most frequently IgM+, IgD+ surface Ig. Inversely, T101 unreactive lymphomas displayed IgM+, IgD+ phenotype. (2) Tp67 antigen (T101, Leu 1) and CALLA (GP 100) were found to be mutually exclusive in these lymphomas. These results suggest that follicular lymphomas could be derived from two distinct germinal center cell populations: IgM+ Ig'D-, Calla+, Leu 1-/T101- and IgM+, IgD+, CALLA-, Leu+/T101+.     

C6 glioma cells differentiated by retinoic acid overexpress the glutamate transporter excitatory amino acid carrier 1 (EAAC1)

The transport of excitatory amino acids (EAA) in CNS is performed by a family of high affinity, sodium dependent carriers. One of these transporters, excitatory amino acid carrier 1 (EAAC1), is known to be regulated by several mechanisms that modify carrier abundance on the plasma membrane. Much less is known on EAAC1 regulation at the level of gene expression. Here we report that, in C6 rat glioma cells, a line recently described to contain neural stem-like cells, EAAC1 is markedly induced by all trans-retinoic acid (ATRA), a well known differentiating agent. Consistently, ATRA stimulates EAA transport, with the maximal effect observed at concentrations>or=1 microM. After 4 days of treatment with 10 microM ATRA, the transport Vmax is fivefold enhanced, Slc1a1 mRNA is increased by 400% compared with control, EAAC1 carrier is sixfold overexpressed and the C6 culture is greatly enriched of cells with bipolar morphology strongly positive for EAAC1 immunoreactivity. Compared with untreated cells, ATRA-treated C6 cells express less Slc1a3 mRNA, for the transporter GLAST, but significantly higher levels of Slc1a2 mRNA, for the transporter GLT-1, although no expression of either protein is detected with Western blot in both untreated and ATRA-treated cells. Consistently, the inhibition pattern of aspartate transport and its stimulation by phorbol esters are indicative of a transport process due to EAAC1 operation. Under the conditions adopted, ATRA treatment causes the induction of proteolipid protein, an oligodendrocytic marker. These results indicate that, in C6 cells, ATRA stimulates the expression of EAAC1, possibly as a step toward oligodendrocytic differentiation, and constitute the first demonstration of the induction of this transporter by a differentiating agent.