Murine Myeloma Protein Research Articles

Calculations of antibody-antigen interactions: microscopic and semi-microscopic evaluation of the free energies of binding of phosphorylcholine analogs to McPC603.

The study of antibody-antigen interactions should greatly benefit from the development of quantitative models for the evaluation of binding free energies in proteins. The present work addresses this challenge by considering the test case of the binding free energies of phosphorylcholine analogs to the murine myeloma protein McPC603. This includes the evaluation of the differential binding energy as well as the absolute binding energies and their corresponding electrostatic contributions. Four different approaches are examined: the Protein Dipoles Langevin Dipoles (PDLD) method, the semi-microscopic PDLD (PDLD/S) method, a free energy perturbation (FEP) method based on an adiabatic charging procedure and a linear response approximation that accelerates the FEP calculation. The PDLD electrostatic calculations are augmented by estimates of the relevant hydrophobic and steric contributions. The determination of the hydrophobic energy involves an approach which considers the modification of the effective surface area of the solute by local field effects. The steric contributions are analyzed in terms of the corresponding reorganization energies. This treatment, which considers the protein as a harmonic system, views the steric forces as the restoring forces for the electrostatic interactions. The FEP method is found to give unreliable results with regular cut-off radii and starts to give quantitative results only in very expensive treatment with very large cut-off radii. The PDLD and PDLD/S methods are much faster than the FEP approach and give reasonable results for both the relative and absolute binding energies. The speed and simplicity of the PDLD/S method make it an effective strategy for interactive docking studies and indeed such an option is incorporated in the program MOLARIS. A component analysis of the different energy contributions of the FEP treatment and a similar PDLD analysis indicate that electrostatic effects provide the largest contribution to the differential binding energy, while the hydrophobic and steric contributions are much smaller. This finding lends further support to the idea that electrostatic interactions play a major role in determining the antigen specificity of McPC603.

Identification of low density lipoprotein as a regulator of Fc receptor-mediated phagocytosis.

Optimal expression of the high-affinity Fc receptor for IgG (FcRI) by the human monocyte cell line U-937 requires the presence of low density lipoprotein (LDL), and neither cholesterol nor high density lipoprotein can provide the component necessary for optimal FcRI expression. Here we show that FcR-mediated phagocytosis also requires LDL. U-937 cells were cultured in medium containing interferon gamma and either fetal calf serum (FCS) or delipidated FCS (DLFCS). The phagocytosis of IgG-coated erythrocytes was measured by a colorimetric assay. U-937 cells cultured in DLFCS medium had less than 16% of the phagocytic activity of cells cultured in normal FCS medium. Phagocytosis of IgG-coated erythrocytes could be inhibited 85% by the addition of murine IgG2a myeloma protein (5 micrograms/ml). U-937 cells cultured in DLFCS medium supplemented with pure cholesterol in ethanol (10 micrograms/ml) had only 30% of the phagocytic activity of cells grown in FCS medium. Addition of very low density lipoprotein (0.2 mg of protein per ml) to DLFCS medium also failed to increase phagocytosis. However, the addition of LDL (0.2 mg of protein per ml) to DLFCS medium restored 90% of the phagocytic activity. Since neither pure cholesterol nor very low density lipoprotein restored normal phagocytic function to U-937 cells despite a normalization of cellular cholesterol content, the restoration of phagocytosis observed with LDL replacement cannot be explained by mere delivery of cholesterol by LDL. Thus, LDL is required for the expression of FcRI and FcR-mediated phagocytosis by U-937 cells and may be an important regulator of phagocytic activity of monocytes and macrophages in vivo.

Relative noncovalent association constant between immunoglobulin H and L chains is unrelated to their expression or antigen-binding activity.

With H and L chains derived from the murine hybridoma and myeloma proteins NQ5-89.4, 93G7, AIDA10/3, AIDA10/16, HyHEL-10, HyHEL-9, HyHEL-8, HyHEL-5, XRPC25, J539, UPC10, 6684, and C101, the relationship between the relative affinity between H-L pairs, their antigen-binding characteristics, and the primary structure of their VH and VL domains was assessed. Using competitive chain reassociation assays in which two different L chains were allowed to compete for a limiting amount of an H chain, it was observed that different pairs of L chains tended to compete to the same degree regardless of which H chain was used as the limiting reagent and regardless of whether they were the autologous or heterologous L chain. In agreement with our previous results, it was also observed that when there was limited diversity between the Vk segments of the competing L chains, the relative competitive ability of an L chain was dictated by the nature of the first residue of the Jk segment, residue 96. However, when a high degree of diversity existed between the Vk segments of the competing L chains, the relative affinity was dictated by the V segment. It was further demonstrated that junctional diversity in the L chain may not necessarily be essential for antibody activity, determined using autologous and heterologous, noncovalently reassociated immunoglobulin molecules in antigen-binding assays. Combined with the results of the competitive reassociation assays, it was evident that no correlation between the competitive ability of these L chains existed or, by inference, the relative mutual affinity between different H-L pairs and their ability to form an antigen-binding site. These results were in agreement with the random rearrangement of VH and VL domain gene segments and argue against any restrictions in the expression of the full repertoire of immunoglobulin molecules due to combinatorial (H-L pairing) mechanisms.

Putative immunoglobulin V H genes of the goldfish, Carassius auratus, detected by heterologous cross-hybridization with a murine V H probe

By using a defined cDNA probe for the V H region of a murine phosphocholine-binding myeloma protein (S107) we have defined a family of distinct cross-hybridizing DNA sequences in genomic DNA of the goldfish. The estimated number of the goldfish putative V H family detectable by the S107 probe is about 36. By using two putative goldfish V H probes to analyze, by hybridization, the relationships among seven of the goldfish genomic clones, we have determined that the putative goldfish V H genes recognized by the S107 probe comprise at least several distinct families that are not closely related.

Combinatorial diversity in the generation of antibody molecules. The complete amino-acid sequence of the variable domain of a monoclonal anti-streptococcal group A polysaccharide antibody.

The first complete amino-acid sequence of the variable region of the gamma 3 heavy chain from a murine anti-streptococcal group A polysaccharide (A-CHO) immunoglobulin (monoclonal antibody 2S1.3) is described. Therefore, in conjunction with the previously published 2S1.3 light chain sequence, a V kappa 25 structure, the entire variable domain of this antibody has been determined. In addition, four partial amino-terminal heavy chain sequences of other antibodies with the same specificity are reported. These heavy chains share a high degree of homology with heavy chains from fructosan-binding murine myeloma proteins with the exception of those positions known to be encoded by the D (diversity) segment in germ line DNA. The light chains associated with the heavy chains reported here are products of the V kappa 25, V kappa 27, and J kappa 5 genes. Up to date three VH and four V kappa subgroups have been shown to contribute genetic material to the assembly of antibodies specific for the A-CHO. Unlike other experimental systems employing structurally completely resolved full antigens the antistreptococcal immune response uses V genes previously shown to be involved in the formation of antibodies with different specificities. This provides further experimental evidence for the physiological relevance of heavy/light chain association as a posttranscriptional diversification mechanism in the generation of the antibody repertoire in addition to those somatic diversifiers acting directly upon the genes encoding the variable regions of individual chains.

Murine IgA binding factors produced by Fc alpha R(+) T cells: role of Fc gamma R(+) cells for the induction of Fc alpha R and formation of IgA-binding factor in Con A-activated cells.

The relationship between the production of a T cell factor having affinity for IgA (IgA-binding factor(s); IgA BF) and the expression of Fc receptors specific for IgA (Fc alpha R) was studied by using murine spleen cells activated with concanavalin A (Con A blasts). Fc alpha R was detected by the cytophilic binding of anti-TNP murine IgA myeloma protein (MOPC 315 IgA) to Con A blasts as determined by an indirect rosette method with trinitrophenylated sheep red blood cells (TNP-SRBC). After 18 hr preculture with IgA, Fc alpha R was expressed on 15 to 20% of Con A blasts, which released IgA BF suppressing the in vitro IgA synthesis of the spleen cells stimulated with pokeweed mitogen (PWM). Without preculture with IgA, there was neither induction of Fc alpha R nor the production of IgA BF from Con A blasts. Fc alpha R was not induced on Con A blasts by IgA if Fc gamma R(+) cells were depleted from the blasts by rosetting with SRBC sensitized with rabbit IgG antibody (EA gamma). Even after preculture with IgA, the suppressive IgA BF was undetectable in the culture supernatant of Con A blasts depleted of the Fc gamma R(+) cell population. By using a double rosette method with EA gamma and trinitrophenylated quail red blood cells, Fc alpha R proved to be co-expressed on Fc gamma R(+) precursor T cells in the Con A blasts. The results suggested that both Fc gamma R and Fc alpha R could be co-expressed on Con A blasts, as is the case with T2D4 Fc gamma R(+), Fc alpha R(+) T hybridoma cells, which are known to produce IgG-binding factor(s) (IgG BF) and IgA BF. The relationship between Fc gamma R and Fc alpha R on a single cell was studied by using monoclonal anti-Fc gamma R antibody (2. 4G2 ). The reactivity of 2. 4G2 antibody with T cell Fc gamma R was proved by the inhibition of EA gamma rosette formation by Con A blasts or T2D4 cells. The addition of 2. 4G2 monoclonal antibody, however, did not affect the induction of Fc alpha R on Con A blasts by IgA. Furthermore, the binding of IgA to Fc alpha R already expressed on L5178Y T lymphoma cell line cells was not inhibited by the monoclonal antibody. The results confirmed that Fc alpha R are distinct from Fc gamma R co-expressed on the same Con A blasts, and that the expression of Fc alpha R on Fc gamma R(+) T cells and their production of suppressive IgA BF may be induced by the binding of IgA to Fc alpha R.

Biosynthesis of arabinogalactan-protein in Lolium multiflorum (Italian ryegrass) endosperm cells. Subcellular distribution of galactosyltransferases.

Intracellular membranes from protoplasts of Italian-ryegrass (Lolium multiflorum) endosperm cells have been fractionated on sucrose density gradients and identified on the basis of putative-marker-enzyme assays. Galactosyltransferases capable of incorporating galactose from UDP galactose into 66% ethanol-soluble products are associated with all membrane fractions. Affinity chromatography of the ethanol-insoluble products on (murine myeloma protein J539)-Sepharose reveals that the enzymes responsible for the synthesis of polymers containing (1----6)-beta-D-galactose residues are associated exclusively with subcellular fractions enriched in Golgi-derived membranes. This suggests that the Golgi apparatus plays an important part in the synthesis of the carbohydrate component of the ryegrass arabinogalactan-protein.

The ability of IgA to inhibit complement consumption by complement-fixing antigens and antigen-antibody complexes.

The polymeric and monomeric (7S) forms of the nitrophenyl-specific MOPC-315 murine IgA myeloma protein were examined for their capacity to inhibit the consumption of guinea-pig complement (C) by the C-activating protein DNP46BSA and by antigen-antibody (Ag-Ab) complexes. On a molar basis, 13S IgA was slightly (2-3 fold) more efficient than 7S IgA in inhibiting C consumption by DNP-BSA. When mixed with IgG antibodies prior to incubation with a non-complement-fixing antigen (TNP-KLH), 13S IgA was considerably more effective than 7S IgA in preventing the formation of Ag-Ab complexes able to fix C. The degree of inhibition observed was related to the concentration of C used in the assay, being greater at lower C concentrations.

Synthetic idiotypes: The third hypervariable region of murine anti-dextran antibodies

Synthetic peptides with the sequence of the third hypervariable region of either M104 or J558 murine myeloma proteins have been used to immunize rabbits. Most of the resulting antisera distinguished the two proteins in a specific manner corresponding to the sequence of the immunizing peptide. Thus synthetic peptides have been used to elicit antisera specific for particular idiotypes. These studies have significant implications for our understanding of the molecular basis of idiotypy.

The use of haptenated immunoglobulins to induce B cell tolerance in vitro. The roles of hapten density and the Fc portion of the immunoglobulin carrier.

The relationship between the Fc region of trinitrophenylated (TNP)-immunoglobulins (Ig), and their ability to induce tolerance was examined. It was found that adult B cells responding to a T-independent (TI) antigen were tolerized by TNP11 human gamma globulin (HGG), but not by TNP10F(ab')2 fragments of HGG. Increasing the hapten density on the F(ab')2 fragments overcame their inability to induce tolerance. Thus, a TNP17-F(ab')2 was an effective tolerogen. Murine myeloma proteins of different IgG subclasses were similarly tested. A TNP12-IgG2a and a TNP11-IgG1 induced tolerance, whereas two TNP11-12-IgG3 did not. However, a more heavily haptenated TNP18-IgG3 was tolerogenic. These results suggest that lightly haptenated immunoglobulins depend upon Fc receptor binding to induce tolerance in adult B cells. Non-Fc receptor-binding carriers are not tolerogenic unless they are more heavily haptenated. Finally, T cell and macrophage depletion experiments suggest that the tolerogens act directly on the B cells.

Human lymphocyte response to CEA: Titration of different CEA samples and neutralization experiments with monoclonal anti-CEA antibodies

By means of the macrophage electrophoretic mobility technique a striking digestive system cancer-associated human lymphocyte response to CEA has been found during a large-scale study including tests in 499 individuals. The question to be answered by this study was whether this response is really CEA-specific. Titration experiments with 3 different CEA preparations in lymphocytes from 5 colorectal cancer patients showed that the threshold dose of CEA necessary to induce lymphocyte responses amounts to 50–100 ng CEA per ml and 106 lymphocytes, regardless of the CEA origin and its state of purity. The CEA specificity of the responses was proved by neutralization experiments with 3 CEA-specific monoclonal antibodies. When allowed to react with CEA before lymphocyte incubation, the MABs prevented CEA from inducing lymphocyte responses. Appropriate murine control myeloma protein did not influence these responses. The reactivity of these lymphocyte samples to a teratocarcinoma extract could not be prevented by treating this material with CEA-specific MABs before incubation. Preliminary attempts to enrich the lymphokine(s) released after CEA stimulation resulted in recovery of the activity within 2 arbitrarily cut Sephadex G-100 fractions comprising the mol. wt range of 3000–47000.

Altered expression of lymphocyte Fc alpha receptor in selective IgA deficiency and IgA nephropathy.

To study the expression of FcR specific for IgA (Fc alpha R) on human peripheral lymphocytes (PBL), PBL from normal donors were incubated with 300 to 500 micrograms/ml MOPC 315 IgA having anti-trinitrophenyl (TNP) antibody activity at 4 degrees C or 37 degrees C for 60 min. Under this condition, less than 2% of total cells could form rosettes with TNP-coated ox red blood cells (TNP-ORBC). When cultured with MOPC 315 IgA at 37 degrees C for 18 hr, however, there was a dose-dependent increase of the rosette-forming cells (RFC) binding TNP-ORBC. Because 15 to 20% of the total cells bound TNP-ORBC but not unsensitized ORBC, the rosette formation appeared to be due to the cytophilic binding of IgA to the cells. The binding of MOPC 315 IgA was competed by TEPC 15 IgA and human myeloma IgA, but not by murine myeloma proteins of other classes, indicating that the receptor is specific for IgA. Fc alpha R was induced on 15 to 20% of fractionated T and B cells, as well as on 15 to 18% of concanavalin A-(Con A) activated lymphocytes when cultured with IgA. The induction of the receptor was dependent on protein and RNA synthesis, but not on DNA synthesis as suggested by the sensitivity to metabolic inhibitors, such as mitomycin C, actinomycin D, puromycin, and cycloheximide. In five patients with selective IgA deficiency (serum IgA, 0 to 4 mg/dl), only 5.1% +/- 1.7 of PBL formed rosettes with TNP-ORBC after culture with MOPC 315 IgA, whereas 12.5% +/- 2.5 of PBL from normal donors (serum IgA, 90 to 330 mg/dl) formed rosettes. Fc alpha R was induced on more than 15% of the cells from these patients, however, when cultured with IgA in the presence of a conditioned medium obtained from mixed lymphocyte culture from two normal donors. The results suggested that the abnormality in the patients' PBL might be in the induction mechanism rather than in the number of precursor cells that could express Fc alpha R in the presence of IgA. On the other hand, Fc alpha R was induced on 10.4% +/- 1.5 of PBL from the patients with IgA nephropathy (serum IgA, 382 +/- 11 mg/dl) when they were incubated with IgA for 1 hr at 37 degrees C. Because Fc alpha R on normal PBL was not induced by 1 hr of incubation with IgA, it appeared that the receptor was already expressed in vivo on the cells of these patients.

Monoclonal antibodies to Toxoplasma cell membrane surface antigens protect mice from toxoplasmosis.

Groups of mice were given an intraperitoneal injection of one of six monoclonal antibodies to Toxoplasma gondii, a mixture of equal amounts of five monoclonal antibodies to T. gondii, or the murine myeloma protein MOPC 21, and challenged with either a highly virulent or moderately virulent parasite strain. Two monoclonal antibodies (FMC 19 and FMC 22) conferred total protection against the moderately virulent challenge, with all mice surviving, whereas 90% of control mice died. FMC 19 and FMC 22 also conferred significant protection against the highly virulent challenge as indicated by a prolonged mean time to death (MTD) of immunized compared with control groups of mice. One monoclonal antibody (FMC 23) and the mixture of five antibodies gave significant protection against the moderately virulent challenge only. Passive immunization with dilutions of FMC 22 antibody indicated that the lowest serum titer needed to confer significant protection to mice against a moderately virulent Toxoplasma challenge was 1/640. Mice challenged with highly virulent tachyzoites that had been preincubated with FMC 22 had a significantly longer MTD than mice challenged with highly virulent tachyzoites that had been preincubated with MOPC 21 or phosphate buffered saline, pH 7.2 (PBS). Immunoprecipitation and autoradiography of radiolabeled tachyzoites confirmed that FMC 19 was directed against a 35,000 molecular weight (mol. wt.) antigen and FMC 22 was directed against a 14,000 mol. wt. fraction. The potential for use of single antigens as protective immunogens in preventing toxoplasmosis is raised.

Ultrastructural and biochemical studies on the immunohistochemistry of Toxoplasma gondii antigens using monoclonal antibodies.

To determine the cellular distribution of Toxoplasma antigens, RH strain tachyzoites were incubated with either one of three monoclonal antibodies (FMC 19, FMC 20, FMC 22) to T. gondii, or one of two controls (the murine myeloma protein MOPC 21, or phosphate buffered saline), and then incubated with peroxidase-labelled goat-antimouse IgG. Diaminobenzidine was added as substrate and electron microscopy was used to localize the reaction. All three antibodies bound to the entire periphery of the tachyzoite surface membrane. To ascertain the chemical composition of the antigens against which seven monoclonal antibodies (FMC 18, FMC 19, FMC 20, FMC 22, FMC 23, 2G11, 3E6) to T. gondii reacted, untreated, pronase-treated, or periodate-treated tachyzoites were incubated with the antibodies or MOPC 21, and then with [125I]-Protein A. The pronase-treated tachyzoites showed reduced binding for six of the antibodies, compared with the reduction in binding of MOPC 21 with the pronase-treated parasites. The periodate-treated tachyzoites had reduced binding for FMC 18 only. The results of these experiments confirm that most Toxoplasma surface antigens are protein in nature, and are consistent with the hypothesis that at least one cytoplasmic antigen is secreted onto the parasite cell surface.

Isolation of plasma membrane from protoplasts of Lolium multiflorum (ryegrass) endosperm cells.

Plasma membranes have been isolated from protoplasts of suspension-cultured ryegrass (Lolium multiflorum) endosperm cells. The protoplast membrane is coated before cell disruption with murine myeloma protein J539, a galactose-binding immunoglobulin A. The plasma membrane is labelled with 125I by using chemically or enzymically catalysed iodination techniques, or, more conveniently, by using 125I-labelled myeloma protein J539, which enables the membrane to be simultaneously coated and labelled. Protoplast lysis is effected by gentle mechanical means after swelling in hypo-osmotic medium. The plasma-membrane fraction is recovered at low centrifugal forces by fractionation of cell lysates on a discontinuous sucrose/sorbitol gradient. The plasma-membrane fraction is enriched 96-fold on a protein basis with respect to the specific radioactivity of 125I-labeled myeloma protein J539 in the homogenate. Electron microscopy showed long membrane profiles often associated with one another.

Cross-reactivity between C-reactive protein and idiotypic determinants on A phosphocholine-binding murine myeloma protein.

Binding of human 125I-C-reactive protein (CRP) to sheep erythrocytes sensitized with pneumococcal C polysaccharide (E-PnC) was found to be Ca++ dependent and inhibitable by phosphocholine, CRP, and HOPC 8. Binding of 125I-HOPC 8 to EPnC was Ca++ -independent but could also be inhibited by phosphocholine, CRP, and HOPC 8. Thus, CRP and HOPC 8, despite a differential Ca++ requirement, share a common binding specificity for phosphocholine. A monoclonal anti-idiotypic antibody (MAB), GB4-10, prepared in A/J mice immunized with BALB/c HOPC 8 inhibited the binding of both 125I-CRP and 125I-HOPC 8 to E-PnC. In addition, both proteins bound to GB4-10 immobilized on polysterene tubes. Interestingly, binding of 125I-CRP to GB4-10 required Ca++. Similar results were also obtained with another MAB (AB1-2) prepared similarly to GB4-10, whereas neither protein bound to a control MAB (EB3-7) against an alpha1 leads to 3 dextran-binding myeloma protein, J558. Binding of 125I-HOPC 8 to GB4-10 could be inhibited by HOPC 8, keyhole limpet hemocyanin-phosphocholine but not phosphocholine but not phosphocholine, and in the presence of Ca++ by CRP. These data indicate that CRP bears antigenic determinants cross-reacting with certain idiotypic determinants on HOPC 8. They also suggest that Ca++ acts as an allosteric effector, perhaps stabilizing the phosphocholine-binding site of CRP.

Immunochemical specificity of the combining site of murine myeloma protein CAL20 TEPC1035 reactive with dextrans.

The immunochemical specificity of the combining sites of murine myeloma protein CAL20 TEPC1035 was studied by quantitative precipitin and precipitin inhibition assays. Myeloma protein CAL20 TEPC1035 precipitated with only three dextrans, B1355S4, B1498S, and B1501S, with high proportions of alpha(1 leads to 3) linkages, but not with any other dextrans, glycogen, and pullulan. Inhibition tests with various sugars show that the combining site of myeloma protein CAL20 TEPC1035 is most complementary to panose, a trisaccharide DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 4)DGlc. Panose was 3.3 times more potent than a tetrasaccharide DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 4)DGlc alpha(1 leads to 4)DGlc and 8, 23, 42, > 42 times more active than maltose, nigerose, isomaltose, and kojibiose, respectively. These findings were paralleled by their binding properties as determined by affinity electrophoresis. The association constants (Ka) of these three dextrans to myeloma protein CAL20 TEPC1035 ranged from 3.8 X 10(3) ml/g to 5.02 X 10(3) ml/g. The association constant of inhibitor (Kia) of panose was 8.19 X 10(3) M-1. Myeloma protein CAL20 TEPC1035 is an antidextran with specificity different from those of other murine myeloma antidextrans and from human antidextrans reported previously and its combining site size is at least as large as a trisaccharide. The binding constant of methyl alpha-D-glucoside (7.2 X 10(2)) was 73% of that of panose and comparable to that of myeloma protein W3129 (9.4 X 10(2)) with a cavity-type site and 600 times lower (1.6 X 10(0)) for QUPC52 with a groove type site, indicating that the terminal nonreducing residue is held in a cavity. Inhibition data with various alpha(1 leads to 4)-linked oligosaccharides also indicate that the internal portions of these inhibitors may react directly with a portion of the combining site. These findings suggest that myeloma antidextran CAL20 TEPC1035 has a partial cavity-type combining site in which the terminal nonreducing dGlc alpha(1 leads to 6) moiety is held in a cavity with the other two sugars forming a groove. However, oligosaccharides with one or more alternating [leads to 3DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 3)DGlcl leads to] units with and without terminal nonreducing DGlc alpha(1 leads to 6) or DGLc alpha(1 leads to 3) side chains remain to be tested to determine whether structures known to be present in the three dextrans which precipitate CAL20 TEPC1035 may not prove to be more active than panose.

Amino acid sequence of the light chain variable region of M511, a phosphorylcholine-binding murine myeloma protein

The complete amino acid sequence of the kappa chain variable region from M511, a phosphorylcholine-binding myeloma protein of BALB/c origin, has been determined. This sequence is compared with another kappa chain from myeloma protein, M 167, which belongs to the same subgroup (VK-24) and also binds both phosphorylcholine and choline. M511 differs from M167 at six positions in the light chain variable domain, all of which are located in framework regions. These substitutions, which require only a single nucleotide change, are distinct from those in mouse lambda chains in which amino acid changes occur only in complementarity-determining regions. These framework differences between M511 and M167 are of a conservative nature regarding their effect on secondary structure and thus on the structure of the binding cavity. These findings add additional features to the problem of antibody diversity but do not in themselves settle the issue of somatic versus germ-line theories.

Binding of murine myeloma proteins of different Ig classes and subclasses to Fc-reactive surface structures in gram-positive cocci.

Twelve different murine myeloma proteins were tested for binding to seventy Gram-positive strains belonging to group A, C and G streptococci and to Staphylococcus aureus. Group A streptococci, known to bind human IgG, were incapable of binding any of eight murine IgG immunoglobulins tested except for one strain that bound an IgG2b myeloma protein. In contrast, group C and G streptococci interacted with murine immunoglobulins of subclasses IgG2a, IgG2b and IgG3, and G strains also to a lesser extent with IgG1. Bovine and equine-group C streptococci did not differ from human group C streptococci in their IgG reactivity. Staphylococcal strains showed a high reactivity with murine myeloma components of IgG subclasses 2a, 2b and 3 and a low but definite binding of an IgG1 myeloma protein. IgA myeloma protein S-122 interacted with nine of fifteen group A streptococci. This binding could not be inhibited by human IgG and the reactivity is thus different from Fc-mediated binding of immunoglobulins. One of three IgA myeloma proteins tested, TEPC 15, bound to staphylococci. The Fc specificity of this interaction was confirmed by chromatography on protein A-Sepharose and by inhibition studies using polyclonal human IgG. The protein A reactivity of this monoclonal protein was detected in IgA aggregates and absent in the monomeric form of IgA.

M467: A Murine IgA Myeloma Protein that Binds A Bacterial Protein

Abstract We have studied the binding of M467, an IgA murine myeloma protein, to flagellin from seven species of Salmonella. It was found that M467 was reacting with antigenic determinants that were common to all the flagellins studied. These determinants were not related to serotypic antigens. Electronmicrographs of unreduced M467 showed a variety of polymeric species bound to flagella in a manner than could produce immobilization as well as agglutination and precipitation through cross-linking of antigenic determinants. Immunodiffusion in agar gel revealed that M467 was recognizing more than one group of peptide determinants on the flagellins studied. Passive hemagglutination inhibition and a solid phase radioimmunoassay provided evidence that there were differences in binding avidities between M467 and the various Salmonella flagellins studied. It was concluded that M467 is binding more than one specific group of antigenic peptide determinants on flagellin molecules. Flagellin from four of the seven species of Salmonella studied were deficient in one or more of these determinants.