Chapter

Combining Regions of Antibodies

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Abstract

When a mammalian organism is immunized with an antigenic determinant, a highly complex humoral immune response is induced. This consists of a large number of antibodies complementary to the antigenic determinant. Such antibodies may vary in class and subclass. Furthermore, within a single subclass, there are differences in the energy of interaction with which each antibody of the population binds the antigenic determinant. These interactions may be viewed at a single point in time, although the antibodies expressed at any one time in the immune response may be only a fraction of the number that the animal is capable of producing. Certainly, in viewing these antibodies over a period of time, there are shifts in the populations produced after the initial immunization.

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... The polyspecificity can be explained by the fact that the paratope consists of ϳ20 amino acids, but only a few of these participate in antigen binding, and water molecules can fill the remaining space between the paratope and epitope. Thus, the same paratope can contain several subsites for different epitopes, and amino acids in the paratope can form strong bonds with peptides unrelated to the immunogen (37,39 ). The polyspecific nature of paratopes toward unrelated peptides has also been demonstrated by Keitel et al. (40 ) and Kramer et al. (41 ), who used x-ray structural data of three peptides complexed with an anti-p24 (HIV-I) MAb. ...
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... Whereas antigen binding does not seem to depend functionally on the Fc fragment, the latter reveals activity which in physiological conditions is triggered by attachment of the Fab fragment to the antigen. Some transfer of information must be postulated to explain such interdependence (1)(2)(3)(4). The structural character of this signaling has been accepted in studies since the very JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2004, 55, 3, 487501 www.jpp.krakow.pl ...
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Excerpt The activity of an antibody, like that of an enzyme, appears to be determined by its primary structure (Haber, 1964; Whitney and Tanford, 1965). Amino acid sequences are thus of considerable importance for an understanding of an antibody's specific activity, as well as for evaluating the genetic basis for the great diversity of antibody molecules formed by an individual organism. Can sufficient amounts of pure (i.e., homogeneous) antibodies be obtained for establishment of amino acid sequences in the variable regions of their polypeptide chains—regions of paramount interest for studies of specificity and evaluation of genetic mechanisms? In seeking an answer we have been examining antibodies to the 2,4-dinitrophenyl group. Large quantities of these antibodies, of the γG immunoglobulin class, are readily isolable, but practically all of the many samples studied over the past 7 years have been heterogeneous with respect to a number of properties: e.g., affinity for DNP ligands...
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13 leven-binding myeloma proteins (LBMP) of BALB/c origin were classified into two groups with different binding specificities; one group of 11 proteins bound beta2 leads to 1 fructosans, a second group of two proteins bound fructosans probably of beta2 leads to 6 linkage. Anti-idiotypic sera prepared to 10 of the proteins in the appropriate strains of mice identified numerous idiotypic determinants. Each protein used for immunization had its own unique individual idiotypic specificities (IdI) and in addition most of the proteins carried two-nine cross-specific or shared idiotypes (IdX) that were found only among LBMP, and not found in 106 non-LBMP. Most of the IdX determinants and only four of the IdI determinants of the beta2 leads to 1 fructosan binding group were located in the antigen-binding site. The multiplicity of antigenic differences in this functionally related group of immunoglobulins reveals an unexpected degree of heterogeneity in V-regions that appears to be unrelated to binding.
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Excerpt It is now generally accepted that the specificity of antibody is consequent to its amino acid sequence and mediated through the effects of sequence on the conformation of the combining site. Evidence for this has been obtained from reversible denaturation experiments on specific antibodies (Haber, 1964; Whitney and Tanford, 1965; Freedman and Sela, 1966) and has been inferred from comparisons of amino acid compositions of purified antibodies (Koshland, 1966), as well as from sequence studies on myeloma proteins (Hilschman and Craig, 1965; Titani et al., 1966; Milstein, 1966). Myeloma proteins have provided an excellent model for the study of antibody structure. These studies have revealed the regions of sequence common to all globulins of a given class as well as regions which are unique to each member. In this way they have pointed to the portions of the molecule which are likely to be responsible for forming the antibody combining...
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The technique of negative staining with phosphotungstate has been used to demonstrate the attachment of rabbit antibody to influenza virus. Depending on the concentration of the reactants, three basically different types of combination can be observed. When high concentrations of antibody react with the virus, the antibody molecules attach through one of their active sites and radiate out from the surface of the particle. When the antibody concentration is relatively low, each molecule attaches to the same virus particle through sites situated at the ends of the molecule; at an intermediate concentration of antibody, aggregation of the virus particles is brought about by antibody molecules forming bridges between adjacent virus particles. The implications of these findings have been discussed.
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1Analysis of the electron spin resonance spectra of different spin-labelled haptens when bound to the Fv fragment from the immunoglobulin A of the mouse myeloma protein MOPC 315 suggests that the combining site is a cleft of overall dimension 11 Å× 9 Å× 6 Å which has considerable structural rigidity.2The 270 MHz proton nuclear magnetic resonance spectrum of the amino acid residues in and around the combining site is obtained by use of paramagnetic difference spectroscopy involving a spin-labelled hapten. There are only the equivalent of about 30 aliphatic and 30 aromatic protons in this difference spectrum.3The C-2 and C-4 proton resonances of three histidine residues in the Fv fragment are observed to titrate with pH. The pKa values of these histidine residues are about 8.1, 6.9 and 6.1. The resonances of the histidine residue with pKa value 8.1 show anomalous behaviour in splitting into two or more components. The values of the chemical shifts of the C-2 and C-4 protons alter slightly in the presence of hapten, particularly for the histidine residues with pKa values of 6.9 and 6.1. The resonances of these two residues are not observable in the presence of the spin-labelled hapten indicating that these two histidines are in the region of the combining site.4The existence of lanthanide binding sites of the Fv fragment, an essential prerequisite in mapping studies, has been demonstrated by measurements of the solvent water relaxation rates in Gd3+ solutions.
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The structure of the Fab' fragment of a human myeloma immunoglobulin was determined by x-ray crystallographic analysis at 2.8- angstrom resolution. The Fourier map of the electron density was correlated with the aminoacid sequence to obtain a three-dimensional model. Four globular subunits, which correspond to the homology regions of the light and heavy chains, are arranged in a tetrahedral configuration. These subunits closely resemble each other, sharing a basic pattern of polypeptide chain folding. In each subunit, long sequences of tightly packed, hydrogen bonded polypeptide chain run parallel to the major axis of the subunit. No helical conformation can be seen. Different patterns of interchain disulfide linkage and unusual intrachain disulfide bonds that have been observed in other immunoglobulins can be explained with this model. The regions of hypervariable sequences in the light and heavy chains occur at one end of the molecule, in close spatial proximity.
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The use of cross-reacting antigens to stimulate antibody responses of limited heterogeneity is described. The cross-reacting haptens 2,4dinitrophenyl (DNP) and 2,4,6trinitrophenyl (TNP), coupled to ovalbumin or bovine IgG, were employed as immunogens in inbred CBA/J and C3H/He mice. Sera from mice primed with DNP-protein and subsequently boosted with TNP coupled to the same carrier were examined by isoelectric focusing techniques. The anti-DNP antibodies in these sera were shown to consist of the products of a small number (usually less than 4) of clones. The frequency of occurrence of anti-DNP antibodies that were indistinguishable by isoelectric focusing techniques was estimated. In 2400 comparisons between antibodies in sera from 140 individual mice, a small number (5 to 7) of indistinguishable antibody pairs was found. It was estimated from these results that not less than about 500 different sorts of antibodies to DNP, strongly cross-reacting with TNP, can be made by inbred mice immunized with these cross-reacting antigens. The problem of natural selection for the ability to produce such a large variety of antibodies is discussed.
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Antisera were prepared in rabbits against anti-p-azobenzoate antibodies of an A/J and a BALB/c mouse and anti-p-azophenylarsonate antibodies of an A/J mouse. After appropriate absorption the antisera reacted with the anti-hapten antibody of the donor mouse but, by sensitive quantitative tests, not at all with other components of the hyperimmune serum or with preimmune serum of the donor mouse. The absorbed antiserum therefore appeared to be specific for idiotypic determinants. Nearly all idiotypic specificities identified in the serum of the donor were also present in the serum of other mice of the same strain, immunized against the same hapten group, but not in mice immunized with a different hapten. In each case the antibodies of the donor mouse reacted most effectively on a weight basis with antiidiotypic antiserum. Cross-reactions were observed among different strains of mice but homologous anti-bodies reacted most effectively with antiidiotypic antisera. C57/BL and DBA antisera contained very low concentrations of specificities present in the A/J and BALB/c antibody populations; antibodies of A/J and BALB/c antisera are more closely related to one another. The results indicate that idiotypic specificity may provide a genetic marker for the variable regions of immunoglobulin polypeptide chains.
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BALB/c mice were immunized with three A-myeloma proteins of BALB/c-2 or BALB/c origin (produced by plasmacytomas MOPC-315, MOPC-460, Adj. PC-22A). Noncross reacting antibodies were formed against Proteins-315 and 460, but the response to Protein-22A was marginal. Proteins-315 and 460 have anti-dinitrophenyl activity, and their reactions with the corresponding BALB/c antibodies were inhibited by dinitrophenyl ligands. It appears that antibodies can be formed in BALB/c mice against unique "idiotypic" determinants in the ligand-binding sites of some BALB/c mycloma proteins.
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Purified preparations of diphtheria antitoxin have been obtained by digestion of the toxin-antitoxin complex with trypsin, followed by fractional precipitation with ammonium sulfate. The various fractions obtained in this way are all 90 per cent or more precipitated by diphtheria toxin but combine with different quantities of the toxin. The fraction precipitated between 0.33 and 0.5 saturated ammonium sulfate is homogeneous by electrophoresis and ultracentrifuge but does not have constant solubility. A small amount of a more soluble fraction has been obtained which does have constant solubility and satisfies the criteria of a pure protein. This protein crystallizes readily in poorly formed thin plates. It is very unstable and reverts to a less soluble non-crystallizable form. It has a sedimentation constant of 5.7 x 10(-13) and a molecular weight of 90,500. It has an antitoxic value of 700-900 flocculation units per mg. protein nitrogen and has an antitoxic value by the protection test of about 700 units per mg. protein nitrogen. The precipitation range of the purified antitoxin with purified toxin is much wider than that with crude preparations.
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Immune complexes formed between a homogeneous rabbit antibody to type III pneumococcal polysaccharide and a series of oligosaccharides of varying size derived from it were prepared and tested for their ability to fix guinea pig hemolytic complement. Antibody and either tetra-, hexa-, or octasaccharide formed only monomeric antibody-hapten complexes and did not show any complement binding. A dodecasaccharide and a 16-sugar residues oligomer formed dimer and trimer immune complexes. These complexes were also unable to fix complement. However, as the size of the sugar oligomers was increased to about 21 sugar residues per oligosaccharide molecule or more, the resulting complexes exhibited substantial complement binding, concomitant with the formation of antigen-antibody aggregates higher than trimers. On the other hand, an independent study carried out with the same material suggested changes in the conformation of the Fc moiety in the antibody molecule upon addition of oligosaccharide ligands as small as a 16-residue unit. Since the resulting complexes hardly ehibited any complement binding, ligand-induced conformational changes in the Fc part of the antibody molecule appears to be an insufficient condition per se for triggering complement fixation.
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The binding of gadolinium [Gd(III)] to a homogeneous rabbit anti-(type-III pneumococcal polysaccharide) IgG (immunoglobulin G) and its Fab (N-terminal half of heavy and light chain) and Fc (C-terminal half of heavy-chain dimer) fragments was demonstrated by measurements of solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 the binding of Gd(III) to the Fc fragment is much tighter (KD approx. 5 micronM) than binding to the Fab fragment (KD approx. 250 micronM). The binding of Gd(III) to the whole IgG molecule (KD approx. 4 micronM) is very similar to that for the Fc fragment alone. This specificity of binding to the Fc region allows the use of Gd(III) as a probe of the Fc conformation. The environment of the Gd(III) in the Fc region of whole IgG is not affected by the presence of octasaccharide derived by hydrolysis of type-III pneumococcal polysaccharide, but the corresponding 28-unit saccharide does cause detectable changes. The addition of 16-unit saccharide to anti-(SIII polysaccharide) IgG in the presence of Gd(III) does not change the solvent water proton relaxation rate, although aggregation does occur. The effects of the 28-unit saccharide may be explained therefore by a change in the tumbling time of the IgG. From a study of the effect of various antigen/antibody ratios, it is concluded that the 28-unit-saccharide-induced changes in the Gd(III) environment in the Fc region are caused by the specific geometrical structure of the antigen-antibody complexes formed, and not simply by occupancy of the combining sites on the antibody.
Article
The preparation and specificity of antibodies specific for the ligand-binding site of HOPC 8, a phosphorylcholine (PC)-binding mouse myeloma protein, are described. Antiserum to HOPC 8, prepared in rabbits, was adsorbed with an HOPC 8-Sepharose immunoadsorbent and anti-binding site antibodies were eluted with PC. These antibodies reacted with HOPC 8 but not other myeloma proteins, including those with PC-binding specificity different from HOPC 8; the specificity of this anti-HOPC 8 antibody for the combining site region of HOPC 8 was shown by the fact that 1) the interaction of the anti-HOPC 8 antibody preparation with HOPC 8 was completely blocked by PC and 2) the antibody preparation failed to bind TEPC 15 in which the combining sites had been blocked by covalently bound PC groups. Moreover, these anti-binding site antibodies did not react with isolated heavy or light chains, indicating the requirement for a heavy-light chain interaction. By contrast an idiotypic antiserum to HOPC 8 prepared in A/J mice did bind affinity-labeled TEPC 15 and the reaction with HOPC 8 was only marginally hapten inhibitable. Both of the idiotypic determinants detected by these two antisera were present on anti-PC antibody raised in BALB/c mice;
Article
This chapter discusses the structure and role of the variable regions of the immunoglobulins (Ig) molecules. Based on amino acid sequence data, hypervariable regions make up a significant part of the variable regions, occupy relatively constant locations in a variety of Ig molecules even from different species, and appear to be intimately associated with the antibody-combining site. In addition, the idiotypic determinants that mark the antigenic individuality of particular Ig molecules are based on the properties of some or all of the hypervariable regions. In contrast, outside the hypervariable regions there is a considerable invariance of sequence, and these invariant sections occur in approximately the same position in Igs of different V region subgroups. The polymeric Ig molecules are intriguing, because one of them, IgM, is structurally the most complex, phylogenetically the most primitive, and ontogenetically the earliest of the Ig molecules. The other polymeric Ig molecule, IgA, evolved relatively recently but retained that portion of the IgM Fc sequences responsible for polymerization and, in addition, developed a mechanism for transport across epithelial cells. The most detailed view of the molecular orientation of the immunoglobulin V region, which is consistent with the known serological and immunochemical data is obtained by X-ray diffraction analyses.
Article
The change in avidity of anti-hapten antibody with time after immunization was studied in mice at the level of the antibody-forming cell. A progressive increase in avidity was seen in both direct and indirect plaque-forming cells. Late (38 days) after immunization with a large dose of antigen there was a preferential loss of high avidity plaque-forming cells and the average avidity decreased. High avidity memory cells were still present since boosting resulted in the prompt appearance of very high avidity plaque-forming cells. There was a strong positive correlation between the avidity of the direct and the indirect plaque-forming cells present in the same spleen. A pattern of change in avidity and heterogeneity of avidity similar to that observed with intact animals was seen in lethally irradiated mice reconstituted with normal spleen and thymus cells.
Article
Immunization of 3 rabbits with (AMP)2-gramicidin S has led to the production of anti-AMP antibodies of the IgG type. Isoelectric focusing of these antibodies and of their light and heavy chains demonstrated that the antibodies were of restricted heterogeneity. In one rabbit, three dominant bands, presumably representing the product of one clone of antibody-producing cells, appeared late after the first boost and continually reappeared after each successive boost. However, the banding patterns seen for the antibodies from the other two rabbits changed after each boost. The anti-AMP antibodies were found to react with DNA and with RNA. Although highly specific for AMP (Ka > 106M−1), they were able to bind several structurally diverse ligands, thus providing evidence that antibody combining sites are multispecific. The order of binding was as follows: AMP > GMP > DNP-gly > hydralazine=menadione > DNP-lys > caffeine > sodium barbital > procaineamide HCl > CMP > TMP > UMP. Binding constants for the first five were > 104M−1, comparable to that reported for the binding of similar ligands by myeloma globulins. The cross reaction with hydralazine might be relevant to the drug's ability to elicit nucleic acid-reactive antibodies in certain individuals. Rabbits immunized with (AMP)2-gramicidin S displayed a delayed hypersensitivity specific for A, indicating a hapten-specific T-cell interaction as well as the B-cell interaction that produced the humoral responses. A delayed skin reaction was also produced by gramicidin S.
Article
Serological test systems were established for determining the heavy-chain variable region (Vh) subgroups of immunoglobulin heavy chains. Myeloma proteins with known Vh subgroups based on amino acid sequence were utilized as the primary basis of reference for analysis by hemagglutination and hemagglutination inhibition. Good agreement between the chemical and serological typing was obtained and nonoverlapping systems established for the three major Vh subgroups. In a survey of 167 myeloma proteins, all except two were exclusively positive in one of the three systems. The two exceptions may represent a fourth subgroup. There was an overall incidence ratio of 1:2:3 for VhI:VhII:VhIII subgroups. Some differences in the overall ratios were encountered within the immunoglobulin classes. Certain advantages of the serological typing antisera were discussed with special emphasis on their use for studies of Vh antigens on the membranes of lymphocytes.
Article
The Fv fragment (Hochman, J., Inbar, D., and Givol, D. ( 1973), Biochemistry 12, 1130) derived from mouse myeloma protein 315, possessing anti-dinitrophenyl (Dnp) activity, is composed of the variable portions of light chain ( VL) and heavy chain (VH) of the intact immunoglobulin. After dissociation of Fv in 8 M urea, VL and VH were isolated and analyzed for their hapten binding properties. VH was found to be an aggregate without anti-Dnp binding activity, whereas VL was a dimer with a molecular weight of 24 000 and possessed two binding sites for Nε-2,4-dinitrophenyllysine with an association constant of 2.3 × 103 M-1. The binding properties of VL dimer were found to be identical with those of the light chain dimer of protein 315 previously reported (Schechter, I., Ziv, E., and Licht, A. (1976), Biochemistry 15, 2785) and exclude the constant part of light chain from participating in the combining site of L dimer. The fine specificity of the anti-Dnp binding site of VL dimer ( VL- VL) or L dimer (L-L) was compared with that of Fv (VL-VH), or of the intact protein (L-H) by analyzing the binding of a homologous series of Dnp ligands. The affinity of protein 315 for Dnp-lysine is approximately 1000-fold greater than that of VL dimer, whereas for DnpOH the affinity of the intact protein is only 6.5-fold greater than that of VL dimer. Thus the subsite for binding the Dnp ring per se can be localized within VL. On the other hand, the interaction with the side chain of Dnp ligands is negligible in VL dimer or L dimer, whereas it is very pronounced in the intact protein, suggesting that VH contributes most of these interactions. The binding of "strange" cross-reacting haptens (Michaelides and Eisen, 1974) like dinitronaphthol and menadione was also localized within VL dimer. Moreover the affinity of VL dimer toward Dnp-lysine, dinitronaphthol, and menadione is similar, whereas the intact protein, or Fv, binds Dnp-lysine much better than they bind dinitronaphthol and menadione. Upon binding of dinitronaphthol or Dnp-caproate to VL dimer, a red shift is observed in their absorbance spectrum. This red shift is similar, although not identical, to that observed with Fv and indicates that in either VL dimer or Fv the ligands interact with a tryptophan residue. VL dimer has only two tryptophan residues one of which (Trp-35L) is a constant residue present in the domain interior, whereas the other tryptophan is localized in the third hypervariable region of VL (Trp-93L) and must therefore be the one interacting with the aromatic haptens. This assignment is in agreement with a model building study of protein 315 combining site (Padlan et al., 1977). The problems of antibody multispecificity and of subsites localized on different chains are discussed.
Article
ONE of the salient questions in immunology concerns the means by which the immune system is capable of recognising an apparently limitless variety of antigenic determinants, including many that presumably did not exist during its evolution. Three major hypotheses have been put forward to explain the diversity of the immune response: a stringent germ line hypothesis, a recombinational germ line hypothesis and a somatic mutation hypothesis (see ref. 1 for discussion). If the assumption is made that an antibody-combining site has a single antigenic specificity, the first of the above hypotheses postulates about 1,000 variable genes and 1,000 constant genes to code for about 106 antigenic determinants. The second and, especially, the third hypotheses require the presence of many fewer genes in the germ line. Even fewer genes would be required if antibody-combining sites were multispecific. Evidence has been put forward to support the concept of multispecificity2. Moreover, the ability of single myeloma globulins to bind structurally diverse molecules3 and the multispecific character of a Bence-Jones4 dimer can be taken as additional supportive evidence. In an earlier paper5, we attempted to investigate the question of multispecificity by studying the binding specificities of a population of antibodies of limited heterogeneity generated by immunisation with a conjugate of adenosine-5'-phosphate and gramicidin S: (AMP)2-gramicidin S (ref. 6). Immunised rabbits produced predominantly three or four globulin proteins specific for AMP. Binding studies on the sera of one of the rabbits, in which there were three major AMP-specific globulins, yielded data that could be interpreted as evidence for multispecificity of antibody-combining sites. We have now succeeded in isolating three homogeneous anti-AMP antibodies from one of the rabbits immunised with (AMP)2-gramicidin S and have studied the ability of each globulin to bind AMP and other ligands. The isoelectric pattern of the specifically purified7 population of antibodies obtained from the serum of rabbit 449 (ref. 5) showed three major bands. The three bands were separated and isolated by preparative isoelectric focusing in polyacrylamide by an adaption of the procedure of Wrigley8, and were shown to be homogeneous by analytical isoelectric focusing. They were designated fractions A, B and C.
Article
The structure of the combining site of the DNP binding IgA mouse myeloma protein MOPC 315 has been determined by a combination of high resolution nuclear magnetic resonance, electron spin resonance, model building and chemical modifications. This approach yields that general dimensions of the site, its polarity and asymmetry features, the assignment of the DNP-contact residues and their three-dimensional coordinates.
Article
The binding of Gd(III) to rabbit IgG (immunoglobulin G) and the Fab (N-terminal half of heavy and light chain), (Bab')2 (N-terminal half of heavy and light chains joined by inter-chain disulphide bond), Fc (C-terminal half of heavy-chain dimer)and pFc' (C-terminal quarter of heavy-chain dimer) fragments was demonstrated by measurements of the enhancement of the solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 there are six specific Gd(III)-binding sites on the IgG. These six sites can be divided into two classes; two very 'tight' sites on the Fc fragment (Kd approx. 5 muM) and two weaker sites on each Fab region (Kd approx. 140 muM). Ca(II) does not apparently compete for these metal-binding sites. The metal-binding parameters for IgG can be explained as the sum of the metal binding to the isolated Fab and Fc fragments, suggesting that there is no apparent interaction between the Fab and Fc regions in the IgG molecule. The binding of Gd(III) to Fab and Fc fragments was also monitored by measuring changes in the electron-spin-resonance spectrum of Gd(III) in the presence of each fragment and also by monitoring the effects of Gd(III) on the protein fluorescence at 340 nm (excitation 295 nm). The fluorescence of Tb(III) solutions of 545 nm (excitation 295 nm) is enhanced slightly on addition of Fab or Fc.
Article
Protein 315 from the mouse plasmacytoma, MOPC-316, has been shown by Haimovich et al. (1970) to be affinity labeled by (BADL). Lysine 52 of the heavy chain was shown to be the residue involved in covalent bond formation with this label. On the basis of this reaction, Haimovich et al. (1972) designated the peptide containing this residue as a “site peptide” and assigned this lysine to the antigen-binding region of the protein.In this communication we report studies on the chemical modification of lysyl residues of protein 315 with maleic anhydride in the presence and absence of the protecting hapten, ϵ-N-dinitrophenyl-aminocaproate, which lead us to conclude that there does not appear to be a protectable lysine in the combining site of this protein. Thus maleylated preparations of protein 315, having essentially every lysyl residue modified, still contain combining sites able to bind hapten with a binding constant identical to that of the unmodified protein. There is essentially no difference between the number of sites remaining in preparations maleylated either in the presence or absence of protecting hapten. In addition, preparations of MOPC-315 protein, maleylated either protected or unprotected, while capbale of binding hapten, do n ot readily incorporate the affinity label BADL, both in terms of rate and extent of incorporation, indicating that lysine 52 of the heavy chain was indeed maleylated.These data indicate that lysine 52, which is involved in affinity labeling with BADL, must be peripheral to the site, since its maleylation does not affect the ability of the site to bind ligand. Further, it cannot be protected against maleytation by ligand.
Article
The entire variable-region sequence of the heavy chain from ABE-47N, a BALB/c inulin-binding myeloma protein, has been determined. This protein is unusual in that the third complementarity region (H3) is extremely short, consisting of at the most three and probably only one amino acid. A comparison of the heavy-chain hypervariable regions from mouse, human, and rabbit proteins shows that the variability in length of H3 is greater than that seen in the first or second hypervariable regions. This variability in H3 length suggests a specialized function for this region.
Article
Lymphocytes from 20 patients with chronic lymphocytic leukemia (CLL) were studied for membrane staining by direct immunofluorescence by employing anti-F(ab')2, anti-VHI, anti-VHII, anti-VHIII subgroup-specific antisera, as well as light chain-specific antisera. Some lymphocyte preparations were also studied in indirect immunofluorescence with an antiserum raised against a fragment (VH) corresponding to the variable region of the heavy chain of a human IgG3 myeloma protein (Kup). Lymphocytes from each CLL patient demonstrated a restriction of VH subgroups expressed on the cell membrane; six were restricted to the VHI subgroup, seven to VHII, and seven to the VHIII subgroup. This restriction gave further evidence for monoclonality of the membrane-bound Ig and the leukemic cell proliferation. Antiserum to the VH fragment stained closely similar percentages of CLL lymphocytes to that obtained with anti-F(ab')2 antiserum. Furthermore, double staining revealed that the same cells were stained with anti-VH antiserum as were stained with anti-F(ab')2 antiserum, i.e., only the B lymphocytes.
Article
The binding site interactions between the phosphorylcholine (phosphocholine)-binding mouse myeloma proteins TEPC 15, W3207, McPC 603, MOPC 167, and MOPC 511 and the isotopically substituted hapten phosphoryl[methyl-13C]choline have been investigated using 13C and 31P nuclear magnetic resonance (NMR) spectroscopy. Each protein exhibits a unique NMR pattern, but extensive similarities in chemical shift parameters upon binding of hapten to immunoglobulin suggest a significant degree of conservation of important hapten-binding site interactions. Moreover, independent binding studies, in conjunction with the NMR data, allow construction of a simple model of the binding sites of these antibodies, analyzed in terms of the relative strength of interaction between hapten and two main subsites. The NMR evidence supports the view that the heavy chains of these proteins dominate in interacting with bound phosphorylcholine; the various subspecificities of these proteins for phosphorylcholine analogues can be accounted for by amino acid changes in the hypervariable regions of the heavy chains.
Article
Excerpt In this paper, we will demonstrate that both the content and the context of immunoglobulin genes change during the differentiation of lymphocytes. By content, we mean the genetic information used for coding immunoglobulin chains. By context, we mean the relative location of this information in DNA. The possibility of such changes has been the subject of much speculation in immunology, but only recently has it become possible to attack the problem directly. The principal technological advance which makes this possible has been the development of procedures for the purification of immunoglobulin mRNA from murine plasmacytomas (Tonegawa and Baldi 1973; Mach et al. 1973; Brownlee et al. 1973; Schechter 1973; Honjo et al. 1974; Stavnezer et al. 1974). Joining of Kappa-chain Genes during Ontogeny Let us begin with changes in context. To study this, we need a way to determine the arrangement of immunoglobulin genes in DNA. Bacterial restriction endonucleases recognize...
Article
A DNA fragment carrying an immunoglobulin gene coding for both variable (V) and constant (C) regions of a mouse lambda light chain was enriched about 15-fold from a total endonuclease EcoRI digest of a plasmacytoma (HOPC 2020) DNA by preparative agarose gel electrophoresis. The DNA fraction was used for cloning of a lambda chain gene in the phage lambdagtWES vector. After screening [Benton, W. D. & Davis, R. W. (1977) Science 196, 180-182] of about 70,000 plaques, each arising from an independent transfection event, we isolated one clone (Ig 303) that contained both a Vlambda and a Clambda DNA sequence. Electron microscopy of R-loops formed between the cloned DNA and purified lambda chain mRNA (HOPC 2020) revealed that the Vlambda and Clambda DNA sequences are separated by a 1250-base DNA fragment.