Chapter

Antibody Combining Regions

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

An immune serum usually consists of a very heterogeneous population of immunoglobulins (Ig’s) the appearance of which in the serum has been induced by antigen. The antigen-ligating function of the Ig molecule (see Figure 1) is confined to the combining regions, which are two symmetrical areas at the solvent-exposed ends of the Fab arms of the Y-shaped Ig molecules. The combining region is situated in the variable (V-region) domain, a compact region consisting of the N-terminal half of the light (L) chain and the N-terminal quarter of the heavy (H) chain that is linked by sulfhydryl bonds. Between the areas of this domain occupied by the L- and H-chain V regions is a cleft exposed to the solvent. Antigens have been shown to bind in, or close to, this cleft (Amzel et al., 1974). An induced antibody population is said to be specific because it usually binds most strongly to the immunizing antigen and with lesser binding energies to certain compounds that resemble the immunogen in structure. Heteroclitic antibodies may be induced that bind more strongly to some determinant other than the immunogen (Mäkelä, 1965). In general, antibody populations show a high degree of specificity, in that they are able to discriminate among chemical compounds differing by as little as a single functional group, between stereoisomers, or between two proteins differing by as little as a single amino acid residue (Reichlin, 1974).

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

Article
Many attemps have been made to predict the position of antigenic sites in proteins from certain features of their primary, secondary or tertiary structures. Recent results indicate that the definition of antigenicity depends on the type of probe used to study it. However, when it is important to predict which short peptides are likely to mimic best certain antigenic sites, it seems that regions of high mobility in the protein are the most likely candidates.
Article
Full-text available
The binding of Gd(III) to rabbit IgG (immunoglobulin G) and the Fab (N-terminal half of heavy and light chain), (Fab')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 μM) and two weaker sites on each Fab region (Kd approx. 140 μM). 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
Full-text available
Rabbit antibody to dinitrophenyl hapten has been elicited by hyperimmunization with a bifunctional cyclic decapeptide antigen, di-dinitrophenyl-gramicidin-S. Examination of the induced antibody by isoelectric focusing, and Sipsian analysis of the haptenbinding data, demonstrated that di-dinitrophenyl-gramicidin-S was capable of stimulating and maintaining the production of a biosynthetically homogeneous antibody in the adult animal.
Article
Full-text available
A mouse myeloma protein with high affinity for 2,4-dinitrophenyl (Dnp) ligands was reacted with the bromoacetyl derivatives of N-Dnp-ethylenediamine and ε -N-Dnp-L-lysine. Up to 1.4 sites per protein molecule were covalently labeled. The labeling reactions were essentially completely blocked by a large excess of Dnp ligands that do not combine covalently (e.g., ε -Dnp-L-lysine). Analyses of the labeled protein revealed that the bromoacetyl derivative of N-Dnp-ethylenediamine reacted exclusively with tyrosyl in the light chain, while the derivative of ε -Dnp-L-lysine reacted exclusively with lysyl in the heavy chain. The findings support the conclusion that both chains are involved in forming specific antibody combining sites.
Article
Full-text available
Crystals of a human γG1 immunoglobulin molecule have been examined in the electron microscope. The molecule appears in projection to have a shape varying between a Y and a T, in agreement with previous electron microscope studies and with the accompanying x-ray diffraction investigation.
Article
Full-text available
Rabbit antiserum was prepared against mouse myeloma Protein-315, an IgA protein with specificiy toward the 2,4-dinitrophenyl group. After absorption of the antiserum with another IgA myeloma protein and with affinity-labeled Protein-315, the antiserum was specific for idiotypic determinants on Protein-315. Monovalent ligands that bind to Protein-315 with high affinity strongly inhibited the reaction of the protein with its anti-idiotypic antiserum. This indicated that the region of the hapten-binding site is a major idiotypic determinant. The myeloma protein is thus similar to rabbit antibenzoate antibody in this respect. These results, considered in conjunction with other data in the literature, indicate that an anti-idiotypic antiserum prepared in an isologous or heterologous species can recognize the same determinant, in this case the region comprising the ligand-binding site. Quantitative aspects of the data indicate that there is competition between the hapten and antiidiotypic antibodies for the site.
Article
Full-text available
Photoaffinity labeling is a recently introduced method for covalently binding chemical tags to the active sites of protein molecules, which is potentially capable of very great specificities of labeling. A labeling reagent is used that is converted by photolysis to an extremely reactive intermediate. According to the expected mechanism, the reagent molecules that are specifically and reversibly bound to the active site at the instant of photolysis react irreversibly in the site before they can dissociate from the site. In two such reagent-protein systems studied in this paper, however, it is shown that, while by the usual criteria photoaffinity labeling appears to have occurred, the expected mechanism in fact does not hold. This was discovered in experiments with scavengers present in the mixtures that were photolyzed. The general properties of, and criteria for, photoaffinity labeling reactions are discussed in the light of these findings.
Article
Full-text available
This paper reports the initial progress in a research programme to identify and obtain the relative orientations, in solution, of the amino acid residues that constitute the combining site of the myeloma protein MOPC 315. This protein has a molecular mass of 150,000, but enzymic digestion yields the Fv fragment of molecular mass 25,000 which still has the combining site intact, as judged by the affinity for dinitrophenyl haptens. Analysis of the e.s.r. spectra of a series of dinitrophenyl spin labelled haptens has allowed the dimensions, rigidity and polarity profile of the combining site to be determined. The combining site is a cleft of overall dimensions 1.1 nm x 0.9 nm x 0.6 nm which has considerable structural rigidity. One of these spin labels has also been used to perturb the n.m.r. spectrum of the Fv and using difference spectroscopy the 270 MHz proton n.m.r. spectrum of the amino acid residues in and around the combining site has been obtained. This spectrum contains only the equivalent of about 30 aromatic and 21 aliphatic protons. Comparison of this difference spectrum with that obtained using a diamagnetic analogue suggests that any conformational changes on hapten binding are mainly localized to the combining site. By the use of (n.m.r.) difference spectroscopy the protons of the three histidine residues in the Fv are observed to titrate with pH and have pKa values of about 8.1, 6.9 and 6.1. The histidine resonances with pKa values 6.9 and 6.1 alter slightly in the presence of haptens and also appear in the spin label difference spectrum, and must therefore be in or near to the combining site. These are assigned to His 102H and His 97L. The existence of lanthanide binding sites on the Fv, necessary for the mapping studies, has been demonstrated by measurements of Gd III water relaxation rates in Fv solutions and also by the changes in the Fv tryptophan fluorescence on addition of Gd III. At pH 5.5 there is one tight binding site for the lanthanides (KD approximately 80 muM) but in the presence of hapten this is weakened 10-20 fold with a reciprocal effect on the hapten binding. Measurements of the Gd III quenching of the e.s.r. spectrum of a spin labelled hapten bound to Fv indicate that the lanthanide site is ca. 1.5 nm from the nitroxide moiety.
Chapter
The specificity and affinity exhibited by the binding sites of antibodies is a result of the interactions of a particular spatial combination of certain residues within the site with the complementary counterpart groups of the hapten. The dynamic equilibrium between hapten and antibody has been shown to involve a single step:
Chapter
In rats injection of alloantibodies (produced in one strain against transplantation antigens of another strain) into F1-hybrids between the two strains will induce the induction of anti-alloantibodies. Radiolabelled anti-alloanti-bodies will bind to normal lymphocytes from rats of the genotype that provided the alloantibody. These lymphocytes are B cells and not T cells, and their idiotypic surface marker has the same antigen-binding specificity as the alloantibody as shown by binding to allogeneic monolayers. However, despite the negative findings in the radioimmunoassay, anti-alloantibody serum can be shown to react with immunocompetent T lymphocytes of the correct parental strain. This was shown both by the capacity of sera to selectively inhibit the binding of T cells to monolayers of allogeneic cells and by the capacity to block the induction of GvH-reactions of parental T cells if inoculated into the F1-hybrids. Adsorption experiments using F1 or parental lymphocytes failed to show that these effects on T cells were caused by antigen-alloantibody complexes but emphasized the antibody nature of these T cell inhibitory factors.
Chapter
The structure of the Fab fragment of McPC 603 protein has been determined by crystallographic analysis to 3.1 å resolution. The site of phosphorylcholine binding was located in a cleft formed by the three heavy chain hypervariable loops and the first and third hypervariable regions of the light chain. There are strong interactions between the phosphate group of the hapten and the side groups of Tyr 33 and Arg 52 both of the heavy chain. Acidic side groups from both light and heavy chains are in the immediate vicinity of the positively charged choline group. The hapten is in van der Waals contact with various portions of the hypervariable loops. Phosphorylcholine occupies only a small portion of the cleft between the variable domains.
Article
Evidence is accumulating that individual antibody species are capable of binding many structurally dissimilar haptens. If individual antibody species arising from an immunization usually do not share their alternate (disparate) specificities, and if cross reactions can be detected only above a reasonable threshold, then it can be shown with a probability model that heterogeneously populated antisera can indeed exhibit a high degree of specificity. Thus, the model substantiates the hypothesis that a limited repertoire of antibody species can, in various portions and combinations, populate specific antisera to an essentially unlimited number of antigens. The model further suggests that the required number of distinct combining regions (VH × VL pairs) may fall in the range of to 106 if nature has followed a conservative course. Specific antibodies from unimmunized animals (“natural” antibodies) can best be explained on the basis of disparate multispecificities. The levels of such antibodies can then be related to the probability model and to estimates of an animal's total antibody combining region repertoire.
Article
This chapter describes the combining sites of anti-2,4-dinitrophenyl (DNP) antibodies. The anti-DNP antibodies formed against dinitrophenylated antigens constitute a highly diversified set of immunoglobulins, composed of various heavy and light chains, diverse allotypes, and combining sites that differ widely in affinities for DNP and for various cross-reacting substances, such as 2,6-DNP, 2,4,6-trinitrophenyl (TNP), and 2- and 4-mononitrophenyl ligands. The anti-TNP antibodies formed against 2,4,6-trinitrophenylated antigens are similarly a highly diversified set of immunoglobulins. Though cross-reactions between DNP and TNP ligands are pronounced, certain characteristics distinguish clearly between populations of anti-DNP molecules and populations of anti-TNP molecules. When the combining sites are saturated by either homologous or by cross-reacting ligands, 70–80% of the tryptophan fluorescence of anti-DNP molecules (rabbit IgG) is quenched. The chapter discusses the results of some studies of the combining sites of two myeloma proteins with considerable affinity for DNP ligands. Because of the homogeneity of the combining sites, the sites of these proteins can be analyzed more thoroughly than those of conventional anti-DNP molecules.
Article
The idiotype present on the Fab of a phosphorylcholine-binding IgA myeloma protein TEPC 15 (T15) of BALB/c origin was found in normal serum of BALB/c mice. Molecules carrying the T15 idiotype in normal serum could be adsorbed with Sepharose phosphorylcholine beads and R36A pneumococci. The T15 idiotype is absent in germ-free BALB/c but appears when the mice are conventionalized. A survey of normal sera of inbred strains for the T15 idiotype showed it to be present in BALB/c, 129, C57L, C58, and ST and absent or in low levels in CBA, C3H, C57BL/6, C57BL/Ka, C57BL/10, SJL, B10.D2, DBA/2, RIII, A, AL, AKR, NZB, and NH inbred strains of mice. The T15 idiotype is associated with some but not all strains carrying the IgCH allotypes found in BALB/c. Linkage of genes controlling the T15 idiotype in normal serum to the IgCH locus of BALB/c was demonstrated in F2 progeny of a BALB/c and C57BL cross, Bailey's recombinant inbred strains, C x BD, C x BE, C x BG, C x BH, C x BI, C x BJ, C x BK, and CB20 congenic strains. Among these strains, only those possessing the IgCH locus of BALB/c including the F2 progeny consisting of BALB/c homozygotes and BALB/c/C57BL heterozygotes and C x BG and C x BJ recombinants showed the T15 idiotype.
Article
When primary immunity is boosted not by the homologous but by a crossreacting vaccine, the newly formed antibodies react better with the primary antigen than with the antigen actually eliciting the response. This phenomenon bears the name of Original Antigenic Sin (1). It is shown that the number of antibody molecules produced against the original and the vaccinating antigen is the same; that each of these molecules is capable of reacting with both antigens; that the activity of an antiserum can be completely absorbed with either antigen; that both residual and adsorbed-dissociated fractions of antibody exhibit the same relative affinities towards the two antigens as did the native serum; that, unlike standard primary and secondary responses, the population of antibody molecules characterizing the Original Antigenic Sin is homogeneous; that each molecule has a lower equilibrium constant (i.e. higher avidity) against the original antigen than against the antigen stimulating the present response; and that all equilibrium constants are typical of secondary antibody. It is concluded that the Original Antigenic Sin is a partial anamnestic response, a related antigen stimulating that sector only of the originally primed cells which is destined to produce cross-reacting antibody. A hypothesis is developed according to which the basic difference between primary and secondary reactivity rests on the presence of a trapping mechanism that allows anamnestic production of antibody against lower doses of the homologous antigen. Such a mechanism is capable of cross-trapping related antigens, thus preventing a standard primary response and allowing manifestations of Original Antigenic Sin.
Article
Excerpt Specifically purified anti-p-azobenzoate antibody, prepared from the serum of an individual rabbit, crystallized spontaneously from cold neutral buffer solution at a concentration of 60 mg/ml. The protein redissolved at 37° and recrystallized at 5° at much lower concentrations. Some of the properties of the crystallized antibody are described here. Immunization Procedure The antigen was prepared by diazotizing p-aminobenzoic acid and coupling to bovine γ-globulin (Fraction II, Pentex Company) at a ratio of 40 mg of hapten per g of protein. The rabbit (X4) was inoculated with a mixture of this and a second antigen, dinitrophenyl bovine γ-globulin, prepared by the method of Farah, Kern, and Eisen (1960). Seven mg of the azobenzoate conjugate and 3 mg of the dinitrophenylated protein were used for each injection. The first three inoculations, spaced one month apart, were made subcutaneously with complete Freund's adjuvant (Difco). The antigen mixture was then administered intravenously once a...
Article
Excerpt For some years, a prevalent view in immunochemistry was to regard the riddle of antibody specificity as entirely dependent on a solution to the problem of specific labeling of active sites. The direct approach of labeling sites has proved feasible and the yield of significant information from this source promises to increase. However, the study of antibody sites may for the present be viewed as complementing the extensive work on myeloma and other immunoglobulin sequences. From these models are derived most of our current insights into the nature of antibody variability. The ultimate synthesis of multiple efforts to clarify the relationship of amino acid sequence to specificity in antibody molecules may have to await the discovery of homogeneous immunoglobulins with antibody activity. Short of that, our ability to label the active sites of a growing assortment of antibodies ought to permit us to explore some questions that cannot be approached...
Article
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...
Article
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.
Article
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.
Article
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...
Article
Proteins 315 and 460 are IgA2 mouse myeloma immunoglobulins which bind the haptens 2,4-Dnp and Menadione, and their derivatives competitively in the combining region. We have studied the binding of these proteins to a series of Dnp and Menadione based solid phase immunoabsorbents with different length spacer groups to which the Dnp and Menadione groups are attached, in order to determine how deep in the combining region each hapten binds. Both protein 315 and protein 460 share similar combining region dimensions with respect to the haptens used. Menadione binds in the combining regions of both proteins at a depth of 22 Å, while Dnp binds at a depth of 8.8 Å in protein 460 and 11 Å in protein 315. The difference between these values, 11–13 Å, measures the separations between the contact sites for these two haptens. In the preceding paper (pp. 929–937) calculations from energy transfer data gave an average minimum distance of 11.4 Å between the contact sites of Dnp and Men in the protein 460 combining regions. Thus, there is agreement between these distances obtained by two independent experimental methods.
Article
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.
Article
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.
Article
The monoclonal IgM (κ) protein EH bound several ligands: nitrophenyl groups, nitronaphthyl groups, denatured DNA, RNA, heparin, dextran sulphate, polystyrene sulphonic acid, a ribitol teichoic acid and a bacterial ribose-phosphate polymer. All ligands were bound by specific interactions, and complement was fixed in reactions with precipitating ligands. The association constant for N-(2,4-dinitrophenyl)-6-amino-caproate (DNP-EAC)§ was 2·5.103M−1 and for flavianic acid (2,4-dinitronaphthol-7-sulfonic acid) 5·105M−1. The active sites were homogeneous, they were located in the Fabμ fragment, and there were 10 sites per IgM molecule for DNP-FAC and flavianic acid. The macromolecular ligand denaturated DNA was bound by more than five sites per intact IgM molecule, and 100 per cent of F(ab)2μ was bound to a DNA-Sepharose column. The tryptophane fluorescence of the immunoglobulin and its Fabμ fragment was quenched by 2,4,-dinitronaphthol (DNN), and DNN underwent a spectral change on binding. DNP-EAC, DNN and flavianic acid inhibited the reaction with the acidic polymers, indicating that the same site reacted with all the different ligands.
Article
Die oxidative Spaltung der aus den Arylaldehyden (Ia), Chloramin und Ammoniak bei -10°C in Methanol gebildeten 2,4,6-Triaryl-1,3,5-triazabicyclo[3.1.0]hexane zu den Diazirinen (IIa) gelingt bei Zugabe von gelbem HgO zur Reaktionsmischung sobald die Triazabicyclohexane auszufallen beginnnen.
Article
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.
Article
THE covalent binding of a haptene to its specific antibody with a view to identifying the section of peptide chains which form the combining site has been pioneered by Singer et al. 1,2, who named this approach affinity labelling. Antibodies were prepared in rabbits against several aromatic haptenes such as benzene arsonate, and affinity labelling was performed by reaction with p-(arsonic acid)-benzene diazonium fluoroborate through the reactive diazonium group. The reaction with specific antibody was considerably more rapid than that with inert IgG because of the concentration of reagent in the combining site. Subsequent separation of heavy and light chains followed by enzymic digestion led to the isolation of one predominant dipeptide (Val-Tyr) from the light chain and another (Thr-Tyr) from the heavy chain; both of these were substituted on the tyrosine residue. No sequence data were available on the peptide chains of rabbit IgG, but from comparison with the known sequences of the light chains of human and mouse IgG it was suggested that the tyrosine residue 86 in the light chain may have been the one labelled.
Article
This chapter formulates the basic concepts for understanding immunologic specificity and stresses the probable importance of apolar interactions in the antigen-antibody reactions. The notion of specificity was nurtured by the commonsense observations made over many centuries that individuals acquired immunity against a particular disease, for example, smallpox, following recovery from that disease. The intensive study of the chemical nature of the combining region of the antibody molecule has begun only very recently. The chapter discusses the aspects of the specific interaction, such as the complementary relationship, the affinity of the specific complex, the contribution of apolar (hydrophobic) interaction, the contribution of columbic interactions, the contributions of hydrogen bonding and the London dispersion force, the steric factor, and the size of the antibody-combining region. It also emphasizes the induction and purification of high-affinity antibody because only with such antibody the interactions reveal their full significance. In this connection the use of relatively large haptenic groups attached to a single kind of protein side chain will provide important advantages. In this way also it may be possible to ascertain the upper limits of the affinity of antigen and antibody and thereby, provide the quantitative basis for elucidating many biological immune processes. The chapter also presents the general considerations of immunologic specificity and molecular structure such as the molecular (organic) crystal as a mode, the haptenic group as a molecular probe, complex formation as the basic process, the free energy of the specific interaction, and intermolecular forces in specific reactions.
Article
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.
Article
In an attempt to account for antibody specificity and complementarity in terms of structure, human κ-, human λ-, and mouse κ-Bence Jones proteins and light chains are considered as a single population and the variable and constant regions are compared using the sequence data available. Statistical criteria are used in evaluating each position in the sequence as to whether it is essentially invariant or group-specific, subgroup-specific, species-specific, etc. Examination of the invariant residues of the variable and constant regions confirms the existence of a large number of invariant glycines, no invariant valine, lysine, and histidine, and only one invariant leucine and alanine in the variable region, as compared with the absence of invariant glycines and presence of three each of invariant alanine, leucine, and valine and two each of invariant lysine and histidine in the constant region. The unique role of glycine in the variable region is emphasized. Hydrophobicity of the invariant residues of the two regions is also evaluated. A parameter termed variability is defined and plotted against the position for the 107 residues of the variable region. Three stretches of unusually high variability are noted at residues 24–34, 50–56, and 89–97; variations in length have been found in the first and third of these. It is hypothesized that positions 24–34 and 89–97 contain the complementarity-determining residues of the light chain—those which make contact with the antigenic determinant. The heavy chain also has been reported to have a similar region of very high variability which would also participate in forming the antibody-combining site. It is postulated that the information for site complementarity is contained in some extrachromosomal DNA such as an episome and is incorporated by insertion into the DNA of the structural genes for the variable region of short linear sequences of nucleotides. The advantages and disadvantages of this hypothesis are discussed.
Article
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.
Article
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.
Article
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.
Article
Nature is the international weekly journal of science: a magazine style journal that publishes full-length research papers in all disciplines of science, as well as News and Views, reviews, news, features, commentaries, web focuses and more, covering all branches of science and how science impacts upon all aspects of society and life.
Article
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.
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.