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Specific penicillin-carrier receptor proteins (CRP) have been isolated from the sera of penicillin allergic rabbits and human subjects in the unconjugated native state in electrophoretically homogeneous form by employing a synthetic polymeric affinity template containing the 7-deoxy analogue of penicillin G. The synthesis of the 7-deoxy analogue has been described. In this affinity system the antipenicillin-antibody is desorbed by 0·9M thiourea and the CRP in 8M urea. The CRP after incubation with penicillin is converted into the full-fledged antigen. Studies on the origin of CRP and the nature of antibody as well as comparative studies on the properties of the rabbit antibody and those of antibodies elicited by a BSA-BPO conjugate are reported.

The availability of an electrophoretically homogeneous rabbit penicillin carrier receptor protein (CRP) and rabbit antipenicillin antibody afforded an idealin vitro system to calculate the thermodynamic parameters of the binding of14C benzyl penicillin CRP conjugate (antigen) to the purified rabbit antipenicillin antibody. The thermodynamic parameters of this antigen-antibody reaction has been studied by radio-active assay method by using millipore filter. Equilibrium constant (K) of this reaction has been found to be 2·853×109M−2 and corresponding free energy (ΔG) at 4°C and 37°C has been calculated to be −12·02 and −13·5 kcal/mole, enthalpy (ΔH) and entropy (ΔS) has been found to be 361 kcal/mole and +30 eu/mole respectively. Competitive binding studies of CRP-analogue conjugates with the divalent rabbit antibody has been carried out in the presence of14C-penicilloyl CRP. It was found that 7-deoxy penicillin-CRP complex and 6-amino penicilloyl CRP conjugate binds to the antibody with energies stronger than that with the14C-penicilloyl CRP. All the other analogue conjugates are much weaker in interfering with the binding of the penicilloyl CRP with the antibody. The conjugate of methicillin,o-nitro benzyl penicillin and ticarcillin with CRP do not materially interfere in the process.

All antigen-antibody (AG-AB) bonds are weak physical bonds; covalent bonds are not encountered. The main bonds involved are: (I) Coulombic bonds; (II) Ca2+-bridges; (III) hydrogen bonds; (IV) Lifshitz--van der Waals bonds. Combinations of III and IV occur as the "bonds" usually alluded to as hydrophobic (H phi) interactions. In primary bonds, mainly types I and IV occur; types II and III are quite rare. Secondary bonds, which evolve after a certain time-lapse (varying from minutes to days), mainly involve type IV bonds and H phi interactions, while hydrogen bonds sensu stricto have been known to play a role in rare instances. In affinity chromatography involving AG-AB interactions, complete elution with mild eluents usually is desirable. It would thus appear essential to let little time elapse between AG-AB complex formation and the elution step, to minimize strengthening of the AG-AB interaction by secondary bond formation. For expeditious elution, it also is important to avoid using dehydrating agents (which tend to decrease the bond distance between AG and AB and thus could strengthen the bond in a number of ways). The degree of involvement of types I and IV bonds as well as of H phi interactions varies considerably among different AG-AB systems. These components thus have to be measured separately in order to determine the optimal conditions for elution. The parameters of the liquid medium that may be modulated to influence dissociation (or association) are, for example, surface tension, pH, ionic strength, dielectric constant, temperature, admixture of dehydrating agents or of chaotropic salts. The influence of variations in each of these parameters on types I, III and IV bonds (and thus also on H phi interactions) will be delineated. Because of the misleading implications of the term "hydrophobic interactions", it seems preferable henceforth to allude to them as "interfacial forces".

The EDP208 pilus contains a major antigenic determinant in the N-terminal dodecapeptide, as shown by E. A. Worobec, A. K. Taneja, R. S. Hodges, and W. Paranchych ((1983) J. Bacteriol. 153, 955-961). This peptide was chemically synthesized, coupled to bovine serum albumin with N-hydroxysuccinimidyl p-azido-benzoate, and used in immunoblot and enzyme-linked immunosorbent assays to show it was capable of reacting with anti-EDP208 pilus antibodies. Antibodies raised against the synthetic peptide conjugate were also capable of reacting with whole pili in these assays. To further examine the specific residues responsible for the antigenicity of this site, several peptide analogs were chemically synthesized. The relative affinity of these peptides for anti-EDP208 pilus antibodies was determined by a competitive enzyme-linked immunosorbent assay using the Fab fragment of anti-EDP208 pilus immunoglobulin G. From these results we established that the antigenic region of this peptide was the N-terminal pentapeptide, N-acetyl-Thr-Asp-Leu-Leu-Ala, and the key residues responsible for the antibody-antigen interaction are the N-acetyl-Thr1, Leu3, and Leu4. Hydrophobic interactions involving the methyl of the acetyl group and the leucine side chains make the largest contributions to the antigen-antibody interaction, while a lesser contribution is made by the Thr1 hydroxyl. The side chains of Asp2 and Ala5 contribute only weakly to the stabilization of the antigen-antibody complex.

Binding of Epstein-Barr-virus-determined nuclear antigen (EBNA) to acid-fixed nuclei could be prevented by exposing EBNA to anti-EBNA antibodies. While EBNA is eluted from acid-fixed nuclei by 0.4 M NaCl, it was not eluted even by 1.5 M NaCl if EBNA bound to acid-fixed nuclei had been exposed to anti-EBNA antibody. In this case the reaction was abolished, and EBNA was at least partially eluted by 0.2 M glycine buffer, pH 2.8, or by 3 M NaSCN. EBNA not exposed to antibody was completely eluted from acid-fixed nuclei by treatment with a solution containing 200 microgram of DNA per 1 ml of isotonic buffer, pH 6.5; however, after treatment with antibody, EBNA was not eluted by DNA.

The specificities of four monoclonal antibodies rho 1D4, 1C5, 3A6, and 3D6 prepared by immunization of rod outer segments containing rhodopsin have been defined using synthetic peptides. All of these antibodies interact within the 18 residues at the COOH terminus of rhodopsin and recognize linear antigenic determinants of 4-11 residues. Twenty-seven synthetic peptide analogs of varying lengths of native sequence or containing single amino acid substitutions at each position of the COOH-terminal 18 residues have provided some insight into the mechanism of antigen-antibody binding. Our results clearly demonstrate that antibodies can be highly specific at key positions as shown by the loss of binding on single amino acid substitutions in the binding site. In contrast single amino acid substitutions at other positions in the binding site only affect affinity for some antibodies. Ionic interactions can dominate immunogenic determinants. Immunogenic determinants are not restricted to highly charged hydrophilic regions on the surface of a protein and may be dominated by hydrophobic interactions. Although certain side chains can dominate the interaction of the antigen with antibody, our results are in agreement with the interpretation that the free energies of all the contact points are additive and a certain free energy must be present to achieve binding. Antibodies with different specificities directed to the same region of the protein antigen can be produced in an immune response. Peptide antigens representing regions of a protein antigen bind best to the anti-protein antibody when the sequence is shortened to contain only those residues binding to the specificity site in the antibody. Cross-reactivity between protein antigens can be explained by conservation of the critical residues in the combining site.

Radial immunodiffusion can be used to determine relative antigen-antibody avidities in exactly the same way as demonstrated previously for quantitative immunoelectrophoresis (Birkmeyer et al., 1981). Antigen-antibody interactions of greater avidity result in a greater value of (delta Area/delta [Antigen]) in plots of immunoprecipitin circle area versus antigen concentration while interactions of equal avidity will yield equal values of (delta Area/delta [Antigen]). This was demonstrated using antigens of different weight ranging from 8000 to 66,000.

Each of several antigen-antibody systems studied has been found to affect the coagulation mechanism in the rabbit, causing a marked shortening of the coagulation time in vitro of samples of whole blood maintained in siliconized glassware. Addition of specific antigen to the blood of actively immunized animals or addition of antigen-antibody mixtures to the blood of normal animals produced the effect. The coagulation time of plasma was not affected, indicating that the phenomenon may be mediated by an effect on platelets. This effect of antigen-antibody interaction may be involved in the production of tissue damage in vivo.

Antigens covalently bound to agarose beads were found to be suitable for microfluorometry on individual beads of fluorescein-conjugated antibodies reacting with these antigens. With this technique, the interaction of conjugates with IgG, IgM and ovalbumin bound to the beads was studied. The system was found to be applicable to specificity tests. It also appeared to be more sensitive for high conjugate dilutions than immunofluorescence methods using tissue sections or cells of defined antigenicity as a substrate.

Studies of the dissociation of complexes using specific insoluble complexing agents under specified conditions, can yield information concerning the nature of the specific interactions contributing to complex formation. For the case of the interaction of specific antibody with antigen conjugates, data is obtained concerning heterogeneity of apparent equilibrium constants and the influence of antigen and immunoadsorbent structure on protein interaction and antigen-antibody dissociation. Such information can be useful for predicting conditions for antiserum purification or antibody recovery. For the case of the cellulose adsorbents chosen, it was found that (1) the adsorbent may contribute strongly to the interaction of ionizable haptens with antibody, with a pH dependency typical of acid catalyzed reactions (2) immunoadsorbent properties can be modified through physical and chemical structural changes (3) the dissociation of antibody from the haptenic and protein immunoadsorbents showed no significant discontinuities in the acid range and (4) antibodies to those haptens chosen which do not ionize in the acid range, do not dissociate appreciably from the immunoadsorbent in the acid range but do at sufficiently high pH.

A general counterpart of the Scatchard analysis has been developed which takes into account the valence of the ligand. Its use is first demonstrated by application to binding data obtained by exclusion chromatography of mixtures of Dextran T2000 and concanavalin A (a bivalent ligand) on a column of porous glass beads (Glyceryl-CPG 170) equilibrated at 5 degrees C with phosphate-chloride buffer (pH 5.5), I 0.5. A recycling partition equilibrium study with Sephadex G-100 as gel phase then provides a quantitative evaluation of the interaction between haemoglobin and a monoclonal mouse antihaemoglobin antibody preparation in 0.1 M phosphate (pH 7.0) in order to emphasize the ability of the present analysis to consider collectively binding results obtained with a range of acceptor concentrations. Finally, the use of the generalized Scatchard analysis to assess acceptor site homogeneity is illustrated by reappraisal of results for the binding of glyceraldehyde-3-phosphate dehydrogenase to erythrocyte membranes.

The immune system may be viewed as the locus of a multitude of molecular interactions involving a limited number of genetically determined structures. Most of these structures act as membrane receptors (i.e., communication organs of cells) or as secreted factors. These are called antibodies (Ab), T cell receptors, or class I and II molecules coded for in the major histocompatibility complex (MHC).

The union of antibody and antigen gives rise to a series of reactions, the qualitative or semiquantitative study of which is the domain of serology.

An improved method of immunoelectrophoresis is described. Infusion of water- soluble polyethylene oxide polymers into agar gel, after the double diffusion had taken place, caused enhanced visibility as well as better resolution of immuno-precipitates into distinct immuno-precipitin bands.

Recent studies have shown that homocytotropic antibody, a specialized immunoglobulin that sensitizes mast cells, and immune aggregates, complexes made up of antigen, antibody and complement, can mediate certain immediate hypersensitivity reactions. Aspects of the immune mechanisms involved and tests for the presence of homocytotropic antibody and immune aggregates are discussed.

Two types of conformationally specific anti-irradiated ovalbumin monoclonal antibodies were prepared in order to study and monitor irradiation-induced structural changes in the ovalbumin molecule. Surface plasmon resonance (SPR) detection was used to investigate the kinetic parameters of the reaction between antibodies and ovalbumin which had been administered with different doses of irradiation (0, 1.5, 2.0, 5.0, 10, 20, 50, and 100 kGy). The results demonstrate that the combination of monoclonal antibodies and the SPR method can be used to characterize the irradiation-induced conformational change with an unlabelled reagent.

The introduction of anti-Rh(D) immunoglobulin in the mid-1960s has protected the vast majority of Rh negative women at risk from pregnancy-induced isoimmunization and the subsequent possibility of haemolytic disease of the newborn, although a disturbingly high incidence of anti-D is still being observed in pregnant women. Increases in the frequency of blood transfusion (and in the volumes used in many instances) have ensured that many hospital patients will be exposed to foreign red cell antigens outside the ABO and Rh systems. In both these groups, antibodies will continue to be made against isologous red cell antigens, and the non-D Rh antigens, as well as those of many other blood group systems, have become increasingly important in transfusion practice. There is a well recognized disparity between the in vitro demonstration of a red cell antigen-antibody reaction and the in vivo results of introducing this antigen in volume into a recipient with the relevant antibody. This paper reviews the currently available data on in vitro and in vivo red cell antigen antibody interaction, and examines the factors which assume importance in an actual or potential haemolytic transfusion reaction.