Structural basis for antibody catalysis of a disfavored ring closure reaction.

The catalysis of disfavored chemical reactions, especially those with no known natural enzyme counterparts, is one of the most promising achievements of catalytic antibody research. Antibodies 5C8, 14B9, 17F6, and 26D9, elicited by two different transition-state analogues, catalyze disfavored endo-tet cyclization reactions of trans-epoxy alcohols, in formal violation of Baldwin's rules for ring closure. Thus far, neither chemical nor enzyme catalysis has been capable of emulating the extraordinary activity and specificity of these antibodies. X-ray structures of two complexes of Fab 5C8 with the original hapten and with an inhibitor have been determined to 2.0 A resolution. The Fab structure has an active site that contains a putative catalytic diad, consisting of AspH95 and HisL89, capable of general acid/base catalysis. The stabilization of a positive charge that develops along the reaction coordinate appears to be an important factor for rate enhancement and for directing the reaction along the otherwise disfavored pathway. Sequence analysis of the four catalytic antibodies, as well as four inactive antibodies that strongly bind the transition-state analogues, suggests a conserved catalytic mechanism. The occurrence of the putative base HisL89 in all active antibodies, its absence in three out of the four analyzed inactive antibodies, and the rarity of a histidine at this position in immunoglobulins support an important catalytic role for this residue.     

Specificity of the antibody neutralization reaction in indicating Shigella antigens

The specificity and sensitivity of the antibody neutralization test in the diagnosis of dysentery have been studied. This test has proved to be highly specific in experimental and clinical trials. The antibody neutralization test is more effective than the methods of the isolation of shigellae. The accuracy of this test can be increased by the elimination of different antierythrocyte agents from the secretions of persons under examination.     

Effect of the metal in the reaction between metallothionein and antimetallothionein antibody

1. An antimetallothionein antibody, raised against Cd-carrying metallothionein, was applied in Western blotting of metallothionein. 2. Treatment of the electroblotted nitrocellulose sheets with metals belonging to the periodic system transition groups Ib and IIb, or with Pb, Ni or Cr, considerably enhanced binding of anti-metallothionein. A similar effect was found when the electroblotted sheets were treated with the strong alkylator N-ethylmaleimide. 3. It seems that the binding of metal to metallothionein modifies the configuration of the antibody binding sites by the formation of metal thiolate complexes. 4. Metal treatment of the nitrocellulose sheets after electroblotting, but before application of the primary antibody, offers a convenient method for use in Western blotting to significantly potentiate the reaction between metallothionein and the antimetallothionein antibody.     

Antigen-antibody reaction by monolayer technique experiments with rabbit IgG antibody and its fragment.

With the monolayer technique, values of about 99.7 Å for rabbit anti-egg albumin IgG antibody, and about 58.9 Å for Fab fragment of IgG were obtained as the thickness of the adsorbed antibody layer. As these values determined in the saturated state were confirmed, it may be reasonable to assume that they correspond approximately to the long axis of both molecules.     

Structural basis for a disfavored elimination reaction in catalytic antibody 1D4.

Murine antibody 1D4 selectively catalyzes a highly disfavored beta-elimination reaction. Crystal structures of unliganded 1D4 and 1D4 in complex with a transition-state analog (TSA) have elucidated a possible general base mode of catalysis. The structures of the unliganded and liganded Fabs were determined to 1.80 and 1.85 A resolution, respectively. The structure of the complex reveals a binding pocket with high shape complementarity to the TSA, which is recruited to coerce the substrate into the sterically demanding, eclipsed conformation that is required for catalysis. A histidine residue and two water molecules are likely involved in the catalysis. The structure supports either a concerted E2 or stepwise E1cB-like mechanism for elimination. Finally, the liganded 1D4 structure shows minor conformational rearrangements in CDR H2, indicative of induced-fit binding of the hapten. 1D4 has pushed the boundaries of antibody-mediated catalysis into the realm of disfavored reactions and, hence, represents an important milestone in the development of this technology.     

Immune reactions in polysaccharide media. The composition of the antigen–antibody complexes in the precipitin reaction

The precipitin reaction is enhanced in the presence of polysaccharides (Hellsing, 1966). This reaction has now been studied in detail with labelled antigen ((125)I-labelled human serum albumin) and antibody ((131)I-labelled rabbit anti-albumin immunoglobulin G). The relative proportions of antigen and antibody in the precipitates are unchanged by the addition of dextran in spite of the increased precipitation. The ratio of antibody to antigen in the soluble immune complexes decreases with increasing polysaccharide concentration. This can be interpreted as a decrease in the aggregate size of the complexes. At the same time the amount of free antigen in the solution increases. The results are consistent with a decrease in solubility, primarily of the large immune aggregates, together with a shift in the equilibrium between small and large complexes. The effect is in accord with a steric-exclusion phenomenon.     

Steric hindrance as a factor in the reaction of labeled antibody with cell surface antigenic determinants.

The binding of rabbit anti-human IgG labeled with 125I, shellfish glycogen or ferritin to human IgG attached to the surface of rabbit RBC with chromic chloride was studied. Maximum binding was noted with 125I labeled antibody. Slightly but consistently less binding was found with shellfish glycogen labeled antibody. The binding of ferritin labeled antibody was strikingly reduced--usually one-third or less of that found with 125I labeled antibody alone. This suggests that under the conditions of these experiments, the attachment of large labels to antibody molecules results in reduced antibody binding to surface antigen. Steric hindrance is probably at least in part responsible for this reduced binding.     

Nonabsorbable rabbit anti-Salmonella typhimurium antibody as detected by the complement-mediated bactericidal reaction

Herzberg, Mendel (University of Florida, Gainesville), Kathryn V. Kenny, and John B. Robbins. Nonabsorbable rabbit anti-Salmonella typhimurium antibody as detected by the complement-mediated bactericidal reaction. J. Bacteriol. 91:1548-1555. 1966.-A portion of antibody active in the complement-mediated bactericidal reaction against Salmonella typhimurium from hyperimmune rabbit serum has been shown to be nonabsorbable by repeated serial absorptions with whole heat-killed or living bacteria. The first two absorptions remove 90 to 95% of the activity, but 1 to 5% cannot be removed by subsequent absorptions. The nonabsorbable antibody appears to be a macroglobulin by density-gradient centrifugation and by comparison of activity and absorbability of purified gamma-M and gamma-G immune globulins. Alternative hypotheses involving low avidity antibody or antibody to minor cell antigenic components are offered in explanation of the phenomenon.     

Plasminogen activation by antiplasminogen monoclonal antibody IV-1C. Properties and mechanism of reaction

In review the results of investigation of plasminogen(Pg) activation by antiplasminogen monoclonal antibody IV-1c have been presented. Antigenic determinant of IV-1c was localized in Val709-Gly718 site of Pg protease domain. IV-1c completely inhibited the Pg activation by streptokinase, but increased the rate of Pg activation by t-PA and urokinase. Catalytic properties of plasmin in complex with IV-1c were studied. It was found that IV-1c induced catalytic activity in Pg-IV-1c complex. It was shown that Pg and IV-1c interacts in complex by two-centre mechanism: IV-1c binds with Pg by paratope and by N-terminal lysine of gamma-chain and Pg binds to IV-1c by one of the lysine binding sites and by V709-G718 site of protease domain. The influence of pH, temperature, 1.5 mM Ca2+, Mg2+, Sr2+, Ba2+, Co2+, Ni2+ cations and 10 mM Cl-, F-, Ac-, SO4(2-), HPO4(2-) anions on lag and fast phases of Pg activation by VI-1c was investigated. It was revealed that Val709-Gly718 site was determining in Pg activation by IV-1c and streptokinase.