Gerald Odstrchel

Kinetic and thermodynamic studies of antigen-antibody interactions in heterogeneous reaction phases—I.: l-Thyroxine (T4) with specific antibody immobilized on controlled pore glass

Abstract The binding reaction of thyroxine (T 4 ) to its specific immobilized antibody (IMA) was treated as a general heterogeneous reaction and kinetic experiments were performed in order to elucidate the reaction mechanism. Except in the complete absence of serum protein, liquid phase diffusion is not the rate determining step. The T 4 binding reaction follows second-order kinetics, in which the two concentration dependent terms are the antibody concentration and the instantaneously free (unbound) T 4 . The rate of this binding reaction can be described by: rate = k [FT 4 ] [IMA]. The [FT 4 ] is a function of the total T 4 and protein concentrations. In the presence of serum protein the rate is much faster when a chemical deblocking agent (merthiolate) is used. The extent of binding does not change much with temperature indicating that the entropic change is the major contribution to the free energy change. The rate of binding, however, changes by an order of magnitude over the range 12–38°C. The apparent activation energies are 9.6 and 16.9 kcal mole in the presence and absence of merthiolate respectively, the difference (7 kcal mole ) being consistent with the binding enthalpy of T 4 to thyroxine binding globulin.

Rapid, quantitative human lymphocyte separation and purification in a closed system.

Abstract A thixotropic, silicone based gel (specific gravity 1.0674) has been developed which permits the rapid isolation of human blood lymphocytes. Gel is incorporated within an evacuated blood draw tube containing crystalline EDTA. Lymphocyte separations are performed within the unopened tube. Quantitative yields of lymphocytes from whole blood are obtained with mononuclear cells comprising 93 percent of the recovered cells. In vitro T and B cell mitogenic responses of recovered lymphocytes are normal thus proving the efficacy of this approach to clinical lymphocyte isolations.

A biased solid-phase radioimmunoassay for thyroxine (T4) using T4-125I presaturated antibodies

An assay for thyroxine (T4) is demonstrated using specific antibodies covalently coupled to controlled pore glass (CPG). In this assay immobilized antibodies saturated with labeled T4 are employed in a preloaded unit tube configuration. These complexes are stable for long periods of time when stored in buffer. This concept results in a highly reproducible and sensitive assay for T4 that requires a single pipetting step.

Characterization of an immobilized antibody-enzyme complex

Antiserum specific for propanediol dehydrogenase, an enzyme found inNeisseria gonorrhoeae cells, has been immobilized to glass. When mixed withN gonorrhoeae cell lysates, the immobilized antibody (IMA) binds the enzyme. Over 70% of the calculated adsorbed activity can be recovered from the immobilized antibody-enzyme (IMA-E) complex. When mixed with bacterial lysates prepared from different organisms having propanediol dehydrogenase-like activity, the IMA specifically adsorbed the enzyme from theN gonorrhoeae lysate. IMA-E complexes have been prepared and their kinetic, temperature and chemical stability, and antigenic properties investigated. These studies demonstrated the feasibility of using an immobilized antibody in the detection of the propanediol dehydrogenase enzyme.

Kinetic and thermodynamic studies of antigen-antibody interactions in heterogeneous phase reaction systems—II.: Interaction of l-triiodothyronine (l-t3) with specific antibody immobilized on controlled-pore glass

Abstract The binding of triiodothyronine (T 3 ) to its specific antibody immobilized on a porous inorganic support (controlled-pore glass) was treated kinetically as a general heterogeneous reaction. The reactions follow apparent second order kinetics, where the rate is proportional to the amount of antibody and to the instantaneous concentration of available unbound T 3 ; this unbound T 3 is inversely related to the concentrations of those serum proteins which normally bind T 3 . In the presence of these proteins T 3 diffusion is not the rate determining step of the reaction. For reaction times in hr the extent of the T 3 binding is inversely related to the protein concentration and the reaction rate does not change greatly with varying protein concentration. For reaction times of weeks the presence or absence of protein or deblocking agent has no effect on the amount of T 3 bound, but the reaction rates are inversely related to the protein concentration. The function of 8-anilinonaphthalene sulfonic acid (ANS) deblocking agent is to effectively displace the T 3 from the TBG fraction of the proteins, which has no effect on the equilibrium point but greatly accelerates the reaction rate. The rate determining step is believed to be the rate of desorption of T 3 from the protein, since the T 3 binding rate to antibody takes place rapidly. The measured enthalpy of the reaction (0–1.3 kcal/mol) indicates that the reaction is principally entropy driven.

Detection and molecular specificity of murine thymocyte receptors for GAT in responder and non-responder mice utilizing a microradioreceptor assay

Abstract A microassay for the detection of murine thymocyte antigen receptors reactive to GAT ∗ has been developed. Immunochemical specificity was demonstrated under competitive binding conditions utilizing labeled antigen. Only GAT could competitively displace 125 I GAT from the thymocyte receptor. Erythrocytes did not bind 125 I GAT whereas myeloma MPC-11 or acute lymphoblastic leukemia CCRF-CEM cell lines bound ≅ 3.5-fold less GAT than thymocytes. The magnitude of thymocyte ligand binding was approximately 4-fold greater at 37°C than at 4°C. Biased displacement studies under conditions which favored phagocytosis indicated that 70% of the cell-associated counts were surface (plasma membrane) bound, making it unlikely that phagocytosis was the source of ligand uptake. Similarly, thymocyte suspensions adsorbed to petri dishes for 45 min at 37°C or assayed in the presence of NaN 3 showed no dimunition in B 0 binding. A rabbit anti-mouse T cell monospecific serum was capable of blocking 125 I GAT binding whereas normal serum was without effect. The specifie binding of GAT to thymocytes occurred through a trypsin-sensitive thymocyte membrane component. In further studies, the equilibrium displacement of 125 I GAT from the T cell GAT receptor by copolymers GA or GT demonstrated that the thymocyte receptor(s) for GAT in unimmunized responder and non-responder mice react with GAT through apparently identical antigenic determinants within the GAT terpolymer. These specificity data corroborate in-vivo functional data and suggest that the T cell antigen receptor for GAT does not regulate the GAT immune response by itself.