First order dissociation rates between a subpopulation of high affinity rabbit anti-fluorescyl IgG antibody and homologous ligand

The first order dissociation rates of liganded subpopulations of purified hyperimmune rabbit antifluorescyl IgG antibodies were determined using the method of Green (1963). The dissociation of radiolabeled fluorescyl ligands was measured in the presence of excess unlabeled homologous hapten. Results with three different antibody preparations indicated dissociation rates of 1.3 × 10[su?3, 2.4 × 10^?4 and 2.2 × 10^?6 sec^?1 for the liganded subpopulations. Assuming an association rate of 4.6 × 10⁸M^?1 sec^?1 (Levison et al., 1971), average equilibrium constants ranging from 3.1 × 10¹¹M^?1 to 2.1 × 10¹⁴M^?1 were calculated. These values are among the highest reported for antibody-hapten interactions.     

Modulation by 310-1310-1310-1stimulation of IgE-mediated14C-serotonin release from rat mast cells

The formaldehyde method was used to examine the effects of clonidine and methoxamine on IgE-mediated¹⁴C-serotonin release from rat mast cellsin vitro. Clonidine (10^?11?10^?8 M) caused dose-related enhancement of the mediator release 7min after the antigen challenge yohimbine (10^?6 M) blocked this enhancement by clonidine (10^?6 M), but prazosin (10^?6 M) did not. Methoxamine did not enhance this immunological release reaction at concentrations up to 10^?6 M. PGE₁ (2×10^?8?2×10^?5 M), isoproterenol (10^?10?10^?8 M), dopamine (4×10^?8?4×10^?8 M) and aminophylline (6×10^?6?6×10^?4 M) caused dose-related inhibition of this mediator release 1 min after antigen challenge. Clonidine (10^?13?10^?12 M), but not methoxamine (10^?8?10^?6 M), reversed dose-dependently this inhibition of mast cells by PGE₁ (2×10^?6 M), isoproterenol (10^?8 M), dopamine (4×10^?6 M); yohimbine (10^?8 M) antagonized this reversing action of clonidine (10^?12 M), but prazosin (10^?10 M) did not. Neither clonidine (10^?14?10^?11 M) nor methoxamine (10^?8?10^?6 M) reversed the inhibitory action of aminophylline (2×10^?4 M). These results suggest that clonidine enhances IgE-mediated¹⁴C-serotonin release from rat mast cells and also antagonizes the inhibition of mast cells by PGE₁, isoproterenol and dopamine, but not by aminophylline in this immunological reaction through α₂-adrenergic receptors, and that the inhibition of adenylate cyclase of mast cells is one of the biochemical actions of α₂-adrenergic mechanisms.     

Monoclonal Antibodies Against Human Chorionic Gonadotropin (hCG): II. Affinity and Ability to Neutralize the Biological Activity of hCG

ABSTRACT: Monoclonal antibodies (MCA) against hCG have been characterized with regard to their affinity and their ability to neutralize the biological activity of hCG in vivo. The production and specificities of these reagents were described in the preceeding paper of this series. Equilibrium association constants (K_a) of the MCA, determined by radioimmunological saturation assays, ranged from less than 1 × 10⁸M⁻¹ up to 3.7 × 10⁹M⁻¹ whereas values for conventional polyclonal antisera against hCG ranged from 8.9 × 10⁹M to 1.8 × 10¹⁰M⁻¹. The ability of MCA to neutralize the biological activity of hCG was tested in a rat bioassay in vivo; 9 of 13 different MCA preparations tested could neutralize hCG. Surprisingly, this property did not correlate with affinity or specificity, and was not restricted to those MCA recognizing the hormone specific β-subunit. It could be demonstrated that determinants on each individual subunit as well as epitopes formed by both subunits are involved in the expression of the biological activity of hCG.     

Characterization of high-affinity antifluorescyl IgM antibodies

High-affinity IgM rabbit antibodies were elicited using the fluorescein hapten system. Purity and identification of IgM and IgG anti-fluorescyl antibodies was determined by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis. Ultracentrifugation studies verified that liganded antibodies were of a high mol. wt (901 kd).Comparative analyses of IgM and IgG anti-fluorescyl antibody active sites substantiated the observation that purified IgM preparations, similar to their IgG counterparts, possessed high-affinity antibody active sites. Dissociation rate data confirmed that some IgM molecules within the purified antibody populations possessed association constants at 10¹¹M^?1, comparable with values of 10¹¹M^?1 or greater obtained for some anti-fluorescein IgG populations studied in this laboratory. A diffusion-controlled association rate of 4.6 × 10⁸M^?1 sec^?1 (Levison et al., 1971) was assumed in dissociation rate calculations. This is the first report of purified IgM antibodies possessing exceptionally high affinities.     

Chemical basis for diversity in antibody specificity analysed by hapten binding to monoclonal anti-4-hydroxy-3-nitrophenacetyl (NP) immunoglobulins

Anti-4-hydroxy-3-nitrophenacetyl (NP)2 monoclonal antibodies were produced by hybrid cell lines obtained by fusion of NSl myeloma cells with lymphocytes from spleens of C57BL/6 mice immunized with NP conjugated with chicken immunoglobulin (NP-CG). Equilibrium constants of four purified immunoglobulins for N¹²⁵IP-ε-aminocaproate, measured by equilibrium dialysis at 4°C, ranged from 1.0 × 10^?8 to 1.0 × 10^?7M. In order to probe fine-specificity differences, binding constants were measured for a group of structural analogs of NP. This study showed that all four antibodies were heteroclitic and bound the iodo group of NIP-ε-aminocaproate with similar affinities, while the affinity constants for the ε-aminocaproate moiety varied widely. The free energies of binding for iodo group and the side-chain moieties were additive, whereas those for the hydroxy and nitro groups were not. Three of the immunoglobulins bound the aromatic ring system of NIP-ε-aminocaproate similarly, but less effectively than the fourth antibody. The data suggest that anti-NP active sites of diverse specificities were generated on the basis of additive interactions with multiple subsites.     

Characterization of monoclonal antibodies against prostaglandin E2: Fine specificity and neutralization of biological effects

The specificity and heterogeneity of the immune response of BALB/c mice immunized with prostaglandin E₂ (PGE₂) coupled to thyroglobulin was studied. All the animals (n = 50) responded to PGB₂, a transformation product of PGE₂. However, following repeated injections most of the animals (n = 30) were also able to respond to PGE₂. Cellular hybridizations were performed and five anti-PGE₂ monoclonal antibodies were isolated and analysed. They are mainly directed against the ring and the ω-chain of PGE₂ but their specificity toward the α-chain is more limited. The association constants are greater than to 1 × 10⁹M^?1. The monoclonal antibody 8E.57.71 (K_a = 1.3 × 10¹⁰M^?1) is particularly convenient for sensitive radioimmunoassays (detection limit 25pg/ml, when iodinated tracer is used). Anti-PGE₂ monoclonal antibodies were found to neutralize the specific binding of [³H]PGE₂ to rat brain hypothalamic receptors and to inhibit the PGE₂ induction of rat fundus muscular contraction.     

Direct quantitative measurement of the kinetics of HLA-specific antibody interactions with isolated HLA proteins

HLA specific antibodies vary in their pathogenicity and this is likely to be the net effect of constant chain usage, quantity, specificity, and affinity. Here we have measured the affinity of human monoclonal antibodies for a range of HLA proteins. Purified antibodies and ligands allowed dynamic interactions to be measured directly by surface plasmon resonance. Physiochemical differences between pairs of ligands were quantified using electrostatic mismatch and hydrophobic mismatch scores.All antibodies were characterized by fast on-rates and slow off rates but with a wide range of association rates (k_on, 3.63–24.25?×?10⁵ per mol per second) and dissociation rates (k_off, 0.99–10.93?×?10^?3 per second). Dissociation constants (K_D) ranged from 5.9?×?10^?10?M to 3.0?×?10^?8?M. SN320G6 has approximately a twenty-fold greater affinity for HLA A2 compared with SN607D8, but has a similar affinity for HLA-A2 and B57. In contrast, SN607D8 has greater than a twofold greater affinity for HLA-A2 compared with A68. Similarly, WK1D12 has about a threefold greater affinity for HLA-B27 compared with B7. The higher affinity interactions correlate with the specificity of stimulating antigen. This is the first study to directly measure the binding kinetics and affinity constants for human alloantibodies against HLA.     

Non-covalent association of heavy and light chains of human immunoglobulin G: Studies using light chain labelled with a fluorescent probe

The fluorescent probe, 5-iodoacetamidofluorescein (5-IAF), was specifically attached to the COOH-terminal cysteine residue of the L chains of two monoclonal human IgKκ proteins. The induced circular dichroism and fluorescence properties of the bound 5-IAF probe were used to study changes in its microenvironment upon reassociation of autologous or heterologous L chains and their domains with Fd' fragments. Recombination of 5-IAF-L with Fd' at pH 5.4 was accompanied by a red-shifted visible difference spectrum, an increase in the magnitude of the induced optical activity and an enhancement of fluorescence. These results were compatible with the transfer of the 5-IAF moeity to a less polar and more asymmetric environment. Equilibrium and kinetic data obtained from difference spectroscopy and fluorescence studies indicated that 5-IAF-L bound with high affinity to Fd' in a 1:1 ratio with a second-order rate constant of 1618 M^?1 sec^?1 at 25°C. Using the same approach, it was shown that the labelled constant region fragment (5-IAF-C_κ) bound to Fd' with an association constant of 5 × 10⁶ M^?1 and a forward rate constant of 30 M^?1 sec^?1. When 5-IAF-C_κ was recombined with a preformed Fd'V_κ complex, however, the intensity of the visible difference spectrum, the fluorescence emission, the binding affinity and the forward rate constant of the reaction, were significantly increased. In contrast, no spectroscopic changes were observed when V_κ was recombined with a preformed Fd'-5-IAF-C_κ complex. These data suggest that: (1) the high affinity interaction between Fd' and L results from the summation of relatively weak interactions between paired domains; (2) the binding of Vκ to Fd' modulates the reactivity of Cγl toward Cκ, probably through conformation changes transmitted via V_H-Cγl contacts; and (3) conversely, once 5-IAF-Cκ is bound to Fd', the addition of the complementary Vκ domains does not induce any detectable change in the vicinity of the fluorescent probe.     

Reconstitution of potato virus X in vitro. I. Properties of the dissociated protein structural subunits.

The functional protein subunits of the X^HR strain of potato virus X (PVX) obtained when the virus was disrupted with lithium chloride sedimented in the analytical ultracentrifuge as a single boundary (s^o_20,w = 2.3 × 10^?13), migrated in polyacrylamide gels as a single band, eluted from Sephadex G-200 as a single peak (Stokes radius = 3.27 nm), and behaved during diffusion coefficient measurements as a monodisperse solute (D_20,w = 5.02 × 10^?7 cm² sec^?1). Amino acid and tryptic peptide analyses showed the molecular weight of these subunits to be 23,000. The dimensions of the functional subunits calculated from these data were consistent with the volume available for the subunit in the intact virus.Results of hydrogen-tritium exchange and circular dichroism studies showed that the subunits possessed appreciable α-helical structure under conditions (20°C, pH 6.10 to 6.50, low ionic strength) optimal for reconstitution of viruslike particles with viral RNA. The properties of functional protein subunits thus differ in several respects from those of denatured subunits prepared with pyridine (Shalla and Shepard, 1970). However, functional subunits were serologically identical to denatured subunits in Ouchterlony double diffusion tests against PVX D-protein antiserum.     

The binding of soluble immune complexes of guinea pig IgG2 to homologous peritoneal macrophages Determination of the avidity constants at 4 °C.

Soluble immune complexes of guinea pig IgG₂ anti-DNP (2,4-dinitrophenyl) antibodies and DNP₁₉BSA (bovine serum albumin) were prepared at a variety of antibody-to-antigen ratios. The mean sizes of the complexes were determined by gel filtration, and equilibrium binding of the complexes to homologous peritoneal macrophages was measured at 4°C. Scatchard plots of the binding data were linear indicating that the complexes were binding to a uniform population of functionally independent membrane receptors. The avidity constants for complex binding to macrophages increased from 15.4 × 10⁷ M^?1 for complexes containing an average of two antibody molecules to 59.0 × 10⁷ M^?1 for complexes containing an average of four, compared with an association constant for monomeric IgG₂ of 0.21 × 10⁷ M^?1. Calculation of the molar-free energy changes accompanying monomer and immune complex binding revealed not only that the intrinsic binding activity of mononer was sufficient to account for the complex binding activities by simple cooperation of linked antibodies, but also that the cross- linking of antibodies with antigen imposed a strain on the antibody-receptor interaction, resulting in complex binding energies that were lower than the values expected from a simple cooperative mechanism. Complex inhibition by monomeric IgG₂ of unrelated antibody specificity was studied, and it was observed that high concentrations of monomer led to an increase in macrophage discrimination between complexes of different size. The relevance of this finding to the in vivo clearance of circulating complexes is discussed.     

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. IX. Australian feral chicken and domestic chicken (Gallus domesticus)

The diffusional water permeability (P _d) of Australian feral chicken and Australian and European domestic chicken red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique. The values of P _d were around 1.7 × 10^?3 cm/s at 15°C, 2.0 × 10^?3 cm/s at 20°C, 2.5 × 10^?3 cm/s at 25°C, 3.7 × 10^?3 cm/s at 30°C, 4.3 × 10^?3 cm/s at 37°C, and 6.1 × 10^?3 cm/s at 42°C, with no significant differences between the three strains of chicken. There was no effect of p-chloromercuribenzene sulphonate on water diffusion. The activation energy of water diffusion was around 37 kJ/mol for all strains of chicken. These results suggest that no changes in the RBC water permeability are correlated with marked alterations in the habitat of chicken introduced to Australia (and that membrane proteins play little role in the diffusion of water across chicken RBC membrane).     

The Complement—Fixing Activity of Immune Complexes Containing IgG Antibodies of Different Functional Affinities: Effects on Superoxide Production by Rabbit Neutrophils

When neutrophil phagocytes are stimulated by IgG containing immune complexes (IgG‐IC), with or without the participation of the complement system, they show a sharp increase in oxygen uptake and begin to release large quantities of superoxide anions (O₂^?) and hydrogen peroxide (H₂O₂) into the surrounding medium. The aim of the present investigation was to provide insights into the production and release of O₂^? by rabbit neutrophils activated with immune complexes (IC) containing IgG antibodies of different functional affinity, opsonized and not opsonized by complement system components. For this purpose, two populations of polyclonal anti‐ovalbumin (OVA) IgG antibodies with different functional affinity, 5 × 10⁸ M^{? 1} and 2 × 10⁷ M^{? 1}, were prepared. The production of O₂^? was measured spectrophotometrically by a method using the superoxide dismutase‐inhibited reduction of ferricytochrome C to the ferrous form. The activation of complement by different IgG‐IC was determined by estimating the total residual haemolytic activity of the alternative and classical pathways in sera treated with different concentrations of anti‐OVA IgG/OVA immune complexes formed at equivalence. The results showed that: 1) antibody functional affinity influenced O₂^? production and the complement‐fixing activity induced by the IC. In general, the higher functional affinity antibodies were more efficient in stimulating the respiratory burst of neutrophils and in activating complement by the classical and alternative pathways than the lower functional affinity antibodies at all IC concentrations tested; 2) complement components incorporated into the immune complex lattice caused an increase in the stimulatory activity of both IgG antibodies to produce O₂^? (? 15% for the IC of IgG with K_a = 5 × 10⁸ M^{? 1} and ? 7% for the IC of IgG with K_a = 2 × 10⁷ M^{? 1}). This effect was dependent on antibody affinity and concentration; 3) there was a direct relationship between the overall level of complement activation, antibody affinity and superoxide production by neutrophils. Thus, we conclude that antibody affinity influences immune complex lattice formation, modulating its three‐dimensional structure and the disposition of Fc fragments interfering with the antibody's biological properties. These results can help understand the precise role of antibody functional affinity in antigen‐antibody complex diseases and define the immunochemical characteristics of pathogenic complexes.     

Genetic characterization of plasmid formation by N- mutants of bacteriophage lambda.

Cells of plants infected with any of three strains of tobacco mosaic virus (the cowpea, common, and a wheat strain) contain, in addition to full-length virus rods, a heterodisperse population of rod fragments. RNA extracted from purified virus showed that some of these rods were of discrete sizes; the RNAs ranged in molecular weight from about 0.28 to 1.7 × 10⁶, containing 3′-ends identical to the original 3′-end of viral RNA, as judged by their capacities to bind a specific amino acid. Only the cowpea strain produced a rod containing a small (0.28 × 10⁶M_r) RNA, which is known to be the mRNA for coat protein. Each strain produced an RNA of 0.68 × 10⁶M_r (intermediate-length RNA) which coded in vitro (using a wheat germ system) for an ～30,000 M_r polypeptide. Other RNAs (0.9 to 1.7 × 10⁶M_r) were less similar in molecular weights among the three strains, but the predominant in vitro product of each was also the ～30,000 M_r polypeptide. peptide maps comparing the translation products of the short and intermediate RNAs of the cowpea strain showed they were distinct polypeptides. We conclude that at least some of the less-than-full-length viral rods found in preparations of the common and wheat strains of TMV, as well as those previously reported for the cowpea strain, represent the encapsidation of subgenomic portions of the viral RNA which are engendered during viral replication.     

Comparative NMR studies of diffusional water permeability of red blood cells from different species. X. Camel (Camelus dromedarius) and alpaca (Lama pacos)

The diffusional water permeability (P _d) of camel and alpaca red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition withp-chloromercuribenzene sulphonate (PCMBS). The values ofP _d were, in the case of alpaca RBC≈4.6×10^?3 cm/s at 25°C, 5.4×10^?3 cm/s at 30°C, 6.6×10^?3 cm/s at 37°C and 7.7×10^?3 cm/s at 42°C. In case of camel RBC the values ofP _d where ≈4.2×10^?3 cm/s and 9.0×10^?3 cm/s at 42°C. Systematic studies on the effects of PCMBS on water diffusion in camel RBC indicated that the maximal inhibition was reached in 45 min with 1–2 mm PCMBS. The values of maximal inhibition were around 47% at 25°C and 68% at 30°C for alpaca RBC and around 62% at 25°C and 56% at 37°C for camel RBC. The basal permeability to water of alpaca RBC was estimated at around 2.6×10^?3 cm/s at 25°C, 1.7×10^?3 cm/s at 30°C and of camel RBC as 1.8×10^?3 cm/s at 25°C and 3.0×10^?3 cm/s at 37°C. The values of the activation energy of water diffusion (E _{a, d}) were around 23 kJ/mol for camel and 34 kJ/mol for alpaca RBC. This suggests that in addition to the number of transport channels other features of the pathways might be important for defining the temperature dependence of the water permeability.     

Characterization of the interactions between immobilized parathion and the corresponding recombinant scfv antibody using a piezoelectric biosensor

A piezoelectric quartz crystal sensor with immobilized parathion was used for real‐time kinetic characterization of the interactions with recombinant anti‐parathion single chain Fv antibody IFRN AA01 (scFv). Parathion was linked to the sensor gold electrodes modified with a self‐assembled layer of aminothiophenol using either bovine serum albumin (BSA) or dextran as spacer molecules. The kinetic dissociation rate constant k_d was 8 × 10^?4 s^?1 for both types of sensors, and the association rate constants k_a, were 590 and 260 mol^?1 1 s^?1 for BSA and dextran‐linked parathion, respectively. The regeneration of BSA‐parathion coated crystals was not successful, however. Those crystals with bound scFv were used to study the formation of scFv dimers. Dextran‐parathion modified crystals were successfülly regenerated using proteinase K. The affinity of scFv to dextran‐parathion was 50 X lower when compared with the affinity of the anti‐parathion monoclonal antibody (IgG, IFRN 1701) the sequence of which served for production of scFv.     

The Production and Characterization of Novel Heavy-Chain Antibodies Against the Tandem Repeat Region of MUC1 Mucin

Camelidae are known to produce immunoglobulins (Igs) devoid of light chains and constant heavy-chain domains (CH₁). Antigen-specific fragments of these heavy-chain IgGs (VHH) are of great interest in biotechnology applications. This paper describes the first example of successfully raised heavy-chain antibodies in Camelus dromedarius (single-humped camel) and Camelus bactrianus (two-humped camel) against a MUC1 related peptide that is found to be an important epitope expressed in cancerous tissue. Camels were immunized against a synthetic peptide corresponding to the tandem repeat region of MUC1 mucin and cancerous tissue preparation obtained from patients suffering from breast carcinoma. Three IgG subclasses with different binding properties to protein A and G were purified by affinity chromatography. Both conventional and heavy-chain IgG antibodies were produced in response to MUC1-related peptide. The elicited antibodies could react specifically with the tandem repeat region of MUC1 mucin in an enzyme linked immunosorbant assay (ELISA). Anti-peptide antibodies were purified after passing antiserum over two affinity chromatography columns. Using ELISA, immunocytochemistry and Western blotting, the interaction of purified antibodies with different antigens was evaluated. The antibodies were observed to be selectively bound to antigens namely: MUC1 peptide (tandem repeat region), human milk fat globule membrane (HMFG), deglycosylated human milk fat globule membrane (D-HMFG), homogenized cancerous breast tissue and a native MUC1 purified from ascitic fluid. K_a values of specific polyclonal anti-peptide antibodies were estimated in C. dromedarius and C. bactrianus, as 7 × 10¹⁰ M^{? 1} and 1.4 × 10¹⁰ M^{? 1} respectively.     

Kinetics of the Reaction Between DNP-Insulin and Homologous Antibody

Kinetic and equilibrium studies were performed on the reaction between monovalent DNP-insulin (DNP-I) and antibodies specific for the 2,4-dinitrophenyl (DNP) determinant. The average intrinsic association constant as determined by fluorescence quenching was 1.2 × 107 M^-1. Kinetic experiments, using a stopped-flow technique, revealed a biphasic behaviour, and the rate constant of association, obtained fran the initial linear portion of the rate data, was calculated to be 4.7 ± 0.5 × 10⁶ M^-1sec^-1.     

Affinity of the interaction between Fcgamma receptor type III (FcγRIII) and monomeric human IgG subclasses. Role of FcγRIII glycosylation

Binding of the Fc region of IgG antibodies to low affinity Fcγ receptors (FcγR) triggers important effector functions in the immune system. The type IIIb FcγR (FcγRIIIb or CD16) is a heavily glycosylated protein anchored to the membrane of neutrophils by a glycosylphosphatidylinositol link. This receptor contributes to cell activation by IgG immune complexes. To better understand the nature of the ligand-receptor association, we have studied the affinity and kinetics of the interaction between human IgG subclasses and two soluble forms of FcγRIIIb (sFcγRIIIb or sCD16) corresponding to the 188 N-terminal amino acids of the extracellular region of the receptor, a glycosylated one made in eucaryotic cells (euc.sCD16) and a non-glycosylated one (proc.sCD16) made in Escherichia coli. Experiments using a BIAcore^TM instrument, to measure protein binding in real time, showed that monomeric human IgG1 and IgG3, but not IgG2, IgG4, IgA and divalent antigen-binding fragments (F(ab′)₂) of IgG1, bound to immobilized euc.sCD16 with an affinity constant (K_A) of 1.3 ± 0.6 × 10⁶ M^?1 and 2.6 ± 0.4 × 10⁵ M^?1, respectively. The affinity constant of proc.sCD16 for human IgG1 was in the same range (1.1 ± 0.2 × 10⁶ M^?1), whereas that for human IgG3 was twofold higher (4.2 ± 0.4 × 10⁵ M^?1). The specificity of the non-glycosylated receptor for human IgG subclasses bound to Sepharose was IgG1 > IgG3 ? IgG4 ? IgG2. Thus, the extracellular polypeptide of FcγRIIIb dictates the interaction of the receptor with IgG subclasses although glycosylation plays an inhibitory role in the interaction with human IgG3.     

Testicular morphology and antispermatogenic effects of 2,4-dichlorophenoxyacetic acid (2,4-D) in male West African Dwarf (WAD) goats

This study investigated the testicular morphology as well as the gonadal and extra-gonadal sperm reserves of West African Dwarf (WAD) goats exposed to graded levels of 2,4-dichlorophenoxyacetic acid (2,4-D). Twenty male WAD goats of five goats per group were used for this study. Goats in groups A, B and C received low [75?mg/kg body weight (BW)], medium (100?mg/kg BW) and high (125?mg/kg BW) dose levels of 2,4-D, respectively. The group D goats served as the control. On day?112, goats in the four groups were sacrificed and the testicular and epididymal sperm reserves were determined. Histopathologic changes in the testis of the 2,4-D-exposed and control goats were also assessed. The mean number of spermatozoa in the testes and the various segments of the epididymides decreased significantly (p?<?0.05) in all the treatment groups relative to the control. Combined testicular sperm reserve per millilitre for the treatment groups (group A—19.61?±?2.63?×?10⁸, group B—12.02?±?1.02?×?10⁸ and group C—9.95?±?0.97?×?10⁸) reduced significantly (p?<?0.05) relative to the mean value (23.52?±?4.43?×?10⁸) of the control—group D. The total epididymal sperm reserve per millilitre in the treatment groups (group A—24.25?±?4.19?×?10⁸, group B—17.18?±?2.57?×?10⁸ and group C—17.88?±?2.89?×?10⁸) was also found to be significantly (p?<?0.05) lower than the mean value (40.85?±?11.24?×?10⁸) for the control—group D. This reduction in the testicular and epididymal sperm counts of the 2,4-D-exposed WAD goats in this study suggest disruption in spermatogenic activity, which may lead to low productivity. Variable degrees of circulatory disturbances were observed in the testis sections of 2,4-D-exposed goats.     

Ligand binding and physicochemical characteristics of an IgM mouse plasmacytoma ABPC-22

Immunoglobulin IgM₂₂, secreted by mouse plasmacytoma ABPC-22, was characterized on the basis of ligand binding and physicochemical properties. Upon purification, only two polypeptides (μ aqnd κ chains) were detected by 6 M urea isoelectric focusing in polyacrylamide and SDS-polyacrylamide electrophoresis (SDS-PAGE). The μ and κ chains possessed pI values of 6.27 and 6.13 respectively, in 6 M urea. The molecular masses of μ and κ chains were established by SDS-PAGE and molecular sieve chromatography to be 70 and 30 kd, respectively. Thus, a molecular weight of 1 × 10⁶ was estimated for non-reduced IgM₂₂, and was confirmed by molecular sieve chromatography and sedimentation equilibrium ultracentrifugation. J chain was detected in freshly purified samples. As shown by competitive hapten inhibition (CHI) of the precipitation assay, using ¹²⁵I-rhodamine B₅BSA as the reference ligand, IgM₂₂ bound a wide range of structurally related and unrelated probes. Competing for the same site were polyaromatic anions (such as pyrene-1-sulfonate, rhodamine B and fluorescein) and weakly inhibitory organic acids (such as acetate and propionate). In addition. Fab fragments inhibited precipitation of ¹²⁵I-rhodamine B₅BSA by IgM₂₂. Equilibrium dialysis studies revealed a K_af 1.5 × 10³M^?1 for the interaction of ¹⁴C-fluorescein with IgM₂₂. A K_a of 3.2 × M^?1 for rhodamine B was determined using a molecular sieve column equilibrated in varying concentrations of rhodamine B. Based on relative K_a values, CHI results and fluorescence binding data with anilinonaphthalene sulfonate (ANS), the nature of the antibody active site was described in detail.