Binding properties of a monoclonal antibody directed toward lead-chelate complexes.

A monoclonal antibody (2C12) that recognizes a Pb(II)-cyclohexyldiethylenetriamine pentaacetic acid complex was produced by the injection of BALB/c mice with a Pb(II)-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to interact with a variety of metal-free chelators and metal-chelate complexes was assessed by measuring equilibrium dissociation constants. The antibody bound to metal-free trans-cyclohexyldiethylenetriamine pentaacetic acid (CHXDTPA) with an equilibrium dissociation constant of 2.3 x 10(-)(7) M. Addition of Pb(II) increased the affinity of the antibody for the complex by 25-fold; Pb(II) was the only metal cation (of 15 different di-, tri-, and hexavalent metals tested) that increased the affinity of the antibody for CHXDTPA. The increased affinity was due primarily to an increase in the association rate constant. The antibody also had the ability to interact with ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), and structurally related derivatives, but with affinities from 50- to 10000-fold less than that determined for CHXDTPA. Addition of metals to EDTA-based chelators reduced the affinity of the antibody for these ligands. However, when DTPA was used as the chelator, addition of Pb(II) increased the affinity of the antibody for the complex by 200-fold. The sensitivity of prototype immunoassays for Pb(II) could be modulated by changing the structure of the immobilized metal-chelate complex and/or the soluble chelator used to complex Pb(II) in the test solution.

Metal binding properties of a monoclonal antibody directed toward metal-chelate complexes.

A monoclonal antibody that recognizes cadmium-EDTA complexes has been produced by the injection of BALB/c mice with a metal-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to recognize 16 different metal-EDTA complexes was assessed by measuring equilibrium binding constants using a KinExATM immunoassay instrument. The antibody bound to cadmium- and mercury-EDTA complexes with equilibrium dissociation constants of 21 and 26 nM, respectively. All other metal-EDTA complexes tested, including those of Mn(II), In(III), Ni(II), Zn(II), Co(II), Cu(II), Ag(I), Fe(III), Pb(II), Au(III), Tb(III), Ga(III), Mg(II), and Al(III) bound with affinities from 20- to 40,000-fold less than that determined for the cadmium-EDTA complex. With the exception of mercury and magnesium, the binding of divalent metal-chelate complexes was well-correlated with the size of the metal ion. The amino acid sequences of the heavy and light chain variable regions were deduced from polymerase chain reaction-amplified regions of the corresponding genes and subsequently used to construct molecular models of the antigen binding region. The key residue for cadmium binding in the model for 2A81G5 appeared to be histidine 96 in the heavy chain.

Hormonal regulation of peripheral blood mononuclear cells in sheep.

The presence of growth hormone receptors (GHR) on sheep peripheral blood mononuclear (PBMN) cells was studied in two ways. The first was to directly measure specific GH-binding sites on PBMN cells drawn from lambs from birth to 5 months of age. The second was to measure the effect of GH on resting and interleukin-2 (IL-2)-activated PBMN cells in vitro and also the effect of other hormones such as prolactin (PRL), insulin-like growth factor-1 (IGF-1), and glucocorticoid. The specific binding of [125I]human GH (hGH) was low on PBMN cells but increased (P < 0.05) with age up to 5 months. Interestingly, serum concentrations of GH-binding protein also increased (P < 0.01) with age and were highly correlated (r = 0.89; P < 0.05) with the specific binding of GH to PBMN cells. The addition of ovine GH (oGH) to PBMN cells in vitro resulted in increased (P < 0.01) proliferation (at a dose of 100 ng/ml). Higher doses of oGH (2 micrograms/ml) also increased PBMN cell proliferation. When PBMN cells were previously stimulated with IL-2, the dose of 100 ng/ml) oGH was no longer able to stimulate proliferation. IGF-1 inhibited (P < 0.04) resting PBMN cell proliferation at 100 ng/ml but had no effect on IL-2-activated PBMN cells. Other hormones such as PRL caused a stimulation of PBMN cell proliferation (P < 0.01) at 100 ng/ml, whereas dexamethasone (dex) inhibited PBMN cell proliferation at doses as low as 63 ng/ml. These studies show that GH, PRL, IGF-1, and glucocorticoids are effective in modulating the proliferation of PBMN cells from sheep and therefore suggest that a modulation of immune system function in vitro by these hormones is consistent with a purpose in vivo.

Concentrations of thymulin in unextracted serum from pigs, sheep and cattle as measured by ELISA.

These studies were conducted to develop an ELISA for measurement of thymulin concentrations in unextracted blood serum (or plasma) from domestic animal species (pigs, sheep and cows). This assay was quite variable (intraassay C.V. of 13.3 and 6.4% at 12.6 and 50.5 pg/mL and interassay C.V. of 24.2%). Serial dilutions of serum from these species produced inhibition curves parallel to the reference standard, suggesting that there were no substances in serum causing non-specific interference in the assay. In addition, none of the other thymic peptides tested resulted in problematic displacement of thymulin binding to the antiserum. Using this assay, it was found that somatotropin (ST) treatment had no effect on serum thymulin concentrations in either pigs or cows. Chromatographic separation of thymulin activity in sheep serum showed three peaks with approximate MW estimates of 95, 80 and 1 kDa. Serum thymulin concentrations in a sheep injected with thymulin was cleared from blood with a half-life (t1/2) of 10.3 +/- 0.6 min. Serum thymulin concentrations increased between birth and 6 mo old in pigs. These data indicate that a rapid and reliable ELISA has been developed to measure thymulin in blood of these domestic animals. This assay should be of value in the study of thymulin function and factors regulating its secretion.

Monoclonal antibodies against surface antigens of Pasteurella multocida strain P-1059.

Four monoclonal antibodies (MAbs) were developed against serotype 3:A, P-1059 strain of Pasteurella multocida. Enzyme-linked immunosorbent assays were used to screen those hybridomas producing antibodies to either a surface protective (2.5 S) or lipopolysaccharide (LPS) antigen. MAbs 6EE11, D7H10, E11E3, and C11H2 were positive against 2.5 S antigen, and two of them, E11E3 and C11H2, were positive for the LPS antigen. MAbs 6EE11 and D7H10 reacted with a major protein band of molecular weight of 35,500, whereas E11E3 and C11H2 recognized a band with a molecular weight of 12,500 of the 2.5 S antigen. Treatment of the 2.5 S antigen with periodic acid abolished epitopes reacting with E11E3 but not with 6EE11. MAb 6EE11 did not recognize any band in Western blot after proteinase K treatment of the 2.5 S antigen, whereas antibody activity of E11E3 did not change. MAb 6EE11 reacted with serotypes 3, 4, 9, 10, 11, 12, and with M-9 strains in the immunofluorescence test. MAb E11E3 was positive only with serotype 3 or 10 strains, excluding M-9 strain. Electron microscopic studies with P-1059 strain indicated that antigens binding to 6EE11 and/or E11E3 were present in the capsule.