The immunochemistry of sandwich ELISAs--V. The capture antibody performance of polyclonal antibody-enriched fractions prepared by various methods.

Studies compare the performance of antibody-enriched serum fractions prepared by various methods, when adsorbed on polystyrene microtiter wells as capture antibodies (CAbs) and tested against multivalent antigens. The criteria of performance in the RIA used included antigen capture capacity (AgCC) and the nmol of functional capture sites per microtiter well (CAbt). Affinity purified polyclonal (pAb) and monoclonal antibodies (mAb) were employed as reference CAbs. AgCC was highest for enriched fractions prepared using caprylic acid and a high-pressure SpG affinity column. The performance of capture antibodies is expressed by an equation which was empirically derived and experimentally tested; CAbt x AgCC/ng adsorbed IgG. In terms of this parameter, CAb-enriched fractions prepared with caprylic acid performed best. The data reported also provide insight into solid-phase ligand immunochemistry. Adsorbed polyclonal CAb performed with remarkable homogeneity in percent bound and in Scatchard plots. Values obtained for CAbt from Steward-Petty plots were directly correlated with the length of the LBR of log-log percent bound plots but indicated that less than 10% of the potential capture sites of polyclonal CAbs remained functional after adsorption; mAb showed a more serious loss of activity. The loss of CAbt was a general phenomenon for all preparations tested although relative to their antibody content, certain antibody-enriched fractions retained a higher proportion of CAbt than their affinity-purified counterparts. Comparative studies in which the activity of adsorbed mAb and pAb was compared to the same antibodies immobilized by a non-adsorptive procedure, indicated that adsorbed CAbs also express lower affinity. The studies we report offer a single parameter criterium for comparatively evaluating CAb performance while simultaneously revealing the need to develop immobilization procedures that can preserve CAbt and antibody affinity so that immunoassays with wide dynamic ranges and high AgCC can be developed without waste of antibody.

A sandwich ELISA for properdin in clinical specimens.

We have developed a symmetrical sandwich ELISA for measuring human properdin (P) in serum by using the globulin fraction from a commercial antiserum as the capture antibody adsorbed on the plastic. The detecting reagent was a glutaraldehyde conjugate of this Ig fraction with alkaline phosphatase. Two types of inhibition were observed in this study. First, inhibition was observed when greater than 2.5 micrograms/ml of the globulin fraction was used to coat the plates. A second type of inhibition was observed for serum dilutions less than 1/400; it was independent of the concentration of capture Ab and did not occur when purified P was assayed. The data generated with this assay could be fitted in log-log mode by a quadratic equation. The coefficient of the linear term in this equation was found to be the same for serum and purified P, within the limits of experimental error. The results for different samples run on the same plate were expressed in terms of the relative concentration of each sample required to produce an OD405 = 0.2. A sample of pooled normal human serum was run on each plate as a reference; it was assigned a titer of 100 ELISA units/ml (EU/ml). The titers of the unknown samples were expressed in terms of EU/ml by reference to this standard. For purified P, the assay could readily detect 10 ng/ml. By comparing purified P with our reference serum pool, we found that 1 EU equals 0.57 microgram. Day-to-day variation for a group of nine normal sera showed a mean difference of -0.85 EU/ml, SD 5.85 EU/ml. The mean titer for these normal sera was 78.9 EU/ml, SD 15.7 EU/ml. In three recovery experiments in which purified P was mixed with pooled normal serum, the recoveries ranged from 96 to 117%. We conclude that the sandwich ELISA constitutes an adequate immunochemical assay for human P in serum specimens.

Effect of reduction and alkylation on structure and function of rabbit IgG antibody--II. Effects on classical pathway C3 convertase formation.

Anaerobic reduction of purified rabbit IgG antibody (Ab) with 1.5 moles of dithiothreitol per mole of Ab at pH 8.0, followed by alkylation, cleaves 39% of the inter-heavy-chain (H-H) disulfide (SS) bonds. This treatment has the following effects on the ability of the Ab to activate the classical pathway of complement. Compared to control Ab, reduced and alkylated (RA) Ab retained 4-5.6% of overall hemolytic activity and 55% of complement-fixing activity at 0 degrees C. Complexes of RA Ab and equivalent amounts of soluble Ag consumed C4, C2 and C3 at 37, 51 and 44%, respectively, of the rate at which these components were consumed by equal concns of complexes containing control Ab. Complexes made with RA Ab bound 18% as much C-1 as those made with native Ab. These data indicate that the principal, if not the only, effect of RA is on C-1 binding. Measurements of the ability of complexes of Ab with cell-bound Ag to bind C-1 showed at most a 20% loss of C-1 binding sites and a ca two-fold decrease in affinity for C-1. Similar results were obtained with purified (activated) C-1 and with native C1 in serum. No significant difference could be detected in the rate of activation of bound C1. Normal rabbit IgG which was reduced and alkylated under the same conditions retained 52% of its H-H SS bonds and 30% of its ability to bind C-1. This finding suggests that the impairment in C-1 binding results from an effect on the C1 binding site itself, rather than from an effect on the ability of the RA Ab to transmit a putative conformational "signal" from the Ag-binding site to the C1 binding site. Finally, our data show that the observed functional effect of reduction and alkylation depends strongly on the assay used to evaluate that effect.

Immunoadsorbents for the isolation of high-affinity anti-hapten antibodies in high yield.

Anti-hapten antibodies with affinities close to 10(8)/M can be isolated in high yield by elution under mild conditions from immunoadsorbents prepared by coupling haptens directly to an aminoethylamino (AEA) derivative of beaded agarose. In experiments with rabbit anti-2,4-dinitrophenyl (DNP) antibody with an average association constant (Ko) of 1.2 X 10(9)/M, elution of antibody bound to a Sepharose-AEA-DNP column produced a yield of 57%. With rabbit antibody to the 5-dimethylaminonaphthalene-1-sulfonyl (DANS) hapten with Ko=2.7 X 10(8)/M and a Sips heterogeneity index a=0.52, a 20% yield of the antibody found in the starting material by quantitative precipitin analysis was obtained by specific precipitation and dissolution of the precipitate with 0.18 M Na DANSulfonate; this antibody had Ko=6.6 X 10(8)/M and a=0.66. Purification of another aliquot of the same antibody preparation by immunoadsorption on DANS-AEA-Sepharose and elution with the same hapten concentration produced a yield of 75% of antibody with Ko=5.7 X 10(7)/M and a=0.29. In 3 production runs with the new method, apparent yields of 181, 101 and 94% (based on quantitative precipitin analysis of the starting material) of anti-DANS antibody were obtained. In 2 of these runs, the immunoadsorbent was further eluted with 0.1 M acetic acid and then with 0.1 M acetic acid in 10% dioxane; additonal yields of 9 and 4% were obtained with acetic acid and further yields of 15 and 11% with acetic acid/dioxane; the antibody eluted with acetic acid was damaged as shown by its failure to mediate passive immune lysis of DANS-coupled sheep erythrocytes, but the antibody eluted with acetic acid/dioxane was recovered intact.

Cooperative binding of a complement component to antigen-antibody complexes. III. Complexes containing IgM antibodies.

The first component of guinea pig complement (C1)2) is bound in a cooperative manner to antigen-antibody complexes containing rabbit IgM antibodies, as was previously shown to be the case for IgG antibodies. The shape of the binding curves is consistent with an allosteric mechanism involving clusters of 10 interacting C1 binding sites. Similar results were obtained with IgM antibodies against an artificial hapten and against a natural constituent of the erythrocyte membrane.

Homotropic cooperative binding of the first component of guinea pig complement to rabbit IgG-erythrocyte complexes: a possible allosteric effect.

Binding of the activated first component of guinea pig complement to immune complexes formed between dinitrophenylated erythrocytes and rabbit IgG antibody to 2,4-dinitrophenylhapten has been studied quantitatively. Cooperative binding was observed; it in volves no interactions between the domains on the erythrocyte surface that bind the activated first component of complement, or between the activated complement molecules in solution. By curve-fitting methods, we find that the data are consistent with an allosteric model, which assumes 10 binding sites per domain, a low allosteric equilibrium constant, and virtually exclusive binding to one of the isomers.