Antigenic precipitin curves

A typical precipitin-curve for the reaction320 between con A and dextran B-1355-S is presented in Fig. 4. Analogous to antibody-antigen precipitin curves, there are three zones lectin excess (all added dextran is precipitated), equivalence (virtually all dextran and lectin are precipitated), and polysaccharide excess (soluble complexes are formed). Optimal precipitation of con A by dextran B-1355-S occurred in 24 h at 25°, between pH 6.1 and 7.2, and was unaffected320 by (buffered) concentrations of sodium chloride of up to 4.2 M. 

The O-phosphonomannan from Pichia pinus effectively inhibited hemagglutination and also precipitated with the lectin both in agar-gel double-diffusion and in solution.123 The flocculation profile was very similar to an antibody-antigen precipitin curve, and proceeded best at 20°, pH 6-8, and an ionic strength >0.1. 

Fig. 3.— Typical Precipitin Curve for an Antigen-Antibody Combination.

One may thus interpret the precipitin curve of a cross-reaction as being due essentially to the presence of a proportion of antibodies having a site capable of accommodating the homologous as well as the crossreacting determinant, the remainder of the antibodies being directed toward determinants that do not cross react. Studies on many types of antigens have amply justified this type of analysis. " ... 

Figure 9-3 Schematic diagram of precipitin curve illustrating zones of antibody excess (A), equivalence (B), and antigen excess (C). The parameter measured may be quantity of protein precipitated, light scattering, or another measurable parameter. Antibody concentration is held constant in this example.

Values were determined at equivalence from precipitin curves on each antigen with the adsorbed antiserum, and each represents the average of three or six replicate determinations which varied 1.2% or less. 

Antibody absorbed was eluted from immunoadsorbent by 5 M guanidine HCl pH 7 and then dialyzed vs. water foBowed by phos riiate/safine buffer pH 7.2. The amount of total antibody was computed from absorbance at 280 nm usi a hictor of 1.38 for absorbance of 1 mg ant3>ody/ml. Precipitin curves were done on the antibody usii BSA and also the homologous native antigen as that on immunoadsorbent. 

The unadsorbed antibody was adsorbed on Sepharose BSA inununoadsorbent, and after elution with 5 M guanidine HCl and dialysis its concentration was calculated from its absorbance at 280 nm. Precipitin curves were done using BSA as antigen and percent of precipitating antibody was estimated. 

Hemocyanin is normally a strong antigen when injected into mammals it produces powerfull antibodies (Schmidt, 1920 Hooker and Body, 1936). Multiple peaks in the precipitin curves of hemocyanin-antihemo-cyanin systems were observed by Hooker and Body (1942) this finding, which can be explained by the dissociation of the protein into smaller molecules, would indicate that the antigenic structure of the dissociated molecule differs from that of the entire one. According to Bartell and Campbel (1959), antigen-antibody combining sites are present on the dissociated molecule which are not available to antibodies in the associated molecule. 

Precipitin tests As the name implies, precipitin tests rely on the fact that when the appropriate ratio of antibody and antigen are mixed together, immune complexes of antibodies and soluble antigens come out of solution, settling to form a visible precipitate. The antibody-antigen precipitate formation can be plotted as a curve, and interaction is maximal at the top of the curve (termed the zone of equivalence) shown in Figure 10.2. This technique can be used to quantify the antibody content of a solution. 

The precipitin line would curve toward the antigen well. 

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