Antibody precipitin

The Mancini and Ouchtedony techniques are the basis of the techniques employed in immunoelectrophoresis. A technique referred to as rockets (59) is named as such because of the appearance of a rocket-shaped antigen—antibody precipitin formed after an antigenic sample is electrophoresed through a gel-containing antibody (Fig. 3c). 

Precipitin Reactions Free soluble antigen in excess of antibody Free antibody Precipitin complex deposited in vascular epithelium... 

The patient s serum or urine sample and a Normal Human Serum Control are electrophoresed on the agarose plate. Antisera then are added to troughs in the plate and allowed to diffuse into the agarose support medium. When a favorable antigen-to-antibody ratio exists, a precipitin arc will form on the plate. Diffusion is halted by rinsing the plate in 0.85 % saline. Unbound protein is washed from the plate by the saline, and the antigen/antibody precipitin arcs are stained with a protein-sensitive stain. The precipitin arcs formed by the patient s sample and the control are compared for a semiquantitative protein analysis. 

Sugg and Hehre43 also obtained precipitin reactions with dextran or with sterile filtrates of sucrose broth cultures of L. mesenteroides (designated for convenience strain A) and not only anti-Leuconostoc sera, but also pneumococcus Types II, XII and XX antisera. Leuconostoc organisms cultured on D-glucose broth neither stimulated the production of dextran-reactive antibodies in rabbits, nor absorbed dextran-reactive antibodies from sera, as did organisms cultured on sucrose. Absorption with the homologous bacteria (Leuconostoc, pneumococcus Types II,... 

Blood film No. of pa tientswith a positive precipitin line Malarial antibody titer IgG (mg/ml) IgM (mg/ml) IgA (mg/ml)... 

With individuals of blood group 0, injection of A and B substances gave a big increase in the amounts of precipitins. For example, the specific antibodies to the blood group A substance could be obtained from the specific precipitate by the use of 15% sodium chloride for dissociating... 

If an antigen-antibody complex has to be precipitated by a secondary antibody (e.g., antigen bound by mAb precipitated by goat anti-(mouse-IgG) antibody), this range of equivalence must be known. For estimation of the range of equivalence the precipitin assay is used. 

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

C Antiserum containing antibody is placed in trough. D Insoluble antigen-antibody complexes form precipitin arcs. 

Fig. 2. Ouchterlony double-diffusion technique. The antigen is placed in the center well, cut in an agarose gel, and different antisera in a range of dilutions are placed in the sunounding wells. Antigen and antiserum diffuse toward each other and form a white precipitin line where an antibody recognizes the antigen.

It was noted in previous reviews (1,3) that injection of urease into rabbits elicited an antibody and that the precipitate formed between urease and its antibody still possessed catalytic activity. This indicates that the antigenic and catalytic regions are not identical. A more recent study employing horse and rat antisera (66) revealed only one major antigenic component in urease preparations and confirmed the lack of complete enzyme inhibition by the precipitin reaction. 

The binding equilibrium expressed as shown above (2.2) is actually a gross oversimplification of the situation. The heterogeneity of the binding sites and multiple valency of individual antibodies lead to formation of secondary bonds that contribute to hysteresis or ripening of the antibody-antigen complex. Its ultimate form is the polymerization of a primary complex, which happens when the antigen is also polyvalent. Formation of the polymer (precipitin reaction) renders such a reaction virtually irreversible. 

Application of 125I Radioimmunoassay to Measure Inhibition of Precipitin Reactions using Carbohydrate-Specific Antibodies, P. H. Boullanger, A. Nagpurkar, A. A. Noujaim, and R. U. Lemieux, Can. J. Biochem., 56 (1978) 1102-1108. 

The anti-S. faecalis antiserum reacted with Lac-BSA amd the glycan as shown in the bottom panels of Figure 8. The amti-lac antibodies from this serum also reacted with these two immunogens yielding a similar pattern as the unfractionated amtiserum. It should be noted that the amtiserum amd amtibody preparations exhibited partial identity with these two antigens. The anti-gal antibodies also reacted with Lac-BSA amd the glycan but the precipitin bamds were quite different. 

Figure 12 Double diffusion experiments using a polyclonal rabbit antibody to the eofi 1-ASP (center well) against four different kASP preparations. Only die unmodified K ccU preparation forms a precipitin line of complete Identity (a), whereas after freezing and ihawing PEG-ASP also reads (b).

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