Precipitation reaction

PRECIPITATIONMolecular Immunogenetics

Vytautas Magnus University..

Immuno-serological tests

Precipitation

Agglutination

neutralization

Precipitation has a clear role in downstream processing for product concentration, and ongoing developments promise a more important role in fractionation. Its advantages include adaptability to continuous processing and larger scales, the wide variety of possible precipitants, and the ability to retain biological activity. The focus here has been on precipitation of proteins, but many of the principles apply to nucleic acid removal and precipitation of lower-molecular-weight biological products, such as pharmaceuticals.

Introduction

Definition

Precipitin: Any antibody which reacts with an antigen to form a precipitate.

The reaction which occurs when specific antibody combines with soluble antigen is known as the precipitation reaction.

This reaction requires that the antigen contain multiple binding sites (epitopes) for the antibody or anti-sera used. This allows for cross-linking to occur with the formation of large macromolecular species. These large complexes become insoluble and subsequently precipitate. The precipitate may be read visually in a gel such as in immunofixation electrophoresis or may be measured by an increase in light scatter in a nephelometer. The end result of the precipitation reaction is to detect the interaction of the antibody with the antigen. Detection may be qualitative (immunofixation) or quantitative(nephelometry).

Difference in the visual appearance of an aggregate and a precipitate.

Precipitation reaction

Precipitation - Principles

The precipitation reaction is an older assay technique that originally relied strictly on visually detection to identify antigen-antibody interactions. Most older assays were performed in gels permeated with antibody. The antigens (in plasma or some other fluid) to be detected were placed into wells cut in the gels and allowed to diffuse into the gel containing the antibody. The antigen antibody complexes formed would then precipitate in the gel and be visible as a white precipitate. Assays for qualitative detection as well as quantitation were available using gel based assays.

The principle of the precipitation reaction that allows for quantitative measurement is called equivalence. This principle is based on the fact that the precipitation reaction will be maximal at the point that antigen (i.e., epitopes) and antibody are in equal numbers. The equivalence point is where maximal cross-linking will occur and the antigen-antibody macromolecule will be the least soluble.

The precipitation reaction can also be performed in liquid solution and is the basis for nephelometry. Since detection of precipitate in solution is inefficient by visual inspection, light scatter measurements can be used to detect the formation of the large antigen-antibody complexes before they become insoluble.

Two techniques utilizing the precipitation reaction will bedemonstrated:1. Nephelometry 2. Immunofixation Electrophoresis

Precipitation - Nephelometry - Introduction

Proteins in solution (such as IgG, transferrin or albumin in serum) will react with specific antisera (i.e., antibodies which bind that protein) and result in the formation of antigen-antibody complexes. The formation of the immune complexes can be detected by measuring the light scattered by the solution when a light source illuminates the sample. At the equivalencepoint the sample will scatter the maximal amount of light. This principle formed the basis for the original end-point nephelometers.

Serum Protein Electrophoresis (SPE) - Introduction

The electrophoretic analysis of proteins is based on their differential migration as charged particles in an electrical field. The individual protein molecules will move toward the electrode of opposite charge to that of the protein molecule itself. The speed is influenced by multiple physical characteristics of the molecules and supporting medium. By using zone electrophoresis the proteins are trapped during migration by the stabilizing medium and can be stained for quantitative analysis.

Immunofixation Electrophoresis (IFE) - Introduction

Immunofixation electrophoresis combines both electrophoreticseparation and immune precipitation of proteins. Identification can thereby be accomplished for a patient's individual proteins present in serum, urine, and other biologic fluid. This test is the confirmatory assay for identifying monoclonal immunoglobulins in patients suspected of B-cell (plasma cell) malignancy and where a gamma migrating paraprotein has been initially identified on SPE.

The diagnosis of a plasma cell malignancy relies on the detection of a monoclonal protein such as IgG Kappa in serum or urine which is referred toas a monoclonal gammopathy. Approximately 5% of individuals over the age of 60 will have a small amount of a monoclonal immunoglobulin presentin serum. In the absence of other features of malignancy this is called monoclonal gammopathy of undetermined significance (MGUS).

monoclonal gammopathy = malignant B-cell clone producing an abnormal immunoglobulin or immunoglobulin fragment. The disease may represent a lymphoma or more commonly a myeloma. The circulating monoclonal immunoglobulins often produce kidney disease and can be used to monitor response to therapy. When the malignant plasma cells are successfullytreated the monoclonal immunoglobulin will disappear from the circulation and will not be visible on the SPE gel.

REFERENCES

1. Baker F.J. Introducion to Medical LaboratoryTechnology;6thed, 1995.Butter worth.2. Cheesbrough Monica. Medical Laboratory Manual forTropical Countries; vol ll 2000.Cambridge Butterworth.Heinemann.Ltd.3. P.Stities Daniel. Basic and Clinical Immunology; 8thed,1994,USA 4.Fischbach Frances, Manual of Laboratoryand Diagnostic tests; 4ed 1992,Lippincott.5. Turgeon L.M, Immunology and Serology in LaboratoryMedicine,2nded, 1996,Mosby.6. Sood Ramnik. Medical laboratory Technology methodsand interpretation.4thed, New Delhi-India, JaypeeBrothers.

THANK YOU…