This document discusses principles of precipitation reactions and their applications in molecular immunogenetics testing. It describes how precipitation occurs when an antibody reacts with a soluble antigen, forming insoluble complexes. This reaction is used in tests like immunofixation electrophoresis and nephelometry. Nephelometry detects antigen-antibody complexes in solution by measuring light scattering. Immunofixation electrophoresis combines protein separation by electrophoresis with precipitation to identify monoclonal proteins. Precipitation reactions are useful for detecting interactions between antibodies and antigens.

1. Molecular Immunogenetics
Vytautas Magnus University..

2. Immuno-serological tests
Precipitation
Agglutination
neutralization

3. 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.

4. 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.

5. 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).

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

7. 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.

8. 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.

9. Two techniques utilizing the precipitation reaction will be
demonstrated:
1. Nephelometry
2. Immunofixation Electrophoresis

10. 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.

13. 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.

15. Immunofixation Electrophoresis (IFE) - Introduction
Immunofixation electrophoresis combines both electrophoretic
separation 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).

16. 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
successfully
treated the monoclonal immunoglobulin will disappear from
the circulation and will not be visible on the SPE gel.

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