• Diagnosticums. Antibody-containing antisera

The suspension of dead (and occasionally living ) microorganisms or their antigens is called diagnosticums. Diagnosticums of killed microbes are fairly stable, retaining their properties for several years and present no risk of contamination. Sometimes bacterial antigens of different microbes may produce false positive results with non-absorbed antisera because they possess relative antigens (e.g., bacteria of genera Escherichia and Shigella are the members of the same family Enterobacteriaceae. Therefore non-pathogenic E.coli may produce false positive results with Salmonella-antiserum). To prevent “cross-reactivity” of identical antigens of various bacteria O-, H- and K-bacterial diagnosticums are prepared.

Erythrocytes sensitized with antigens are called erythrocytic diagnosticums. Sheep red blood cells which possess high adsorptive capacity are the most commonly used erythrocytes for preparing erythrocytic diagnosticums. Diagnosticums contain known antigens (bacterial, viral, etc.) and are usually used to detect unknown antibodies in the patient’s serum. Sometimes antibodies can be adhered to the surface of erythrocytes. Preparation, that contains erythrocytes coated with known antibodies is called erythrocytic antibody-containing diagnosticum.

NOTE: The antigens labeled with fluorochromes, enzymes and radioactive agents will be discussed later (see LESSON 14).

Several antibody-containing antisera are employed to determine the unknown antigen. There are different types of diagnostic antibody-containing antisera. For example, agglutinin-containing antisera (agglutinating antisera) for agglutination tests, precipitin-containing antisera (precipitating antisera) for precipitation tests, etc. These diagnostic antibody-containing antisera are usually prepared by hyperimmunization of animals (rabbits, goats or donkeys) with certain antigen (agglutinogen or precipitinogen, etc.) and by concentration of specific antibodies collected from the animal serum several weeks later.

The titer of the agglutinating antiserum (agglutinin-containing antiserum) is the minimum concentration of serum antibodies (=maximum antiserum dilution) which can interact with the corresponding antigen to produce readily visible clumps.

The titer of the precipitating antiserum, in contrast to the titer of agglutinating antiserum, is determined by the maximum dilution of the antigen which is precipitated by the specific antiserum.

This is explained by the fact that the antigen participating in the precipitation reaction has an infinitesimal magnitude and that in a volumetric unit of the serum there are much more antigens than antibodies.

NOTE: One should not mix up the terms “SERUM” and “ANTISERUM”!!! Antiserum contains known antibodies and is used for diagnosis, treatment or immunoprophylaxis. It is obtained by immunizing animals with corresponding known antigen.

The term “serum” is commonly used for patient’s tested serum with the “unknown antibodies”. These antibodies are identified by diagnosticums with known antigens in the serologic tests.

The agglutinating antiserum, obtained by immunizing a rabbit (or other animal) with the whole bacterial cells, is called native non-absorbed antiserum. This antiserum contains antibodies which can false react with identical antigens of other microorganisms (these antibodies are usually termed «cross-reactive antibodies»). Possible cross-reactions between related antigens can limit the test’s specificity. That is why the immune pre-absorbed antisera are more preferable. The Castellani technique is used to prepare these antisera. Antibodies, which can cause false-positive result with the related antigens are adsorbed on the identical antigens (agglutinogens) of another microbes and then eliminated from the serum. Therefore, the pre-absorbed antiserum is more specific and it can couple only with certain antigen (or antigens). There are monovalent pre-absorbed antisera, which can react only with one antigen or receptor, and polyvalent pre-absorbed antisera, that contain more than two types of antibodies and are able to bind with several antigenic determinants of the microorganism.

NOTE: Monoclonal antibodies are excellent tools for identification of antigens because heterogeneity of antibodies is absent.

NOTE: Do not confuse monovalent antisera and monoclonal antibodies. Monoclonal antibody is the immunoglobulin manufactured by cloned cell cultures (hybridoma technology). (For Hybridoma Technology see Relevant LECTURE).