16.2.1: Introduction

This type of serologic testing employs known antiserum (serum containing specific known antibodies). The preparation of known antibodies is prepared in one of two ways: in animals or by hybridoma cells.

1. Preparation of known antisera in animals.

Preparation of known antiserum in animals involves inoculating animals with specific known antigens such as a specific strain of a bacterium. After the animal's immune responses have had time to produce antibodies against that antigen, the animal is bled and the blood is allowed to clot. The resulting liquid portion of the blood is the serum and it will contain antibodies specific for the injected antigen.

However, one of the problems of using antibodies prepared in animals (by injecting the animal with a specific antigen and collecting the serum after antibodies are produced) is that up to 90% of the antibodies in the animal's serum may be antibodies the animal has made "on its own" against environmental antigens, rather than those made against the injected antigen. The development of monoclonal antibody technique has largely solved that problem.

2. Preparation of known antibodies by monoclonal antibody technique.

One of the major breakthroughs in immunology occurred when monoclonal antibody technique was developed. Monoclonal antibodies are antibodies of a single specific type. In this technique, an animal is injected with the specific antigen (see Fig. \(\PageIndex{1}\), step 1) for the antibody desired. After appropriate time for antibody production, the animal's spleen is removed. The spleen is rich in plasma cells and each plasma cell produces only one specific type of antibody. However, plasma cells will not grow artificially in cell culture. Therefore, a plasma cell producing the desired antibody is fused with a myeloma cell ,a cancer cell from bone marrow which will grow rapidly in cell culture, to produce a hybridoma cell (see Fig. \(\PageIndex{1}\), step 2). The hybridoma cell has the characteristics of both parent cells. It will produce the specific antibodies like the plasma cell and will also grow readily in cell culture like the myeloma cell. The hybridoma cells are grown artificially in huge vats where they produce large quantities of the specific antibody (see Fig. \(\PageIndex{1}\), step 3).

Fig. \(\PageIndex{1}\): Production of Monoclonal Antibodies, Step-1	Fig. \(\PageIndex{1}\): Production of Monoclonal Antibodies, Step-2	Fig. \(\PageIndex{1}\): Production of Monoclonal Antibodies, Step-3
Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0

Monoclonal antibodies are now used routinely in medical research and diagnostic serology and are being used experimentally in treating certain cancers and a few other diseases.

3. The concept and general procedure for direct serologic testing.

The concept and general procedure for using antigen-antibody reactions to identify unknown antigens are as follows:

• Concept:

  This testing is based on the fact that antigen- antibody reactions are very specific. Antibodies usually react only with the antigen that stimulated their production in the first place, and are just as specific as an enzyme-substrate reaction. Because of this, one can use known antiserum (prepared by animal inoculation or monoclonal antibody technique as discussed above) to identify unknown antigens such as a microorganism.

• General Procedure:

  A suspension of the unknown antigen to be identified is mixed with known antiserum for that antigen. One then looks for an antigen-antibody reaction.

Examples of serologic tests used to identify unknown microorganisms include the serological typing of Shigella and Salmonella, the Lancefield typing of beta streptococci, and the serological identification of Neisseria gonorrhoeae and Neisseria meningitidis. Serological tests used to identify antigens which are not microorganisms include blood typing, tissue typing, and pregnancy testing.

4. Examples of direct serologic testing to identify unknown antigens

As stated above, this type of serologic testing uses known antiserum (antibodies) to identify unknown antigens. Six such tests will be discussed and four looked at in lab today.

• Serological Typing of Shigella
• Serological Typing of Streptococci
• Serological Testing to Diagnose Pregnancy
• COVID-19 Rapid Antigen Tests
• Identification of Microorganisms Using the Direct Fluorescent Antibody Technique
• Identification of SAR-CoV-2 RNA Using RT-PCR

Contributors and Attributions

• Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)