Chapter 32: Serological Tests and Its Basis
Serology is an in-vitro study of serum where we see antigen and antibody reactions.
Serological tests are used for:
- Diagnosis of the diseases.
- It helps in treatments.
- Guide for the prognosis of the disease.
- Antigen and antibody can be quantitated.
Serological tests may be:
- Specific for the diagnosis of viral, and bacterial infection e.g. Widal test for Enteric fever and test for Brucellosis.
- Non- specific tests give evidence for some diseases like Syphilis and SLE.
Serological tests can be:
The main use of Serological Test
- Identification of microorganisms.
- Detection of antibody.
- Detection of autoantibody.
- Detection of offending drugs.
- Immunoassay of hormones. These serological means can measure the minute amount of hormones.
- Detection of immunologic abnormalities.
Disadvantages of Serological Tests
- These tests have little value in the early phase of the disease that is 7-10 days. Usually, these tests are positive after 10 days.
- Changing titer (rising) is more important for confirmation of the disease. If the titer is low and there is no rising (change) titer after 5-7 days, then this test has no value.
- The best sample is serum.
- Other fluids, which can be used, are:
These serological tests are based on antigen and antibody reaction. These are:
- Flocculation-modification of precipitation
- Fluorescent antibody test
- ELISA (Enzyme-linked immuno-absorbed assay)
- Radioimmunoassay (RIA)
The antigens are in a particulate form where antigen and antibody form clumps. This is a direct measurement of antibody binding to antigen. It is used in the quantitative serologic assay. Antibodies are called agglutinin.
The antigen may be present on bacterium which forms an agglutinate with the antibody. While antigen on RBC forms haemagglutination.
The antigen may be bound to RBC and antibody may be detected this is called passive haemagglutination.
- For ABO blood grouping.
Widal test for diagnosis of enteric fever etc.
This reaction of Ag & Ab is just like agglutination with the difference that here antigens are insoluble form. The antibodies giving precipitation are called precipitation.
Precipitation may be seen:
- In liquid media.
- In semisolid media.
Precipitation in Liquid Medium
Antigens and antibodies are present in liquid form and precipitate depend upon the concentration of antigens and antibodies. By fixing the number of antigens and with a serial dilution of serum (Ab), then the antibody can be quantitated.
Procedure: This is shown diagrammatically.
- Add Antigen to tubes.
- Add serum 0.1ml and 0.3ml normal saline (1:4 dilutions).
- Add 0.1ml normal saline to all other tubes.
- Now take 0.1ml from tube 1 and add to tube 2.
- Then take 0.1ml from Tube 2 to tube 3. In these ways, there will be serial dilution.
- Now the last tube showing a small amount of precipitate is the titer. e.g. in this case 1:32.
Precipitation in Semisolid Media
This is done in Gel (agar) and this method is called Ochterlony Gel diffusion, which was described by Swedish scientists. This can be used as:
- Qualitative or
This may be done as:
- Single radial immunodiffusion(SRID)
- Double radial immunodiffusion (DRID)
Single Radial Immunodiffusion (SRID)
This method can measure up to 1-3μg/ml. In this method antigen is placed in the central well of Gel-plate and antibody are mixed in the Gel. Now antigen diffuses in the medium and gives rise to a line of visible precipitation by a combination of Antigen and Antibody where they meet at the zone of equivalence.
Now a plot can be graphed by doing this procedure with a known concentration of antibody.
Double Radial Immunodiffusion (DRID)
In this method gel medium doest not contains any antigen or antibody. Three wells are made, where two antigens are placed in two wells, and in the third well antibody is added. Now Ag & Ab diffuse out in the gel-medium and give rise to a line of precipitate at the zone of equivalence.
Fluorescent Antibody Technique
Coons described this in 1940.
Antibodies are labeled by fluorescent material, then antigen is added. These are washed and seen with the help of a fluorescence microscope. If the antibody is not bound to the antigen, then no fluorescence will be seen.
Stain materials used are:
This method may be:
Direct Fluorescent method
The biopsy material is taken on the slide which contains antigen. Now add labeled antibody () and after incubation, wash the slide. Now see under the fluorescent microscope and in positive cases fluorescence will be seen.
Indirect Fluorescent Antibody
In this case, there is a double layer of antibodies. One is labeled and the other is unlabelled. First biopsy (antigen) is treated with an unlabelled antibody, then do the washing of the slide. Now add labeled antibody which will attach with antigen + antibody complex and fluorescence will be seen.
Advantages of Indirect F.A.
- There is better fluorescence.
- This test is more sensitive.
ELISA(Enzyme-linked Immuno Absorbent Assay)
Principle: ELISA relies upon the “capture” antibody fixed to the plastic plate. Antigen or antibody-coated beads or wells are taken. Suppose there are antigen-coated wells. Now add serum of the patient after washing and add enzyme-linked antibody to these wells, after incubation washes the wells. Now add substrate, color develops. Stop the reaction and read by photometer, which will measure absorbance according to the intensity of color, which depends upon the number of antibodies bound to the antigen.
The enzyme used is Peroxidase and alkaline phosphatase.
Now, this procedure can be modified by fixing the antibody to pallets and adding antigen with an enzyme.
- These are very sensitive and safe.
- There is no radiation hazard.
- These are economical and simple instruments are needed.
Principle: RIA is used for the detection of molecules (analyte) in the circulation. It depends upon the availability of an antibody that specifically recognizes the analyte. This is basically a competitive assay. A fixed amount of antibodies is added and which compete for the analyte, either in the sample or added to the sample in a radiolabeled form (e.g. bound to I125). Analyte-antibody complexes form and are precipitated by physicochemical means. The radioactivity in the precipitate is measured by Gamma-Counter.
A high level of radioactivity reflects the low concentration of an analyte in the serum sample, while low radioactive count indicates a high level of an analyte.
RIA methods are also used to measure small quantities like hormones even in pictogram levels.
Principle: This method is used to separate proteins in serum in the electrical field. Also used to separate different types of Haemoglobins. Proteins migrate within the electrical field according to their charge. This migration in turn is influenced by pH and the optimum separation achieved by Buffer at pH 8.6.
Once separated on gel or fitter, these are stained with dye.
Albumin is the major constituent of serum proteins and at this pH migrates fast as a negatively charged molecule towards the anode.
Electrophoresis used for:
- To find hypergammaglobulinemia.
- To find the oligoclonal band in myelosclerosis.
- Abnormality in other proteins.
- It can be used for Haemoglobinopathy.
Serum protein can be quantitated in the densitometer, which plot graph according to the concentration of each protein.
For separation of immunoglobulin, modification of electrophoresis is used as immunoelectrophoresis.