Immunological and Serological Reactions - labsstudies

Immunological and Serological Reactions

Immunological and Serological Reactions

Definitions of Terms in Immunological and Serological Reactions


is the binding of antigens and antibodies that results in the formation of visible particles is known as agglutination.


Involves reactions of soluble antigens that form insoluble deposits when they react with antibody (precipitate).


Involves reactions of soluble antigens, which form loose clumps of precipitate when reacting with antibody (floccules).

Immunochromatographic test (ICT)

Is the Runs solvent down the length of the chromatography paper or percolates upward.

Antibody molecules form immune complexes by reversibly combining with antigens. Serology, a subfield of immunology, is based on the detection and measurement of these reactions.


is the study of antibodies and antigens Reactions.

The immune response involves the production of antibodies (substances that protect the body from the antigen). However, there are times when antibodies are ineffective (e.g. Hay fever, rash).


Is the study of body defense mechanism.


Application of Immunological and Serological Reactions

 Agglutination test used in:

  •  Bacterial identification, such as Salmonella and Shigella serotyping with known antisera.
  •  Serological infection diagnosis, such as the Widal test for Typhoid.

Precipitation test used in:

  •  Bacterial identification, such as the detection of group-specific polysaccharides substance in the Lancefield group of streptococci.
  •  Detection of antigenic components of bacteria, such as Bacillus anthracis, in infected tissues.
  •  Toxin and antitoxin standardization.
  •  Antibody demonstration in serum VDRL, Widal test.

 flocculation test is used in:

  •  Cardiolipin antibody detection in Syphilis, such as RPR.

 Immune-chromatographic test (ICT) is used in:

  • Detection of  Hepatitis B.
  • Salmonella Typhi antigen detection in faces.
  •  Pregnancy test for HCG detection.
  • Specific Treponema Antibody in Syphilis.


Principles of Immunological/Serological Reactions

Agglutination test 

When a specific antigen is mixed with its antibody in the presence of electrolytes at a suitable temperature and pH, the particles clump or agglutinate.

Agglutination tests are commonly used because they are less expensive and easier to perform than other serological tests.

The agglutination test principle

The visible clumping together of bacteria, cells, or particles caused by an antigen combining with its specific antibody is known as agglutination.

The resulting clumps are known as agglutinates.

Antibodies that are specific Surface antigen antibody binds to antigen and agglutinates cells.

A known antigen (agglutinogen) suspension is used in tests to detect antibody (agglutinin) in a patient’s serum.

If the serum contains the corresponding antibody, the antigen particles become agglutinated. In general, a known antigen suspension or a specific antibody is added to detect antibody in patients’ serum.

Agglutination tests are available:

  1. on slides
  2. in tubes
  3.  microtitration plates.

1. Agglutination tests on slides

These are quick, simple techniques that produce results in minutes or even seconds. They are not, however, as sensitive as tube or microtitration techniques. Their specificity is determined by the reagent used. The agglutination process can be active or passive.

  • Slide tests for active agglutination

These are tests in which a bacterial antigen is directly agglutinated with its corresponding antibody. For example, using a specific antibody to agglutinate salmonellae, shigella, or Vibrio cholera on a slide. Slide agglutination tests are difficult to standardize and control when used to identify bacteria from cultures.

  • Passive agglutination slide tests

These are tests in which a specific antibody or known antigen is attached to inert particles or cells. When a known antigen or antibody combines with its corresponding antibody or antigen in a specimen, the particles or cells are only used to demonstrate that an antigen antibody reaction has occurred.


2. Agglutination tests in tubes

Agglutination occurs in a larger volume of fluid and thus in a more fully controlled environment in tube tests. Tube tests are more sensitive than slide tests in most cases. In this tube agglutination test, serum is serially diluted and then the antibody level is determined by adding a standard antigenic suspension. The temperature and time of agglutination must be accurate, and a control tube containing only the antigen suspension must be included to check for reagent autoagglutination.

Tube tests are primarily used in the investigation of enteric fever (Widal test) and brucellosis to detect and measure serum antibodies.

Antibody detection in serum (antibody titer) To diagnose microbial diseases based on their antibody response, the serum antibody level must typically increase three or fourfold. This is because the patient’s serum may already contain agglutinating antibodies from a previous infection or from natural or acquired immunization. As a result, two specimens must be tested (paired sera). The first was collected within 5 days of the onset of symptoms, and the second was collected 5-10 days later.

3. Agglutination tests using microtitration

These procedures are carried out in microtitration plates. They have now largely replaced several tube agglutination tests because they are more sensitive, less expensive, easier to perform, and usually provide faster results.

Flocculation test

  • A soluble antigen reagent is used to form flocculates by reacting with antibodies in the patient’s serum (Clumps of precipitate)

Precipitation Tests

o When a suitable antigen combines with its antibody in the presence of electrolytes (NaCl) at a suitable temperature and pH, an insoluble precipitate is formed.

Immunochromatographic examination (ICT)

The antibody in the specimen first comes into contact with a specific antigen bound to colloidal gold particles, and then binds to the antigen.

 The antibody-antigen colloidal gold complex migrates up the strip and is captured by a specific antigen line, resulting in a pink line in the test area.

 A further pink line, i.e. positive control, is produced above this, indicating that the test was successful.


Antigen tests

Antigen tests frequently allow for an early or presumptive diagnosis of an infectious disease by:

  •  Identifying a pathogen isolated by culture
  •  Pathogen identification in different patient samples, etc. Tests for antibodies.

These tests are primarily used to:

  • Diagnosis a microbial disease when the pathogen or microbial antigen is not present in routine specimens or is difficult to isolate and identify using other available techniques.
  •  Testing donor blood for various infectious diseases
  • Measuring antibody liters to assess the efficacy of a given treatment.
  •  To detect autoimmune disorders, for example.

Secondary binding examinations

Secondary binding examinations detect and quantify the outcomes (secondary effect) of antigen-antibody interactions.

Precipitation of soluble antigens, lumping (agglutination) of particulate antigens are some of the consequences.

Activation of the complement system and neutralization of bacteria, viruses, or toxins.

They are typically less sensitive than primary binding tests, but they may be simpler to carry out.

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