Immunodiffusion and HIV Testing

Antigen-Antibody Reactions

• Antigen-antibody reaction occurs between an antigen and the antibody elicited by that antigen or by a closely related antigen (Lock and Key Concept).
• If the antigen is particulate, the reaction is called agglutination.
• If the antigen is in solution, the reaction is precipitation.
• If the reactants are invisible, substances like fluorescent dyes or enzyme are added to make the reaction visible.

Types of Antigen-Antibody Binding Reactions

• Agglutination
• Precipitation
• Antibody-labeled assays
  • Enzyme-Linked Immunosorbent Assays (ELISA)
  • Immunofluorescence
  • Radioimmunoassay (RIA)

Agglutination Reactions

• Definition: An antigen-antibody reaction in which the antigen is in the form of particles, such as microorganisms or cells, or latex particles coated with the antigen.
• These particles become clumped, i.e., agglutinated, when mixed with its specific antibody.

Types of Agglutination

• Direct Agglutination
  • Slide method (Qualitative agglutination tests)
  • Tube method (Semi-Quantitative agglutination test)
• Passive Agglutination
  • e.g., Latex agglutination
• Antiglobulin test
  • e.g., Coomb's test
• Co-Agglutination

Slide Method (Qualitative Agglutination Tests)

• A rapid test commonly used in blood grouping and useful in identification of organisms isolated from clinical specimens.

Tube Method (Semi-Quantitative Agglutination Test)

• Determines the amount of antibodies in the serum of patients.
• Example: Widal test used to detect antibodies against Salmonella in the serum of patients suspected to have enteric fever.

Passive Agglutination

• An agglutination test done with a soluble antigen coated onto an inert particle, such as latex or RBCs, for measurement of antibodies to this antigen.
• Examples: Rheumatoid factor latex test, C-reactive protein (CRP), and anti-streptolysin "O" (ASO) test.

Antiglobulin Tests

• Antiglobulin agglutination test, also known as Coomb's test, is used for:
  • Detection of anti-Rh Ab
  • Autoimmune hemolytic anemia

Coomb's Test

• Principle: Incomplete Antibodies
• Rh IgG antibodies are incomplete (unagglutinable) and can only coat Rh positive RBCs.
• However, they cannot bridge between RBCs to cause visible agglutination, known as the prozone phenomenon.
• Coomb's test can be performed in two ways:
  • Direct Coombs Test
  • Indirect Coombs Test

Coagglutination (CoA)

• Killed Staphylococcus aureus can be used to detect antibodies nonspecifically through the process of coagglutination.
• If Staphylococci are coated with the antigen, they will agglutinate.
• This test can be used in various specimens like serum, urine, and cerebrospinal fluid (CSF).

Precipitation

• In these reactions, the antigen is soluble, and the reaction can be done in agar gel.
• Single Radial Immunodiffusion:
  • Ab is put in gel, and the Ag is in wells punched in the agar.
  • The diameter of the ring is proportional to the concentration.
  • It can measure the immunoglobulin level, i.e., Quantitative.

Western Blot Test

• Used for detection of specific HIV antibodies and to confirm a positive ELISA test.
• HIV proteins (antigens) are separated electrophoretically on a nitrocellulose strip.
• The strip is incubated with the patient's serum.
• If serum contains HIV antibodies, they will bind with the corresponding viral antigens.
• These are then detected by using an enzyme-linked antihuman IgG, as in ELISA.
• A positive colorimetric reaction forms bands on the nitrocellulose paper strip corresponding to specific HIV antigens.

Double Diffusion (Elek's Test)

• Used to prove toxigenicity of diphtheria bacilli.
• It is a gel diffusion, toxin-antitoxin precipitation test.
• Antitoxin-soaked strip of filter paper is placed on the surface of a 20% serum agar medium.
• The plate is heavily inoculated by streaking a line of inoculums perpendicular to the strip.
• The appearance of a white line of toxin-antitoxin precipitate at a 45° angle, within 24-48 hours, is diagnostic of a toxigenic organism.

Assays Based on Complement

• Complement Fixation
  • This is an antigen-antibody reaction that occurs in the presence of a third component known as complement.
  • Considered now a historical test.

Antibody-Labeled Assays

• Enzyme-Linked Immunosorbent Assays (ELISA)
  • This method depends on the conjugation of an enzyme with either antigen or antibody, then the enzyme activity on a substrate is used as a quantitative measure.
  • Indirect Method:
    • A known antigen is fixed to a solid phase (plastic cup or microplate) and incubated with the test serum and washed to remove excess unattached antibody.
    • Then reincubated with enzyme-labeled antiglobulin.
    • The latter will attach to the antibody bound to the fixed antigen.
    • After washing, a specific substrate is added, and the enzyme activity is measured (the degree of color change).

Immunofluorescence

• Direct Immunofluorescence
  • Labeled antibodies are added to the unknown antigen fixed on a slide, left to react, and the excess antibody is washed.
  • Then the slide is examined under the fluorescent microscope.
  • The appearance of a fluorescent site indicates the antigen-antibody reaction.
• Indirect Immunofluorescence
  • To detect antibodies in the serum of the patient, the serum is added to known antigen fixed on a slide, left to react, and the excess antibody is washed.
  • A second labeled anti-immunoglobulin antibody directed toward the first antibody is added.
  • Then the slide is examined under the fluorescent microscope.

Radioimmunoassay (RIA)

• Although it is an extremely sensitive and specific technique, it requires specialized equipment, special precautions, and licensing due to the hazards of radiation.
• Used for the detection of unknown Ag or Ab.

This quiz covers various immunodiffusion techniques, including Single Radial Immunodiffusion, and their application in HIV testing, including the Western Blot Test.