Serology for Medical Laboratory Students

Serology for Medical Laboratory Students

• Serology is the scientific study of blood serum and immune reactions in human blood.
• The key principle in serology is the antibody-antigen reaction.
• Antigens are substances that provoke the body to produce antibodies.
• Antibodies are substances that fight invading organisms.
• Serological tests analyze samples to detect either antigen or antibody.

Learning Objectives

• Students should be able to list various serological tests.
• Students should understand the principles behind serological tests.
• Students should be able to describe various serological techniques.
• Students should be able to identify the advantages and disadvantages of different serological techniques.
• Students should be able to identify factors affecting antigen-antibody reactions.

Outline

• Introduction to Serology
• Immunological techniques (primary, secondary, tertiary binding)
• Factors affecting antigen-antibody reactions

Introduction to Serology

• Serology is the study of blood serum and immune reactions.
• It uses antibody-antigen reactions to detect or measure antigens or antibodies in body fluids.
• It assists in diagnosing infections, autoimmune diseases, and blood types.
• Serum is the fluid portion of blood after clotting.
• Plasma is the fluid portion of anticoagulated blood.

Diagnostic Identification

• Diagnostic identification of antibodies in serum aids in identifying infections.
• Antibodies are formed in response to infections.
• Antibodies also form against other foreign proteins and one's own proteins.

Diagnostic Identification of Antigens

• Antigens are whole organisms, structural components of organisms (e.g., cell wall, capsule, flagellar), products of microorganisms (e.g., toxins, enzymes), tumor markers, infection products (e.g., antigen-antibody deposition), allergens, drugs, hormones, blood cells, and self-antigens.

Immune System

• The immune system is composed of structures, cells, and soluble constituents, enabling the host to respond to foreign stimuli.
• Secondary immune responses occur when the body encounters an antigen again.
• Specificity is the particular affinity between antigen and its accompanying antibody.

Application of Serological Tests

• Serological tests are used for:
  • diagnosing suspected infections
  • detecting rheumatic illnesses
  • determining blood type
  • diagnosing immunodeficiencies (lack of antibodies)

Types of Serological Tests

• Qualitative tests determine the presence or absence of antibodies or antigens in serum.
• Quantitative tests use serial dilutions to measure the concentration of antibodies and/or antigens.
  • Results are reported as antibody titer.
• Antigen tests assist in early diagnosis of infectious diseases through identifying pathogens.
• Antibody tests are useful when pathogens or microbial antigens are absent or hard to identify, to screen donor blood, and monitor treatment effectiveness.

Immunological Techniques

• Three main groups are:
  • Primary binding: Measure/visualize antigen-antibody complexes directly.
    • Examples: ELISA, RIA, Western blotting.
  • Secondary binding: Detect consequences of antigen-antibody interactions.
    • Examples: Precipitation, agglutination, complement fixation.
  • Tertiary binding: Measure consequences of immune responses in living organisms.
    • Examples: Measurement of protective antibody effects.

Primary Binding Tests

• These tests directly measure antigen-antibody binding.
• Using labels like radioisotopes, fluorescent dyes, enzymes aids in identifying reactants.
• Examples of these tests include ELISA and RIA.

Immunofluorescence Tests (FAT)

• These tests detect antigens using fluorescent dyes to show specific antigens and antibodies bonding.
  • Direct FAT: Probes directly react with the antigen in the sample.
  • Indirect FAT: Probes react with the antibody that has bound to the antigen in the sample.

Secondary Binding Tests

• Precipitation: Soluble antigens react with antiserum in electrolytes (e.g., NaCl) forming insoluble precipitates.
• Agglutination: Antibodies cause cells (such as bacteria, red blood cells) to clump.
• Neutralization: Antibodies block harmful substances.
• Complement fixation: Antibodies use complement to destroy cells.

Agglutination Tests

• Detects antigens, antibodies in serum.
  • Slide Agglutination Tests: Direct/passive agglutination; quick, inexpensive.
  • Tube Agglutination Tests: More sensitive, control conditions are vital.
  • Microtitration Agglutination Tests: More sensitive than tube tests, economical, used in place of tube tests.
  • Hemagglutination tests use antibodies against red blood cells (e.g., hemagglutination inhibition, indirect hemagglutination, reverse passive hemagglutination).

Measurement of Antibody Titer

• Used to diagnose infections by measuring the antibody response.
• Testing two specimens (paired sera) taken at different stages of infection is vital to see if there is an increase in antibody level.

Prozone Effect

• Prozone phenomenon: High antibody titer initially masking agglutination, only detected in higher dilutions.

Precipitation Tests

• Used to detect and measure antigens and antibodies using solution or semisolid media (agar, agarose).
  • There are three main types: Tube precipitation, Gel diffusion, Counter immunoelectrophoresis.

Complement Fixation Tests (CFT)

• Detects antibodies that do not agglutinate or precipitate, but can be linked with complement fixation.
• Used in labs with the capacity to standardize and control reagents for accuracy.

Tertiary Binding Tests

• Measuring the consequences of immune responses in a living organism.
  • Studying the effects of antibodies in vivo is essential.

Factors Affecting Antigen-Antibody Reaction

• Specificity of antigen-antibody reaction.
• Cross-reactivity.
• Temperature.
• pH.
• Ionic strength(s).
• Antigen concentration.
• Antibodies concentration.
• Intermolecular specificity.

Radioimmunoassay (RIA)

• Extremely sensitive technique used to measure extremely low concentrations of antigen or antibody (e.g., hormones, drugs).
• Uses radioactively labeled compounds to measure the sample.
• Liquid-phase and solid-phase assay methods are possible.

Western Blot Test (WB)

• Assay technique for proteins.
• Very specific, confirmatory test for protein detection.
• Detects specifically sized proteins in a complex mixture through electrophoresis gel separation and protein transferring to a blotting membrane to probe and examine the protein.

Southern Blotting

• Detects specific DNA sequences through gel electrophoresis and blotting to a membrane, probing, and washing to identify complementary DNA sequences.

Northern Blotting

• A technique used to detect and quantify mRNA in a sample.
• It involves isolating RNA, separating it by size, transferring it to a membrane and using a probe to detect the specific mRNA.

Other Techniques Mentioned

• ELISA
  • Qualitative/Quantitative.
  • Enzyme-linked immunosorbent assay- uses enzymes to detect and quantify antibodies and antigens.
• Immunodiffusion/Immunoelectrophoresis
• Other complement fixation test methods

Review Questions

• Compare precipitation and agglutination tests.
• How does the prozone reaction impact test results?
• Discuss the advantages and disadvantages of serological tests contrasted with other laboratory methods for infectious disease evaluation.

This quiz explores the fundamental concepts of serology, including antibody-antigen reactions and various serological tests. Students will learn about the principles, techniques, and factors influencing immunological responses. It's essential for aspiring medical laboratory professionals seeking a comprehensive understanding of serological applications.