Serology and Immunodiagnostic Methods

Ag-Ab Reactions

• Ag-Ab reactions are used for diagnosing infectious diseases
• The union of an antigen (Ag) with its specific antibody (Ab) is the basis of these reactions
• Reactions are best performed when Ag and Ab concentrations are optimal
• The type of reaction depends on the nature of both Ag and Ab used
• These reactions are used to detect either the Ag (direct diagnosis) or the Ab (indirect diagnosis)

Types of Infectious Diseases

• Bacterial: extracellular and intracellular bacteria
• Viral: DNA or RNA viruses, enveloped or non-enveloped
• Fungal: unicellular or multicellular molds
• Protozoal infections
• Helminthes

Immune Response to Microorganisms

• Bacteria:
  • Extracellular: antibodies target capsular polysaccharides, bacterial enzymes (e.g., ASO), exotoxins (e.g., diphtheria, tetanus), and bacterial endotoxins. Complement fixation (CF) antibodies are involved.
  • Intracellular: cell-mediated immunity (CMI) is the primary response.
• Viruses:
  • Viremic viruses: neutralize via IgM antibodies (recent infection), IgG antibodies (later infection), and IgA antibodies. Complement fixation antibodies are also involved. Cell-mediated immunity (CMI) is the primary type of immunity to viral infections.
• Fungal infections: cell-mediated immunity (CMI) is the main immune response because fungal infections are mainly intracellular

Post-Infection Immunity

• Viral infection: mostly long-lasting immunity
• Bacterial infection: shorter-term immunity
• Protozoa infection: shorter-term immunity
• Helminthes infection: no protective immunity

Diagnosis of Infectious Diseases

• Epidemiological data (time, location, vector, etc.)

• Clinical features (symptoms, signs)

• Laboratory findings

  • Routine examination of blood, urine, feces
  • Biochemical examinations

• Serological diagnosis

  • Etiological examinations (direct exam, pathogen isolation)
  • Molecular biological examinations (PCR, LCR, etc.)
  • Immunological examinations
  • Endoscopy examinations
  • Image examinations

Laboratory Investigation of Microbial Infections

• Microscopy
• Culture techniques
• Biochemical reactions
• Serological identification
• Molecular biology techniques
• Bacteriophage typing

Serological Diagnosis of Infections

• Diagnosis of infectious diseases based on antigen-antibody (Ag-Ab) reactions
• Ag-Ab reaction: union of an antigen (Ag) with its specific antibody (Ab)
• Best detected when each (Ag and Ab) is in optimal concentration; reaction type depends on nature of both agents

Types of Ag-Ab Reactions (In Vitro)

• Agglutination
• Precipitation
• Complement fixation test (CFT)
• Neutralization
• Immunofluorescence test (IF)
• Enzyme-linked immunosorbent assay (ELISA)
• Radioimmunoassay (RIA)
• Immunochromatography (ICT)
• Immuno-blotting assays (Western blotting)

Sensitivity of Serological Tests

• Gel diffusion: 30 µg/mL
• Ring precipitation: 18 µg/mL
• Bacterial agglutination: 0.05 µg/mL
• Complement fixation test: 0.05 µg/mL
• Passive hemagglutination: 0.01 µg/mL
• Hemagglutination inhibition test: 0.005 µg/mL
• Immunofluorescence: 0.005 µg/mL
• ELISA: 0.0005 µg/mL
• Bacterial neutralization: 0.00005 µg/mL

Application of Ag-Ab Reactions (Serology)

• Diagnosis of many infectious diseases (direct antigen detection or measuring immunity to infection), especially for non-culturable or slowly growing microorganisms.
• Estimation of severity or stage of diseases.
• Determination of response to treatment
• Epidemiological studies
• Diagnosis of congenital infections
• Screening of blood and tissue donations
• Non-infectious diagnostics (tumors, autoimmune diseases, endocrinology, etc.)

Agglutination

• Visible clumping of particulate (insoluble) antigen (Ag) with its specific antibody (Ab)
• Forming a visible lattice structure
• Can be done on slides or in tubes
• Can be direct or indirect using a carrier (e.g., latex, RBCs, staph protein A)

Examples of Agglutination Reactions

• Direct slide detection of bacterial or viral antigens in a lesion or culture.
• Direct tube agglutination (i.e., Widal test) for typhoid fever diagnosis (now largely replaced by latex).
• Indirect latex tests commonly used in microbiology labs.
• Indirect passive hemagglutination tests (e.g., TPHA) for syphilis diagnosis.
• Brucella slide or tube agglutination test (prozone phenomenon)

Hemagglutination

• Agglutination reaction involving red blood cells (RBCs)

Passive Hemagglutination

• Uses coated RBCs

Agglutination Inhibition

• A test to detect the presence of an antigen in a sample by inhibiting agglutination.

Latex Reactions for Detection of Ags or Abs

• Latex particles coated with antigens for detection of antibodies (i.e., antibodies that bind to antigens on the latex particles)
• Latex particles coated with antibodies to detect antigens (i.e. when antigens bind to antibodies on latex particles)

Why are Latex Tests Common?

• Easy manufacture, use, and have clear visualization
• No complex instrumentation needed.
• Coat with any soluble antigen (Ag) to detect antibody (Ab)
• Coat with antibody (Ab) to detect antigen (Ag)

Cryptococcal Antigen Latex, MRSA Latex, HCG Latex Agglutination Test, Pregnancy Test Latex, Toxoplasmosis - Latex Agglutination

Prozone Phenomenon

• Absence of agglutination in early tubes (high Ab concentrations)
• Appears as false-negative result
• Occurs with following tubes as concentration decreases (higher dilution)
• Overcome by using higher dilutions

Advantages of Agglutination

• Widely used for diagnosis and screening
• Simple and easy
• Inexpensive
• No instrumentation required
• Somewhat sensitive

Disadvantages of Agglutination

• Low specificity (false positives)
• Confirmation tests needed
• Low sensitivity (false negatives)
• Effect of vaccine-induced antibodies
• Subjective errors in interpretation
• Antibody production may be late in infections

Precipitation Tests

• Reaction between soluble antigens (Ag) and specific antibodies (Ab)
• Can occur in solution or in gel
• Ring test for detecting infectious antigens (e.g., in cerebrospinal fluid (CSF))
• Slide test for detecting pneumococcal capsules
• Used for quantifying immunoglobulins in patient sera (less common) - using single or double radial immunodiffusion or immunoelectrophoresis.

Single Radial Immunodiffusion

• Antibody (Ab) in gel; antigen (Ag) diffuses into gel
• Precipitation ring diameter proportional to antigen concentration

Double Radial Immunodiffusion

• Separates antigens by isoelectric point and molecular weight within a gel.
• Fusion lines demonstrate antigen identity, non-identity or partial identity.

Explanation of the reaction: RADIAL IMMUNODIFFUSION

• Antibody incorporated into agar; antigen diffuses into the gel
• Precipitation ring forms from the antigen and antibody interaction

Complement Fixation Test (CFT)

• Detects complement-fixing antibodies (IgM or IgG)
• Uses guinea pig complement and antigen in Wassermann tubes; serum is heated to destroy internal complement proteins.
• RBCs and anti-RBCs are added as indicators; no hemolysis indicates the presence of antibody, and complement has been fixed.
• Testing for syphilis, chronic gonorrhea, or influenza
• Laborious; rarely used

Advantages of CFT

• Antibody does not persist long (recent infections)
• Stable antigen
• High specificity
• Quantitative results

Disadvantages of CFT

• Requires time
• Not standardized
• Relies on high technical skills
• False positive risks
• Anti-complementary issues

Neutralization Tests

• Antibody (Ab) neutralizes antigen (Ag) effect (e.g., bacterial toxin or virus).
• Anti-Streptolysin O (ASO) titre test for post-streptococcal complications (e.g., rheumatic fever) used in vitro.

Enzyme-Linked Immunosorbent Assay (ELISA)

• Detects unknown antigens (Ag) or antibodies (Ab)
• Monoclonal antibody (mAb) or recombinant antigen fixed to microtiter plate wells.
• Test sample added, incubated, and washed
• Secondary enzyme-linked antibody (conjugate) applied.
• Substrate added to visualize reaction
• Color reaction proportionate to the unknown amount; results measured spectrophotometrically.
• Alkaline phosphatase often used as the conjugate
• Various formats exist (direct, indirect, sandwich, competitive)

ELISA Formats

• Direct: labeled antibody directly binds to the antigen
• Indirect: labeled antibody binds to another antibody already bound to the antigen
• Sandwich: two antibodies are used, each binding to a different epitope on the same antigen
• Competitive: antigen in the sample competes with labeled antigen for antibody binding

Advantages of ELISA

• Simple (small volumes, simple washing)
• Easy reading (naked eye, spectrophotometry)
• Rapid (few hours)
• High sensitivity (detection levels of 0.01-1 µg/mL)
• Commercially available reagents

Radioimmunoassay (RIA)

• Uses radioisotopes (e.g., Iodine-125) as labels, for antigen (Ag) or antibody (Ab) conjugates
• Quantifies proteins with incredibly small amounts (e.g., hormones)
• Less common nowadays due to radiation safety concerns

Immunofluorescence Assay (IFA)

• Antibody (Ab)-antigen (Ag) reaction, utilizing fluorescent dyes (e.g. fluorescein or rhodamine).
• Visualized with a fluorescent microscope
• Direct: use labeled antibody to detect antigen in tissue samples
• Indirect: Use labeled secondary antibody to detect primary antibody binding to the antigen in tissue samples

Immuno-blotting Assays (WB)

• Analyzes and detects proteins through separation with antibody-protein complex formation, visualized by appropriate methods, and is used in confirming diagnosis of HIV.
• Separates proteins based on isoelectric point and molecular weight through electrophoresis
• Results visualized via staining and photography
• Method for detection of proteins.
• Frequently used for confirmatory testing for HIV

Flow Cytometry

• Identifies and enumerates cells with specific antigens; uses a laser beam to illuminate cells, measuring scattered light for cell size, and stimulating fluorophore on cell surfaces for measurement of fluorescence, using specialized equipment and specialized analysis software

Immunochromatography (ICT)

• Rapid assay (sometimes called lateral flow assays) using sandwich ELISA principles on nitrocellulose or chromatographic paper
• Strips or cartridges use monoclonal antibodies (mAbs)
• Test zone (T) is specific antibody or antigen
• Control zone (C) validates test components; only a C zone detects a negative test
• Positive test: Colored band appears at both T and C zones
• Negative test: Colored band appears only at C zone

Rapid Tests Advantages

• Simple to use
• Immediate results
• Inexpensive
• Used for screening
• Conducted at home/clinic

Rapid Tests Disadvantages

• Confirmation sometimes needed
• Subjective results
• Qualitative not quantitative
• Less sensitive than ELISA

This quiz explores the fundamentals of antigen-antibody reactions and their applications in serologic testing and epidemiology. Delve into topics like sensitivity levels in tests, the agglutination process, and specific applications in disease diagnosis, including the VDRL test for syphilis. Perfect for anyone looking to enhance their knowledge in serology.