Immunological Assays and Techniques: Agglutination, Precipitation, ELISA, and Immunofluorescence

What are the biological consequences of antigen-antibody interactions?

Agglutination, precipitation, complement activation, neutralization, opsonization, and diagnostic applications.

What is agglutination?

Clumping reaction when antibodies cross-link particulate antigens such as red blood cells or bacteria.

Which immunoglobulin is more efficient in agglutination, IgM or IgG?

IgM, because it has more binding sites (pentameric).

What is precipitation?

Formation of insoluble immune complexes between soluble antigens and antibodies at optimal ratios.

What are the three zones of precipitation?

Antibody excess zone, equivalence zone (max precipitation), antigen excess zone.

What is the equivalence zone?

The antigen-antibody ratio is optimal for cross-linking; all complexes precipitate out.

What is a titer?

The highest dilution of serum that still gives a positive reaction (e.g., visible agglutination).

What is the prozone effect?

False negative result due to excess antibody preventing cross-linking.

What is passive agglutination?

A test where soluble antigens are attached to carrier particles (e.g. latex beads) to detect antibodies.

What is reverse passive agglutination?

A test where antibodies are attached to carriers to detect antigens in serum.

What is a limitation of agglutination tests?

Positive result may indicate past infection; false negatives possible from low antibody production.

What is precipitation-based assay principle?

Antigen and antibody interact to form a visible precipitate when at optimal ratio.

What is an immunodiffusion test used for?

Identifying antigenic relationships using precipitation lines.

What are lines of identity in Ouchterlony test?

Antibodies recognize identical epitopes; lines fuse.

What are lines of non-identity in Ouchterlony test?

Antibodies recognize different epitopes; lines cross.

What are lines of partial identity in Ouchterlony test?

Antigens share some epitopes; a spur forms.

What is radial immunodiffusion?

Quantitative test; antigen diffuses in agar with uniform antibody to form precipitation rings proportional to concentration.

How are unknown antigen concentrations determined in radial immunodiffusion?

By comparing ring diameter to standard curve.

What are monoclonal antibodies?

Identical antibodies produced from a single clone; recognize the same epitope.

How are monoclonal antibodies produced?

By fusing B cells with myeloma cells to create hybridomas.

What is hybridoma technology?

Method to produce immortal cells that secrete monoclonal antibodies.

What is immunofluorescence?

Use of fluorescent-labeled antibodies to detect antigens or antibodies in samples.

What is direct immunofluorescence?

Labeled antibody binds directly to target antigen.

What is indirect immunofluorescence?

Unlabeled primary antibody binds antigen; labeled secondary antibody binds primary for detection.

What is the advantage of indirect immunofluorescence?

Signal amplification and flexibility.

What is flow cytometry?

Technique that detects fluorescence on individual cells to identify and count specific populations.

What markers can be detected in flow cytometry for T cells?

CD3, CD4, CD8.

What is ELISA?

Enzyme-Linked Immunosorbent Assay; detects presence of specific antibodies or antigens using enzyme-conjugated antibodies and color change.

What is the principle of ELISA?

Antigen-antibody binding detected by enzyme reaction producing a colored product.

What are the main steps of ELISA?

1) Bind sample, 2) Add primary antibody, 3) Add enzyme-linked secondary antibody, 4) Add substrate, 5) Measure color.

What is Western blot used for?

Detection of specific proteins separated by electrophoresis using labeled antibodies.

What is Radioimmunoassay (RIA)?

A sensitive assay where labeled and unlabeled antigens compete for antibody binding; radioactivity inversely proportional to antigen concentration.

What is complement fixation assay used for?

Detecting antibodies by observing if complement is consumed, preventing hemolysis of sensitized RBCs.

What does lack of hemolysis indicate in complement fixation?

Presence of antigen-antibody complexes; positive test.

Which assays are based on competition principle?

Radioimmunoassay (RIA) and some ELISAs.

Rank the assays by sensitivity from highest to lowest.

RIA > ELISA > Western blot > Flow cytometry > Agglutination > Precipitin > Complement fixation.

What is a false positive?

Test shows positive when the patient is not infected (e.g., cross-reactive antibody).

What is a false negative?

Test shows negative when the patient is infected (e.g., low antibody levels).

What does a positive agglutination test indicate?

Presence of specific antibody or antigen, but not necessarily active infection.

What is an advantage of ELISA over RIA?

No radioactivity, safer, easier to perform.

What is a disadvantage of precipitation tests?

Low sensitivity; require high concentrations of antigen and antibody.

What is FITC?

Fluorescein isothiocyanate, green fluorescent tag used in immunofluorescence.

What is TRITC?

Tetramethylrhodamine isothiocyanate, red fluorescent tag used in immunofluorescence.

What is meant by 'equivalence' in immunodiagnostic tests?

Point where antigen and antibody concentrations are optimal for complex formation.

What type of virus is HIV and what makes it unique?

HIV is a retrovirus with an RNA genome that uses reverse transcriptase to convert RNA into DNA, allowing it to integrate into the host genome.

What are the key structural components of HIV?

Lipid envelope, gp120 (binding), gp41 (fusion), capsid proteins (p17, p24), RNA genome, reverse transcriptase.

What does gp120 do?

Binds to CD4 receptors on T helper cells.

What does gp41 do?

Mediates fusion between viral and host membranes.

Why is HIV highly immunoevasive?

Reverse transcriptase has a high mutation rate, constantly changing viral epitopes.

What cells does HIV primarily infect?

CD4+ T helper cells, macrophages, and other immune cells.

What happens to CD4+ T cells during HIV infection?

They are destroyed, leading to immune system collapse.

Why don’t people die directly from HIV?

Death results from opportunistic infections due to immune system failure (AIDS).

What is a provirus?

Viral DNA integrated into the host genome.

What triggers HIV replication after latency?

Activation of infected T helper cells.

What is ELISA used for in this lab?

Detecting anti-HIV IgG antibodies in patient serum.

What is the first step of ELISA?

Antigen coating — HIV antigens bind to the plate.

What happens after antigen coating?

Patient serum is added; antibodies (if present) bind the antigen.

What is the role of the secondary antibody?

Binds to human IgG and is linked to an enzyme (peroxidase).

What enzyme is used in this ELISA?

Horseradish peroxidase.

What happens when substrate is added?

The enzyme converts it into a colored product (brown).

What indicates a positive ELISA result?

Color change in the well.

Why is ELISA highly sensitive?

Enzyme amplification produces strong signals even from small amounts.

What is the purpose of washing steps?

Removes unbound molecules to reduce background noise.

Why is blocking normally used (even if skipped here)?

Prevents nonspecific binding to plate surfaces.

What is the negative control in this lab?

PBS (no antibodies expected → no color).

What is the positive control?

Sample known to contain anti-HIV antibodies → should turn color.

Why are controls essential?

They confirm whether results are valid or due to error.

Why can the same pipette be used across wells?

Because tips are changed or rinsed to prevent contamination.

What does lysozyme do?

Breaks down peptidoglycan in bacterial cell walls.

Where is lysozyme commonly found?

Saliva, body fluids, and immune cells.

How is lysozyme activity measured in this lab?

By the diameter of clearing zones in agar.

What does a larger clearing zone indicate?

Higher lysozyme concentration.

What is a standard curve used for?

Determining unknown concentrations from known standards.

What happens in Ouchterlony diffusion?

Antigen and antibody diffuse toward each other in agar.

What forms when antigen and antibody meet?

A precipitin line.

What does a single line indicate?

One antigen-antibody system.

What is a reaction of identity?

Lines fuse → antigens are identical.

What is non-identity?

Lines cross → antigens are unrelated.

What is partial identity?

Lines fuse with a spur → antigens share some epitopes.

What is the equivalence zone?

Optimal antigen-antibody ratio → maximum precipitation.

What is agglutination?

Visible clumping of particles due to antigen-antibody binding.

What must antigen and antibody be for agglutination to occur?

Multivalent (multiple binding sites).

What is a lattice?

Network of antigen-antibody complexes causing clumping.

What is the prozone effect?

Too much antibody → no visible agglutination.

What is the postzone effect?

Too much antigen → no visible agglutination.

Which antibody is best at agglutination?

IgM (more binding sites).

What is endpoint titer?

Last dilution where agglutination is still visible.

What diffuses in RID?

Antigen diffuses into antibody-containing agar.

What forms in RID?

A circular precipitin ring.

What does ring size indicate?

Antigen concentration.

Why is RID quantitative?

Ring diameter squared correlates with concentration.

What is required to determine unknown concentration?

A standard curve.

Where is the equivalence zone in RID?

At the precipitin ring.