Practical Course 1: Antibody Titer

Practical Course 1: Antibody Titer

• Antibody titer is used to identify antigen-antibody interactions through precipitation methods.

COVID-19 Antigen and Antibody Detection

• COVID-19 has various antigens detected, including spike (S), envelope (E), membrane (M), hemagglutinin-esterase (HE), and nucleoprotein (N).
• Genomic RNA is also used for detection.
• RT-PCR is a technique used to detect genomic RNA.
• Antibodies against COVID-19 are also detectable.

Polyclonal and Monoclonal Antibodies

• Polyclonal antibodies recognize various epitopes on an antigen.
• Monoclonal antibodies recognize a unique epitope on an antigen.
• Epitopes are specific regions on an antigen recognized by antibodies.

First Phase: Ag-Ab Reaction

• Polyclonal antiserum and monoclonal antibody react with the antigen, leading to the formation of complexes.
• This phase involves the initial binding of antibodies to antigens.

Second Phase: Visualization of Ag-Ab Reaction

• This phase involves the visualization of the formed immune complexes. There is no detail given on procedures in this section.

Immunoprecipitation

• When soluble antibody binds to a soluble antigen, lattice formation occurs, leading to precipitation.
• This results in visible immune complexes that fall out of solution.

Immunoprecipitation: Conditions

• High concentrations of both antigen and antibody are needed.
• Polyclonal antibodies and polyvalent antigens (multiple epitopes) are beneficial.
• Equimolar concentrations of both are ideal for optimal precipitation.

Measurement of Precipitation by Light Scattering

• Antigen-antibody complexes precipitate and form a turbid solution.
• Turbidimetry measures the turbidity/cloudiness of the solution by measuring the light passing through it.

Passive Immunodiffusion

• Antigens and antibodies migrate in agar gel towards each other in optimal proportions to form a precipitate.
• This is a passive process, where no external force (like electrical charge) is applied.

Ouchterlony Gel Diffusion

• Holes are punched in agar gel.
• Known antibody/antigen is put in the center well.
• Samples are put in outer wells.
• Bands form where the antigen and antibody meet.
• This method is used to determine the presence of antigens/antibodies, rather than specific concentrations.

Ouchterlony Immunodiffusion

• This method involves punching wells in agar gel, using a well for the antigen in the center and wells for samples containing specific antibodies that match. Antibodies and antigens diffusing from wells in gel, precipitate to form observable lines if matched. This defines presence of antigen/antibody without determining concentration.

Immunoprecipitation in Solid Phase

• Antigen is at the center of an agar-based slide or plate.
• Known serum samples are added around the antigen in varying dilutions.
• Based on the formation of precipitation/solid lines, the titer (dilutions) of serum antibodies can be determined.

Immunoprecipitation in Liquid Phase

• Beads are added to wells that contain antibodies against rabbit IgG.
• Rabbit serum and pure rabbit IgG is added.
• Blocking buffer is added to all wells to ensure no interference with precipitation.
• Antibody–antigen complexes are measured for absorbance.

Measurement of Antibody

• Presence or absence of antibody target can be measured by observing either a high absorbance result from non-bound target-beads or low absorbance result from antibody-bound target-beads.

Further Testing Protocol

• A protocol for testing antibody (e.g., rabbit IgG) is detailed showing dilutions, a control serum sample and anti-IgG con sepharose (antibody), blocking buffer, and measurement by plate reader.
• Results are measured for absorbance at 405nm using a plate reader.