Practical Course 1: Antibody Titer

Practical Course 1: Antibody Titer

• This course focuses on identifying antigen-antibody interactions using precipitation methods.
• The goal is to determine antibody titers, which measure the concentration of antibodies in a sample.

COVID-19 Antigen and Antibody Detection

• Antigen Detection: Identifies viral proteins (e.g., spike protein, membrane proteins) to indicate an active infection.
• Antibody Detection: Detects antibodies produced by the body in response to the virus, indicating past or present infection.
• RT-PCR: Used to detect viral RNA.

Polyclonal and Monoclonal Antibodies

• Polyclonal Antibodies: Recognize various epitopes (antigenic determinants) on the antigen.
  • The antibodies are diverse in binding sites.
• Monoclonal Antibodies: Bind to a single unique epitope on the antigen.
  • The antibodies are identical and specific.

First Phase: Ag-Ab Reaction

• This is the initial phase of antigen-antibody (Ag-Ab) interaction.
• Polyclonal antibody (antiserum) typically reacts with multiple sites on the antigen.
• Monoclonal antibodies recognize and bind to a single epitope.

Second Phase: Visualization of Ag-Ab Reaction

• This phase follows the antigen-antibody reaction and reveals the results of the interaction.

Immunoprecipitation

• Occurs when soluble antibodies bind to soluble antigens, forming lattice complexes that precipitate out of solution.
• The precipitation creates a visible reaction.
• The immune complexes, formed in soluble phases, are now outside of the solution.

Immunoprecipitation: Conditions

• High concentration of antigen and antibody: Essential for efficient precipitation.
• Polyclonal antibody: More diverse binding to multiple epitopes.
• Polyvalent antigen: Contains multiple epitopes.
• Equimolar concentrations of antigen and antibody: Ideal conditions for maximum precipitation.

Measurement of Precipitation by Light Scattering

• Antigen-antibody complexes, when formed at a high rate, precipitate out of solution.
• Turbidimetry measures the turbidity (cloudiness) of the solution by measuring the amount of light that passes through the solution.

Passive Immunodiffusion

• Antigens and antibodies migrate in an agar gel.
• They encounter each other in optimal proportions, forming a precipitate where they meet.
• The method is passive because it does not use external forces or enhancements.

Ouchterlony Gel Diffusion

• Wells are punched into an agar gel.
• Known antibody or antigen is added to the central well.
• Unknown samples are added to surrounding wells.
• Bands, where the optimal antigen-antibody reaction occurs, form in the gel.
• It is a qualitative test to determine the presence of specific substances, not their concentration.

Ouchterlony Immunodiffusion

• A diagram shows the setup and how the antigen and antibody diffuse and form a reaction.
• It shows the zone of antigen excess, optimal precipitation, and antibody excess.

Immunoprecipitation in Solid Phase

• A schematic illustrates an experiment using various dilutions of antibody in a gel-based reaction for antibody titer determination.

Immunoprecipitation in Liquid Phase

• A protocol describes a liquid-phase experiment.
• Different amounts of rabbit serum are mixed with antibodies.

Antibody Target Identification

• If no antibody is present, high absorbance will be recorded (beads are dispersed).
• When antibodies are present, absorbance will decrease because the antigen-antibody complexes aggregate the beads (producing agglutination).

Determining Antibody Concentration

• A step-by-step procedure and materials list are shown, including the dilutions of rabbit IgG and the readings obtained.
• The measurements are used to determine the antibody concentration using a plate reader.
• A graph shows the decreasing absorbance of the well readings as the concentration of antibodies increases in each well.