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Introduction to Immunoanalysis

• Immunoanalysis is the study of immune responses and the use of antibodies in diagnostic techniques.

Learning Objectives

• Describe the primary and secondary immune responses.
• Define the terms: plasma, serum, antiserum, clone, polyclonal antibody (PAbs), immunogen, and adjuvant.
• Understand how polyclonal antibodies are generated.

Serum, Antiserum, and Cloning

• Serum is the liquid portion of clotted blood.
• Antibodies within serum that bind to a specific antigen are known as antiserum.
• Antibody cloning is the process of producing identical copies (clones) of a specific antibody.

Adaptive (Humoral) Immune Response

• The primary immune response occurs when the host 첫 접촉 with an antigen (Ag).
• IgG levels appear after approximately 7 days, rise rapidly, and then gradually decrease.
• The secondary immune response occurs after subsequent exposure to the same antigen (Ag).
• The lag period is shorter, and the response and duration are longer than the primary response.

Immune Response - Cell Types

• The primary immune response involves short-lived plasma cells.
• A secondary exposure to the antigen leads to memory B cells, differentiating into plasma cells to produce antibodies.
• Long-lived plasma cells found in bone marrow generate antibodies continuously.

Immune Response - Immunoglobulins

• The primary immune response shows an initial increase in IgM and a later increase in IgG.
• The secondary immune response shows significantly higher IgG levels and a faster response due to memory cells.

Polyclonal Antibodies (PAbs)

• Polyclonal antibodies (PAbs) are produced when several different plasma cell lines (clones) produce antibodies against different epitopes of an antigen.
• They are heterogeneous; each clone secretes different antibodies against different epitopes.

Group Activity – Generating and Purifying Abs

• This section describes the process of immunization, a key factor for generating antibodies.
• The purification of Abs.

Polyclonal Antibody Production – Host

• Laboratory-scale production uses small volumes of serum from various animals (e.g., rabbits, chickens).
• Large-scale production uses larger volumes from animals like sheep, goats, and pigs.
• Serum is extracted from animals after immunization with an appropriate antigen.

Polyclonal Antibody Production – Adjuvants

• Immunogens include proteins, polysaccharides, lipids, nucleic acids, conjugates, microorganisms, and mammalian cells.
• Adjuvants enhance sustained immune responses by acting as depots to slowly release antigen.
• Examples include Freund's adjuvant and alum.

Adjuvants Enhance to Production of Abs

• Visual representations of vaccine development with and without adjuvants.
• Immunization process with and without adjuvants.

Polyclonal Antibody Production

• Injecting an antigen into a rabbit triggers a response producing antibodies.
• Activated B cells differentiate into plasma cells, producing polyclonal antibodies.
• Collect antiserum containing polyclonal antibodies from the immunized rabbit.

Polyclonal Antibody Production

• Standard immunization protocols are designed to maximize the immune response.
• Factors to consider include species, form, and dose of immunogen, choice of adjuvant, and immunizations' route, number, and timing.

Polyclonal Antibody Purification

• Purification methods involve concentrating antibodies from serum, typically using ammonium sulfate precipitation.

Purification of Polyclonal IgG

• Purify polyclonal IgG using immunoaffinity chromatography with cyanogen bromide-activated Sepharose.
• Sepharose is a matrix to which the specific target antigen is attached to bind the antibody.
• Unbound materials washed away, antibodies eluted using elution buffer.

Immunoaffinity Chromatography – Antigen

• Antibodies binding to the antigen are retained by the beads.
• Other antibodies are eluted.
• Antigen-specific antibodies eluted with acidic solutions.

Immunoaffinity Chromatography – Ab

• Antibodies are immobilised on a Sepharose matrix, binding to Protein A through the IgG Fc region.
• Other proteins eluted.
• IgG eluted using an acidic solution.