Improved antibody coating protocol using a second antibody antiserum. Application to total thyroxin immunoassay

Area of Science:

• Biotechnology
• Immunochemistry
• Assay Development

Background:

• Antibody immobilization is crucial for immunoassays.
• Existing antibody coating protocols can be complex and lack long-term stability.
• A simplified and stable antibody coating method is needed for various diagnostic applications.

Purpose of the Study:

• To present a complete antibody coating protocol for dry antibody coated tubes.
• To improve and simplify a previously described antibody immobilization principle.
• To develop a drying procedure for long-term storage stability of antibody coated tubes.

Main Methods:

• Modification of an antibody immobilization principle for polystyrene tubes.
• Coating tubes with rabbit gamma-globulins followed by sheep anti-rabbit IgG antiserum.
• Direct addition of antigen-specific antibody antiserum to the tubes.
• Washing, blocking, and a novel drying procedure for long-term stability.
• Application in developing a radioimmunoassay (RIA) for total thyroxine.

Main Results:

• The modified protocol simplifies antibody immobilization and enhances tube stability.
• The developed RIA for total thyroxine demonstrated comparable characteristics to commercial kits.
• Assay performance metrics included a detection limit of 4 microg/L and a dynamic range up to 210 microg/L.
• Low coefficients of variation (CV) for within-run (2.7-5.7%) and between-run (5.1-7.3%) assays were achieved.
• High recovery (84.4-112%) and low cross-reactivity for T3 (1.9%) were observed.

Conclusions:

• The modified protocol offers an improved and simplified method for preparing dry antibody coated tubes.
• The protocol ensures long-term storage stability, suitable for high antibody loading immunoassays.
• The developed thyroxine RIA highlights the efficacy of the protocol for diagnostic applications.