Bispecific-Ab-based immunoassay of thyroid-stimulating hormone

Area of Science:

• Biochemistry
• Immunology
• Assay Development

Background:

• Thyroid-stimulating hormone (TSH) is a crucial biomarker for thyroid function.
• Existing enzyme-linked immunosorbent assays (ELISAs) for TSH can be complex and require specific conjugate preparation.
• There is a need for more sensitive and streamlined TSH detection methods.

Purpose of the Study:

• To develop a bispecific F(ab')2 fragment targeting both TSH and alkaline phosphatase (AP).
• To create a sandwich ELISA for TSH utilizing AP polymers and the bispecific fragment.
• To improve assay sensitivity and reduce non-specific protein interactions.

Main Methods:

• Preparation of a bispecific F(ab')2 fragment via disulfide bond exchange between anti-TSH and anti-AP IgG1 mAb F(ab')2 fragments.
• Polymerization of AP using glutaraldehyde.
• Development of a sandwich ELISA employing AP polymers and the bispecific F(ab')2 fragment for TSH detection.

Main Results:

• The assay eliminated the need for covalent AP-mAb conjugate preparation.
• Non-specific protein interactions were significantly minimized.
• TSH sensitivity increased proportionally with AP polymerization degree, achieving a 0.5 muU/ml lower detection limit with AP trimer.
• The bispecific F(ab')2 fragment enabled adjustable assay sensitivity through AP monomer/polymer use.

Conclusions:

• A novel and sensitive sandwich ELISA for TSH detection was successfully developed.
• The bispecific F(ab')2 fragment strategy simplifies assay construction and enhances performance.
• This approach offers a versatile method for regulating assay sensitivity in TSH diagnostics.