3-(4-Hydroxyphenyl)propionic acid: the forgotten detection substrate for ligand-binding assay-based bioanalysis.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Immunology

Background:

• Ligand-binding assays are crucial for routine bioanalysis.
• Conventional chromogenic substrates in immunoassays often exhibit narrow dynamic ranges.
• Horseradish peroxidase (HRP) with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is a common substrate in preclinical immunoassays.

Purpose of the Study:

• To enhance the dynamic range and sensitivity of a preclinical immunoassay for human IgG detection.
• To evaluate the replacement of a chromogenic substrate with a fluorogenic alternative.

Main Methods:

• A preclinical immunoassay for detecting human IgG was modified.
• The conventional chromogenic substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was replaced with the fluorogenic 3-(4-hydroxyphenyl)propionic acid.
• Assay performance was evaluated using sera from horses, mice, and monkeys.

Main Results:

• The fluorogenic substrate replacement resulted in improved sensitivity, reaching 0.47 ng/ml (MRD 10) and 1.02 ng/ml (MRD 50).
• Dynamic ranges were significantly broadened to 3.3 orders of magnitude (MRD 10) and 3.6 orders of magnitude (MRD 50).
• Enhanced sensitivity and dynamic range were observed across different animal sera without specific assay optimization.

Conclusions:

• Fluorogenic substrate substitution offers a straightforward method to improve immunoassay performance.
• This approach provides superior sensitivity and dynamic range compared to conventional colorimetric ELISAs, other ligand-binding assays, and LC-MS.
• The method is effective for bioanalysis in preclinical settings, applicable to various species without extensive optimization.