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Fluorescent nucleotides: application in a fluorescence immune assay

R R Kardost, E W Voss

Molecular Immunology

|January 1, 1982

Summary

A new fluorescence immunoassay uses a novel fluorescent nucleotide probe to detect anti-nucleotide antibodies. This assay shows significant fluorescence enhancement upon antibody binding, offering a sensitive method for nucleotide detection.

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Area of Science:

Biochemistry
Immunology
Analytical Chemistry

Background:

Anti-nucleotide antibodies play roles in various autoimmune diseases.
Accurate detection of these antibodies is crucial for diagnosis and research.
Existing assays may have limitations in sensitivity or throughput.

Purpose of the Study:

To develop a novel fluorescence immunoassay for quantifying anti-nucleotide antibody activity.
To synthesize and characterize a fluorescent nucleotide probe with low intrinsic fluorescence.
To validate the assay's performance against established methods.

Main Methods:

Synthesis of a fluorescent nucleotide probe by conjugating AmNS (1-naphthylamine-5-sulfonic acid) to nucleotide phosphates.
Utilizing carbodiimide activation for covalent conjugation.
Measuring fluorescence quantum yield changes upon antibody-antigen interaction.
Correlating results with a modified radioimmune Farr assay.

Main Results:

The synthesized fluorescent nucleotide probe exhibited low quantum yield in aqueous solution (approx. 0.025).
Significant fluorescence enhancement (12-35 fold) was observed upon binding of the probe to homologous anti-nucleotide antibodies.
No significant fluorescence enhancement occurred with non-specific proteins or sera.
The fluorescence assay provided quantitative nucleotide inhibition data and correlated well with the radioimmune Farr assay.

Conclusions:

A sensitive and specific fluorescence immunoassay for anti-nucleotide antibodies has been developed.
The novel fluorescent nucleotide probe enables detection through enhanced fluorescence upon antibody binding.
This assay offers a viable alternative to traditional methods for nucleotide antibody detection.

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