ELISPOT analysis method for detecting IFN-gamma and IL-2 secretion and detection kit thereof

The dual-enzyme, dual-factor ELISPOT assay, combining specific concentrations of IFN-γ and IL-2 capture antibodies with blue-red chromogenic solution, solves the problems of high detection sensitivity and cost in existing technologies, achieving efficient and low-cost dual-factor detection suitable for vaccine development and clinical diagnosis.

CN121595880APending Publication Date: 2026-03-03THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
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Patent Information

Application Number
CN202511735571.4
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2025-11-25
Publication Date
2026-03-03

AI Technical Summary

Technical Problem

Existing technologies lack highly sensitive and low-cost two-factor ELISPOT detection methods, and commercial kits suffer from long lead times, instability, and high prices, making it difficult to meet the needs of scientific research and vaccine development.

Method used

The dual-enzyme, dual-factor ELISPOT assay was employed, using specific concentrations of IFN-γ and IL-2 capture antibodies for coating, followed by colorimetric reactions with blue and red chromogenic solutions. Data processing was performed using a CTL enzyme-linked spot analyzer to optimize incubation temperature and time. Outsourced antibody and chromogenic solution were used to ensure the sensitivity and specificity of the assay.

Benefits of technology

It achieves highly sensitive and low-cost two-factor detection, saving experimental time and sample volume, improving the reliability and comparability of detection, and is suitable for vaccine development and clinical diagnosis, reducing dependence on commercial reagent kits.

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Abstract

The invention provides an ELISPOT (Enzyme-Linked Immunospot Assay) analysis method for detecting IFN-gamma and IL-2 secretion, which comprises the following steps: activating an ELISPOT plate, washing, adding IFN-gamma and IL-2 capture antibody coating with specific concentration, and staying overnight at 4 DEG C; recovering and counting target sample cells, resuspending the target sample cells according to a certain concentration, and adding the target sample cells into coated plate holes; incubating for more than 16 hours in an environment of 37 DEG C and 5% COC; washing the incubated plate, adding IFN-HRP and IL-2-ALP detection antibodies, and incubating at room temperature for 2 hours; after washing, adding a blue developing solution for developing for 10-15 minutes; adding a red developing solution, incubating for 10-15 minutes, and stopping developing; according to the method, T cell subset identification and multifunctional analysis can be carried out, in the early stage of vaccine research and development, due to the fact that target positive population is small, the number of obtained PBMC cells is small, two-factor detection can detect different cell factor reaction frequencies and locate different cell populations and subtypes at the same time, and comparison of two-factor data is beneficial to finding out population immune differences; and scientific research and vaccine development are promoted.
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