The study on the TNF-α/hs-CRP double-labeled time-resolved fluorescence immunoassay for early screening and diagnosis of sepsis

doi:10.11958/20220176

Abstract

Abstract:

Objective To investigate the value of double-labeled time-resolved fluorescence immunoassay (TRFIA) for quantitatively detecting serum levels of tumor necrosis factor-α (TNF-α) and high-sensitivity C-reactive protein (hs-CRP). Methods Anti-TNF-α and hs-CRP monoclonal antibodies were coated on the 96-well plate, meanwhile prepared the europium (Eu³⁺) and samarium (Sm³⁺)-detection antibody conjugates, and then established the double-antibody sandwich TRFIA method and assembly into a kit, and finally evaluated the detection performance of this kit, such as sensitivity, linear range, spike recovery rate. Results A new method for detecting serum TNF-α and hs-CRP levels by TRFIA was successfully established and assembled into a kit. The linear range of the prepared TRFIA kit for TNF-α was 0-100 ng/L, sensitivity was 0.05 ng/L, the linear range for hs-CRP was 0-100 mg/L, sensitivity was 0.02 mg/L. The spiked recovery rate of TNF-α was between 92.00% and 107.00%, and that of hs-CRP was between 95.00% and 106.82%. There was no obvious cross-reaction with the symptom detection indexes. The TNF-α CV of intra-assay was between 4.57% and 9.24%. The inter-assay was between 5.13% and 9.27%. The hs-CRP CV of intra-assay was between 5.12% and 7.69%, and the inter-assay was 6.07%-10.00%. Additionally, the kit can be stored stably at 4 ℃ for half a year and at 37 ℃ for 7 days. The detection threshold of TNF-α was 0.44 ng/L, and the detection threshold of hs-CRP was 1.41 mg/L. The test results of the kit were consistent with the clinical situation, and the coincidence rate reached 100%. Conclusion The double-labeled TRFIA method can quantitatively detect TNF-α and hs-CRP levels, which has the advantages of high sensitivity, high specificity, convenience and fast. This TRFIA kit provides a new detection method for the early screening, efficacy evaluation and prognostic assessment of clinical samples of sepsis.

Key words: sepsis, tumor necrosis factor-alpha, high-sensitivity C-reactive protein, time-resolved fluorescence immunoassay

CLC Number:

R446

Figures/Tables 4

References 16

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