ABSTRACT
BACKGROUND: AKI is related to severe adverse outcomes and mortality with Coronavirus Disease 2019 (COVID-19) patients, that early diagnosed and intervened is imperative. Neutrophil gelatinase-associated lipocalin (NGAL) is one of the most promising biomarkers for detection of acute kidney injury (AKI), but current detection methods are inadequacy, so more rapid, convenient and accuracy methods are needed to detect NGAL for early diagnosis of AKI. Herein, we established a rapid, reliable and accuracy lateral flow immunoassay (LFIA) based on europium nanoparticles (EU-NPS) for the detection of NGAL in human urine specimens. METHODS: A double-antibody sandwich immunofluorescent assay using europium doped nanoparticles was employed and the NGAL monoclonal antibodies (MAbs) conjugate as labels were generated by optimizing electric fusion parameters. Eighty-three urine samples were used to evaluate the clinical application efficiency of this method. RESULTS: The quantitative detection range of NGAL in AKI was 1-3000 ng/mL, and the detection sensitization was 0.36 ng/mL. The coefficient of variation (CV) of intra-assay and inter-assay were 2.57-4.98 % and 4.11-7.83 %, respectively. Meanwhile, the correlation coefficient between europium nanoparticles-based lateral fluorescence immunoassays (EU-NPS-LFIA) and ARCHITECT analyzer was significant (R2 = 0.9829, n = 83, p < 0.01). CONCLUSIONS: Thus, a faster and easier operation quantitative assay of NGAL for AKI has been established, which is very important and meaningful to diagnose the early AKI, suggesting that the assay can provide an early warning of final outcome of disease.
Subject(s)
Acute Kidney Injury/diagnosis , Europium , Fluoroimmunoassay/methods , Lipocalin-2/urine , Metal Nanoparticles , Acute Kidney Injury/virology , Animals , Antibodies, Monoclonal/isolation & purification , COVID-19/complications , Enzyme-Linked Immunosorbent Assay , Humans , Lipocalin-2/immunology , Mice , Recombinant Proteins/isolation & purification , Reproducibility of Results , SARS-CoV-2ABSTRACT
Objective To prepare and identify mouse monoclonal antibodies against human vasorin (VASN) protein using electrofusion method. Methods The mice were immunized with human recombinant protein VASN-His, and then the cells were fused by electrofusion apparatus. Indirect ELISA was used to screen the positive hybridoma cells which could bind natural protein VASN. The titer and affinities of the antibodies were detected by ELISA, and Western blotting was used to determine whether the antibody could recognize VASN protein in HepG2 cells. Results The fusion rate reached 0.31% when the ratio of spleen cells and Sp2/0 myeloma cells was 2:1, the alternating electric field intensity was 50 V, 2 MHz for 20 seconds, and the direct current pulse intensity was 500 V for 0.5 second. Two mouse anti-human VASN monoclonal antibodies (4H1and 8B9) were obtained, with the highest titer of 1:256 000 and the highest affinity constant (Ka) of 4.9×106 L/mol. Western blotting showed that both monoclonal antibodies could specifically recognize VASN in HepG2 cells. Conclusion Two mouse anti-human VASN monoclonal antibodies have been successfully prepared by the cell electrofusion method.
