Establishment and preliminary clinical evaluation of a high-sensitivity chemiluminescence immunoassay for serum IL-12p70 using fab' antibody fragments and acridinium ester

• Innovative and reliable method for quantifying interleukin-12p70 (IL-12p70) in human serum. • We employed the following techniques: Fab′ antibody fragments, biotin-streptavidin amplification, and acridinium ester chemiluminescence. • The assay employs a unique sandwich format with an acridinium ester-labeled full IL-12p70 antibody and a biotin-labeled p40 fragment. • It demonstrates high sensitivity, specificity, and stability with superior performance to existing methods. • Excellent correlation with flow cytometry validates the assay's clinical utility for serum analysis. This study aimed to establish a detection method for serum interleukin 12p70 (IL-12p70) using digested Fab' antibody fragments and acridine esterification chemiluminescence immunoquantitative analysis. The double-antibody sandwich method was used with the complete IL-12p70 antibody labeled with acridine ester and an enzymatically digested antibody fragment of p40 labeled with biotin. A sandwich complex then formed with the analyte IL-12p70, in which biotin was captured by magnetic solid-phase particles coated with streptavidin, which glowed under alkaline hydrogen peroxide excitation. The luminescent signal was quantitatively analyzed. The stability of the labeled antibodies and the performance of the new quantitative method was evaluated. The clinical analysis results were compared with flow cytometry fluorescence to verify the clinical performance. Acridine ester sulfonamide and liposoluble biotin showed good stability as antibody labels. The limit of blank was 0.1 pg/mL and the limit of detection was 0.5 pg/mL, determined according to CLSI EP17-A2 with n = 60 replicates per batch with two independent batches. The linearity was 0.5–5000 pg/mL, and the precision was < 5.5 %. The results of the new method and flow cytometry fluorescence were highly correlated ( P < 0.05). Interfering substances, including triglyceride, hemoglobin, bilirubin, rheumatoid factor, heterophilic human anti-mouse antibodies, and the structural analogs IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and IL-23 had no significant effect on the results. A method for detecting IL-12p70 in serum was established based on immunoquantitative analysis using Fab' antibody fragments, biotin and streptavidin amplification effects, and acridine ester chemiluminescence. The method exhibited high sensitivity, strong specificity, and a wide detection range, indicating its strong clinical application prospects.