Background: To establish a fast and simple quantitative method for detection of heart-type fatty acid-binding protein (H-FABP) in serum based on a background fluorescence quenching immunochromatographic assay.
Methods: A detection card based on the double-antibody sandwich double-antibody method with background fluorescence quenching was developed for quantitative measurement of H-FABP in serum. The optimal concentrations of control for coating the test and control lines were determined as well as the concentrations of gold-labeled antibodies used in preparing the detection system. The detection method for H-FABP in serum was established and validated using real-world clinical samples.
Results: The optimal concentrations of labeling antibody and coating antibody were 5.0 μg/mL and 1.0 mg/mL, respectively. The test card had a sensitivity of 1.15 ng/mL over a linear concentration range of 0-100 ng/mL. Based on three batches prepared for testing the card, the relative standard deviation (RSD) within batches was less than 15% without a significant difference (P=0.942). The detection method was tested against common interfering substances in serum, such as bilirubin, triglyceride and serum anticoagulants ethylenediamine tetraacetic acid (EDTA), heparin, and sodium citrate, and no significant cross-reaction was detected. The test method was further validated with 50 clinical serum samples, and the test results were comparable with standard reference detection methods with good correlation (R=0.95).
Conclusion: Our study presents a new method with strong specificity and sensitivity for the detection of H-FABP in serum, which could promote H-FABP detection in a broad range of applications.
Uvod: Cilj ovog rada je bio da se izradi brz i jednostavan kvantitativni metod za detekciju srčanog vezujućeg proteina za masne kiseline (H-FABP) u serumu na osnovu fluores-centnog imunohromatografskog određivanja.
Metode: Detekciona karta se zasniva na duploj antibodi sendvič duploj-antibodi metodi zasnovanoj na fluorescenciji i razvijenoj za kvantitativno merenje H-FABP u serumu. Određene su optimalne koncentracije za presvlačenje linija testa i kontrola kao i koncentracije antitela označenih zlatom pri pripremi samog sistema. Uspostavljen je detekcioni metod za H-FABP u serumu i validiran primenom priznatih svetskih kliničkih metoda.
Rezultati: Optimalne koncentracije označenih anititela i pre-svučenih antitela bile su 5,0 μg/mL odnosno 1,0 mg/mL. Test karta imala je osetljivost 1,15 ng/mL u opsegu linearne koncentracije od 0–100 ng/mL. Na osnovu pripremljene tri grupe uzoraka za ispitivanje karte dobijena je relativna standardna devijacija (SD) između grupa manja od 15% bez značajnih razlika (P = 0,942). Metod detekcije ispitivan je naspram uobičajenih interferujućih supstanci u serumu, kao što su bilirubin, trigliceridi i serumski antikoagulansi etilendiamin tetrasirćetna kiselina (EDTA), heparin i natrijum citrat i pri tom nije nađena bilo kakva interferencija. Metoda je zatim validovana na 50 kliničkih serumskih uzoraka, pri čemu su rezultati bili uporedivi sa standardnim detekcionim metodama i dobrom korelacijom (R = 0,95).
Zaključak: Naša izučavanja predstavljaju novu metodu sa snažnom specifičnošću i osetljivosti za detekciju H-FABP u serumu, pa se može preporučiti za širu upotrebu.
Keywords: background fluorescence quenching; heart-type fatty acid binding protein; immunochromatography.