A-353 Investigation of the Immunochromatographic Method using 150 nm Gold Colloids (2)-Comparison with the ALP-based Chromogenic Detection Method
Authors: Asahi M , Hirano A , Otsuka M , Kubota R 
Published: September 2023

Abstract

Background
Currently, standard test kits based on the immunochromatography principle use 40 nm gold colloids as the labeling material. However, some kits use enzymes such as horseradish peroxidase and alkaline phosphatase (ALP) to improve sensitivity. Among the various labeling substances, 150 nm gold colloidal nanoparticles are thought to enable higher sensitivity in immunochromatography than the commonly used 40 nm gold colloids. This study aims to compare a detection method using 150 nm gold colloidal nanoparticles as a labeling material with a colorimetric detection method using ALP-labeled antibodies, which was previously studied in our laboratory.

Methods
1) Detection method using 150 nm gold colloidal nanoparticlesThe BioReady high sensitivity gold conjugation kit for lateral flow (nanoComposix) was used to prepare gold colloid-labeled antibodies. Solid-phase antibodies, such as anti-human albumin monoclonal antibody (abcam) or anti-human troponin I monoclonal antibody (Merck Millipore), were applied onto a nitrocellulose membrane (HF075, Merck Millipore) and then transferred onto a test plate. The sample (human serum albumin or troponin I standard) was diluted with phosphate-buffered saline (PBS), and the gold colloid-labeled antibody and blocking solution (EzBlock Chemi, ATTO) were added and mixed. The mixture was added dropwise onto the test plate and incubated for 15 min. Subsequently, the results were recorded. 2) Chromogenic detection using ALP-conjugated antibodiesThis method was performed according to a method previously investigated in our laboratory. An anti-human albumin polyclonal antibody (DAKO) was applied onto a nitrocellulose membrane and then transferred onto a test plate. The membrane was mixed with the PBS-diluted sample, ALP-labeled anti-human albumin monoclonal antibody, and blocking solution. The mixture was added dropwise onto the test plate and incubated for 5 min. Subsequently, a drop of the chromogenic reagent was added directly onto the test plate, and the results were recorded after 10 min.

Results
Based on the immunochromatography results using 150 nm gold colloidal nanoparticles, black lines were detected in the judgment zone at albumin concentrations of 100, 10, and 3 ng/mL. Moreover, albumin concentrations of up to 3 ng/mL were detectable. Subsequently, the colorimetric detection results using ALP-labeled antibodies indicated that albumin concentrations of up to 100 ng/mL were detectable, with the exception of the 10 ng/mL concentration. Similarly, when troponin I was analyzed using 150 nm gold colloidal nanoparticles, black lines were detected in the judgment zone at troponin I concentrations of 30, 10, and 5 pg/mL.

Conclusion
In this study, the application of troponin I in point of care testing (POCT) was evaluated, indicating that troponin I was detectable at concentrations of up to 5 pg/mL. Because the detection sensitivity of commercially available immunochromatographic kits for troponin I is approximately 30 pg/mL, the developed method is six times more sensitive. Moreover, the developed method was demonstrated to be more sensitive than the chromogenic method using ALP-labeled antibodies.