An Enzyme Immunoanalytical System Based on Sequential Cross-Flow Chromatography

Abstract

A new enzyme immunoanalytical concept that can be used for point-of-care testing has been investigated. Enzyme as a tracer requires a separate reaction step for signal generation, which follows the completion of immune complex formation with analyte (e.g., Hepatitis B surface antigen) in a sample. This has been a major factor limiting its utilization within the laboratory. We carried out such sequential processes employing chromatographic analysis, using two crosswise-arranged membrane pads in vertical and horizontal directions. The vertically arranged pads were the same as those in the usual format for pregnancy testing, for instance, with the exception of the use of horseradish peroxidase (HRP) as tracer. By placing the horizontally arranged pads on each lateral side of the signal generation pad in the vertical arrangement, they were employed to supply substrate to the enzyme present in the immune complexes. The substrate flow was initiated after the antigen-antibody bindings to produce a signal, which was typically a color change in proportion to the analyte concentration. Under optimal conditions, the use of HRP labeling increased the detection capability of the assay approximately 30 times compared to that of gold colloids. Potential advantages of using the concept investigated are (1) provision of a rapid and simple immunoassay, (2) satisfaction of a clinical need for highly sensitive determination of analyte, and (3) utilization of relatively inexpensive, portable quantitation means.

Lateral Flow Immunoassay Using Europium Chelate-Loaded Silica Nanoparticles as Labels

“…The major reasons for this limited use of LFIAs are the low signal intensity (1 ) and poor quantitative discrimination (2, 3 ) of the color-formation reaction based on label accumulation (4,5 ). Although researchers have tried to use instrumental means to generate quantitative results (6,7 ), the relatively low signals obtained often make such efforts meaningless (1 ). To address this limitation, a variety of reporters have been developed, including colored particles (8,9 ), carbon black (10 ), and fluorophores (11,12 ).…”

Immunogold–silver staining-on-a-chip biosensor based on cross-flow chromatography

“…As reference, the conventional rapid test kit employing colloidal gold (30 nm diameter) as a tracer was constructed as previously described [12]. Identical samples to those used for the IOC were loaded on each kit and laterally migrated for 15 min.…”

“…The same analytical concept of cross-flow immunochromatography [12] can be applied to IGSS so that this staining procedure can be conducted anywhere the specimen is furnished, particularly outside of the laboratory (Fig. 2).…”

Moving Enzyme-Linked ImmunoSorbent Assay to the Point-of-Care Dry-Reagent Strip Biosensors

“…Combining the high sensitivity of ELISA and the unique above mentioned advantages of the IST, the enzyme based immunochromagographic strip test (EIST) has shown the promising to detect proteins at very low concentration levels. The EIST was pioneered by different groups [13][14][15][16][17]. Early EIST was based on the measurement of the height of the formed color bands of the enzymatic product on the strip [13,14].…”

“…Early EIST was based on the measurement of the height of the formed color bands of the enzymatic product on the strip [13,14]. The performance of the EIST was improved by designing cross-flow plastic chips and analysis of the intensities of the formed bands [16,17]. However the quantification of the analytes was usually performed on either the array scanner or complex and expensive instrumentations, which limits its in-field and POC applications.…”