A nanogram-level colloidal gold single reagent quantitative protein assay

Abstract

We have developed a nanogram level quantitative protein assay based on the binding of colloidal gold to proteins adhered to nitrocellulose paper. The protein-gold complex produces a purple color proportional to the amount of protein present, and the intensity of the stain is quantified by densitometry. Typical assays require minimal starting material (10 -20 μl) containing 2 -5 μg protein. A small volume (2 μl) of protein solution is applied to nitrocellulose paper in a grid array, and dried. The nitrocellulose is incubated in colloidal gold suspension with gentle agitation (4-16 h), rinsed with water and scanned. Densitometric analysis of the scanned images allows quantitation of the unknown sample protein concentration by comparison with protein standards placed on the same nitrocellulose grid. The assay requires significantly less sample than conventional protein assays. In this report, the Golddots assay is calibrated against weighed protein samples, and compared with the Pierce Micro BCA protein assay kit. In addition, the assay is evaluated with several known proteins with different physical properties.

A modified protein assay from microgram to low nanogram levels in dilute samples

“…Among them, solid phase assays are either less sensitive [3, 7] or require additional steps and reagents [4–6]. Liquid phase assays on the other hand, require spectrophotometry, disallowing the ease of densitometry [8, 9].…”

“…In this study, we examined the performance of our chosen combination staining (AB+CG) on five proteins of different molecular weight by APN. The poor and inconsistent staining of low molecular weight proteins like insulin (MW 5.8 kDa), ribonuclease-A (MW 13.7 kDa), and lysozyme (MW 14.3 kDa) (Table 1), is not uncommon [3, 7]. This could be due to poor binding of small size proteins to NC membranes [18], dyes [4] or its passage through the membrane and into the filtrate [5, 13].…”

“…The combined effect of amido black followed by colloidal gold was found to be the most suitable combination staining of protein dots in terms of sensitivity and specificity. Like several other solid phase [3–7, 13] and liquid phase [1, 2, 8–11] assays, our improved assay is also susceptible to protein-to-protein variations. The protein assay presented in this paper, however, is unaffected by detergents that are normally used in solubilization of membrane proteins, included in 2-dimensional SDS-PAGE sample buffer [15].…”

Embedded Immunodetection System for Fecal Occult Blood

“…Prior to the development of image analyses for rapid tests, the analytical method relied only on visual qualitative or semi-quantitative analyses, in which the standard colorimetric card and the color of the T line on the rapid test are compared [ 7 , 8 ]. These methods limit the scope of clinical applications because the basis for judging the results is direct observation using the naked eye, which may lead to subjective misjudgment.…”

Optimization of an Optical Inspection System Based on the Taguchi Method for Quantitative Analysis of Point-of-Care Testing

“…However, the color of test line was lighter under lower levels of hCG concentration. The results of the test line were compared with the standard chroma card [ 4 – 6 ]. The color difference between the test line and standard chroma card was not clearly determined at lower levels by the naked eye, and the color discrimination was affected by the external environment and human factors.…”