Abstract
BackgroundSandwich enzyme-linked immunosorbent assay (ELISA) is one of the most frequently employed assays for clinical diagnosis, since this enables the investigator to identify specific protein biomarkers. However, the conventional assay using a 96-well microtitration plate is time- and sample-consuming, and therefore is not suitable for rapid diagnosis. To overcome these drawbacks, we performed a sandwich ELISA on a microchip.Methods and FindingsThe microchip was made of cyclic olefin copolymer with straight microchannels that were 300 µm wide and 100 µm deep. For the construction of a sandwich ELISA for procollagen type I C-peptide (PICP), a biomarker for bone formation, we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-PICP antibody on the surface of the microchannel. After the infusion of the mixture of 2.0 µl of peroxidase-labeled 2nd anti-PICP antibody and 0.4 µl of sample to the microchannel and a 30-min incubation, the substrate for peroxidase was infused into the microchannel; and the luminescence intensity of each spot of 1st antibody was measured by CCD camera. A linear relationship was observed between PICP concentration and luminescence intensity over the range of 0 to 600 ng/ml (r2 = 0.991), and the detection limit was 4.7 ng/ml. Blood PICP concentrations of 6 subjects estimated from microchip were compared with results obtained by the conventional method. Good correlation was observed between methods according to simple linear regression analysis (R2 = 0.9914). The within-day and between-days reproducibilities were 3.2–7.4 and 4.4–6.8%, respectively. This assay reduced the time for the antigen-antibody reaction to 1/6, and the consumption of samples and reagents to 1/50 compared with the conventional method.ConclusionThis assay enabled us to determine serum PICP with accuracy, high sensitivity, time saving ability, and low consumption of sample and reagents, and thus will be applicable to clinic diagnosis.
Highlights
Many plasma proteins that have originated from various tissues or blood cells as a result of secretion or leakage can be used as biomarkers, and many studies have demonstrated that plasma protein levels reflect human physiological or pathological states and can be used for disease diagnosis and prognosis [1,2,3]
We demonstrated the possible application of microchips for monitoring serum procollagen type I C-peptide (PICP) levels and its potential for point-of care testing (POCT)
The cyclic olefin copolymer (COC) microchip was fabricated by injection molding with a nickel mold, and the microchip surface was treated with polymer solution containing p-nitrophenyl ester which binds to amino groups in proteins for 1st antibody fixation on microchannel surface (BSX2321, SUMITOMO BAKELITE Co., Ltd, Tokyo, Japan)
Summary
Many plasma proteins that have originated from various tissues or blood cells as a result of secretion or leakage can be used as biomarkers, and many studies have demonstrated that plasma protein levels reflect human physiological or pathological states and can be used for disease diagnosis and prognosis [1,2,3]. The double antibody sandwich technique, the so-called sandwich enzyme-linked immunosorbant assay (ELISA), is frequently used for antigen measurement [4,5]. This assay is useful for detecting a specific antigen when only a small amount of it is available and purified antigen is unavailable. Sandwich enzyme-linked immunosorbent assay (ELISA) is one of the most frequently employed assays for clinical diagnosis, since this enables the investigator to identify specific protein biomarkers. The conventional assay using a 96-well microtitration plate is time- and sample-consuming, and is not suitable for rapid diagnosis To overcome these drawbacks, we performed a sandwich ELISA on a microchip
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