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Kinetic ELISA in microfluidic channels.

Yanagisawa N, Dutta D - Biosensors (Basel) (2011)

Bottom Line: It has been shown that this format of the immunoassay allows very reliable quantitation of the target species using inexpensive glass microchips and a standard epifluorescence microscope system coupled to a CCD camera.For the viral antibodies assayed here, the limit of detection (LOD) for the analyte concentration in our microchips was established to be 3-5 times lower than that obtained on commercial microwell plates using a fiftieth of the sample volume and less than a third of the incubation time.This benefit is primarily realized as the observed variation in the background fluorescence (signal at the start of the enzyme reaction period) was significantly larger than that in the rate of signal generation upon repeating these assays in different microchannels/microchips.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Wyoming, Laramie, WY 82071, USA. nyanagis@uwyo.edu.

ABSTRACT
In this article, we describe the kinetic ELISA of Blue Tongue and Epizootic Hemorrhagic Disease viral antibodies in microfluidic channels by monitoring the rate of generation of the enzyme reaction product under static conditions. It has been shown that this format of the immunoassay allows very reliable quantitation of the target species using inexpensive glass microchips and a standard epifluorescence microscope system coupled to a CCD camera. For the viral antibodies assayed here, the limit of detection (LOD) for the analyte concentration in our microchips was established to be 3-5 times lower than that obtained on commercial microwell plates using a fiftieth of the sample volume and less than a third of the incubation time. Our analyses further show that when compared to the end-point ELISA format, the kinetic mode of this assay yields an improvement in the LOD by over an order of magnitude in microfluidic devices. This benefit is primarily realized as the observed variation in the background fluorescence (signal at the start of the enzyme reaction period) was significantly larger than that in the rate of signal generation upon repeating these assays in different microchannels/microchips. Because the kinetic ELISA results depend only on the latter quantity, the noise level in them was substantially lower compared to that in its end-point counterpart in which the absolute fluorescence measurements are of greater significance. While a similar benefit was also recorded through implementation of kinetic ELISAs on the microwell platform, the improvement in LOD registered in that system was not as significant as was observed in the case of microfluidic assays.

No MeSH data available.


Related in: MedlinePlus

Temporal variation in the fluorescence signal for Epizootic Hemorrhagic Disease Virus (EHDV) antibody samples in (a) microchannels (b) microwells with an active ELISA surface. (c) Calibration curves for the kinetic and end-point ELISAs in microchips and microwell plates for EHDV antibody samples. (d) Limit of detection (LOD) curves for the kinetic and end-point ELISAs in microchannels and microwells for the EHDV antibody samples. Notice that the x-coordinate in (c) and (d) has been chosen to be the reciprocal of dilution factor rather than the dilution factor itself. Our analyses show that if the latter choice is made, the linear variation between the x and y data points is compromised.
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biosensors-01-00058-f003: Temporal variation in the fluorescence signal for Epizootic Hemorrhagic Disease Virus (EHDV) antibody samples in (a) microchannels (b) microwells with an active ELISA surface. (c) Calibration curves for the kinetic and end-point ELISAs in microchips and microwell plates for EHDV antibody samples. (d) Limit of detection (LOD) curves for the kinetic and end-point ELISAs in microchannels and microwells for the EHDV antibody samples. Notice that the x-coordinate in (c) and (d) has been chosen to be the reciprocal of dilution factor rather than the dilution factor itself. Our analyses show that if the latter choice is made, the linear variation between the x and y data points is compromised.

Mentions: In Figure 2(d), we have presented the signal to noise ratio (S/N) in our BTV antibody ELISA experiments as a function of the sample dilution factor. The noise level in these evaluations was assumed to be three-times the standard deviation in the magnitude of the signal which establishes the limit of detection for these curves to be the dilution factor when S/N = 1. For all of the measurements presented in this figure, the noise level was estimated based on five independent experiments both for the end-point and kinetic assays. Based on these measurements, the limits of detection (in terms of dilution factors) were established to be 420.1 ± 7.9 and 21.1 ± 4.4 for the kinetic and end-point ELISAs, respectively, in the microfluidic channels. The 95% confidence intervals for these LOD values are estimated to be 420.1 ± 9.8 and 21.1 ± 5.5, respectively, based on the two-sided t-distribution. This corresponds to a nearly 20-fold reduction in this quantity on adopting the kinetic version of the assay over its end-point format in microchips. Moreover, the data shows a significantly greater reproducibility in the former mode of the immunoassay yielding a relative standard deviation of 1.9% in the limit of detection (LOD) as compared to 20.9% for the latter case. For the microwell based assays, the LODs for the kinetic and end-point ELISAs were estimated to be 142.3 ± 13.4 and 44.2 ± 4.1, respectively. In this situation, the 95% confidence intervals for these quantities turn out to be 142.3 ± 16.7 and 44.2 ± 5.1, respectively, based on the two-sided t-distribution. This corresponds to about a 3-fold improvement in the smallest concentration that could be reliably quantitated and the relative uncertainly in this figure of merit on adopting the kinetic version of the assay over its end-point format in microwells. On comparing the kinetic ELISAs in microchannels to those in microwells, the former platform has been shown to yield a 3-fold lower LOD with at least 5-fold higher precision. But for the end-point immunoassays the trend reverses and the latter platform was observed to perform twice as well as the microfluidic devices. For the EHDV antibodies, similar trends to those reported above were again noticed for the immunoassays (see Figure 3). In the case of the microfluidic experiments, the LOD in terms of the dilution factors were estimated to be 307.3 ± 5.9 and 18.8 ± 6.3, respectively, for the kinetic and end-point version of the ELISAs. The corresponding numbers for the microwell plates were 63.1 ± 17.2 and 29.3 ± 8.1, respectively. Based on the two-sided t-distribution, the 95% confidence intervals for these four LODs can be shown to be 307.3 ± 7.3, 18.8 ± 7.8, 63.1 ± 21.4 and 29.3 ± 10.1, respectively. Therefore, the improvement in LOD yielded by the microchips over the microwell plates was a factor of 4.9 for kinetic EHDV antibody ELISAs while the latter platform performed only about 1.5 fold better than the microchips in the case of the end-point assays. As before, the best reproducibility was obtained for the microchip based kinetic ELISAs with relative standard deviation of 1.9%.


Kinetic ELISA in microfluidic channels.

Yanagisawa N, Dutta D - Biosensors (Basel) (2011)

Temporal variation in the fluorescence signal for Epizootic Hemorrhagic Disease Virus (EHDV) antibody samples in (a) microchannels (b) microwells with an active ELISA surface. (c) Calibration curves for the kinetic and end-point ELISAs in microchips and microwell plates for EHDV antibody samples. (d) Limit of detection (LOD) curves for the kinetic and end-point ELISAs in microchannels and microwells for the EHDV antibody samples. Notice that the x-coordinate in (c) and (d) has been chosen to be the reciprocal of dilution factor rather than the dilution factor itself. Our analyses show that if the latter choice is made, the linear variation between the x and y data points is compromised.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264342&req=5

biosensors-01-00058-f003: Temporal variation in the fluorescence signal for Epizootic Hemorrhagic Disease Virus (EHDV) antibody samples in (a) microchannels (b) microwells with an active ELISA surface. (c) Calibration curves for the kinetic and end-point ELISAs in microchips and microwell plates for EHDV antibody samples. (d) Limit of detection (LOD) curves for the kinetic and end-point ELISAs in microchannels and microwells for the EHDV antibody samples. Notice that the x-coordinate in (c) and (d) has been chosen to be the reciprocal of dilution factor rather than the dilution factor itself. Our analyses show that if the latter choice is made, the linear variation between the x and y data points is compromised.
Mentions: In Figure 2(d), we have presented the signal to noise ratio (S/N) in our BTV antibody ELISA experiments as a function of the sample dilution factor. The noise level in these evaluations was assumed to be three-times the standard deviation in the magnitude of the signal which establishes the limit of detection for these curves to be the dilution factor when S/N = 1. For all of the measurements presented in this figure, the noise level was estimated based on five independent experiments both for the end-point and kinetic assays. Based on these measurements, the limits of detection (in terms of dilution factors) were established to be 420.1 ± 7.9 and 21.1 ± 4.4 for the kinetic and end-point ELISAs, respectively, in the microfluidic channels. The 95% confidence intervals for these LOD values are estimated to be 420.1 ± 9.8 and 21.1 ± 5.5, respectively, based on the two-sided t-distribution. This corresponds to a nearly 20-fold reduction in this quantity on adopting the kinetic version of the assay over its end-point format in microchips. Moreover, the data shows a significantly greater reproducibility in the former mode of the immunoassay yielding a relative standard deviation of 1.9% in the limit of detection (LOD) as compared to 20.9% for the latter case. For the microwell based assays, the LODs for the kinetic and end-point ELISAs were estimated to be 142.3 ± 13.4 and 44.2 ± 4.1, respectively. In this situation, the 95% confidence intervals for these quantities turn out to be 142.3 ± 16.7 and 44.2 ± 5.1, respectively, based on the two-sided t-distribution. This corresponds to about a 3-fold improvement in the smallest concentration that could be reliably quantitated and the relative uncertainly in this figure of merit on adopting the kinetic version of the assay over its end-point format in microwells. On comparing the kinetic ELISAs in microchannels to those in microwells, the former platform has been shown to yield a 3-fold lower LOD with at least 5-fold higher precision. But for the end-point immunoassays the trend reverses and the latter platform was observed to perform twice as well as the microfluidic devices. For the EHDV antibodies, similar trends to those reported above were again noticed for the immunoassays (see Figure 3). In the case of the microfluidic experiments, the LOD in terms of the dilution factors were estimated to be 307.3 ± 5.9 and 18.8 ± 6.3, respectively, for the kinetic and end-point version of the ELISAs. The corresponding numbers for the microwell plates were 63.1 ± 17.2 and 29.3 ± 8.1, respectively. Based on the two-sided t-distribution, the 95% confidence intervals for these four LODs can be shown to be 307.3 ± 7.3, 18.8 ± 7.8, 63.1 ± 21.4 and 29.3 ± 10.1, respectively. Therefore, the improvement in LOD yielded by the microchips over the microwell plates was a factor of 4.9 for kinetic EHDV antibody ELISAs while the latter platform performed only about 1.5 fold better than the microchips in the case of the end-point assays. As before, the best reproducibility was obtained for the microchip based kinetic ELISAs with relative standard deviation of 1.9%.

Bottom Line: It has been shown that this format of the immunoassay allows very reliable quantitation of the target species using inexpensive glass microchips and a standard epifluorescence microscope system coupled to a CCD camera.For the viral antibodies assayed here, the limit of detection (LOD) for the analyte concentration in our microchips was established to be 3-5 times lower than that obtained on commercial microwell plates using a fiftieth of the sample volume and less than a third of the incubation time.This benefit is primarily realized as the observed variation in the background fluorescence (signal at the start of the enzyme reaction period) was significantly larger than that in the rate of signal generation upon repeating these assays in different microchannels/microchips.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Wyoming, Laramie, WY 82071, USA. nyanagis@uwyo.edu.

ABSTRACT
In this article, we describe the kinetic ELISA of Blue Tongue and Epizootic Hemorrhagic Disease viral antibodies in microfluidic channels by monitoring the rate of generation of the enzyme reaction product under static conditions. It has been shown that this format of the immunoassay allows very reliable quantitation of the target species using inexpensive glass microchips and a standard epifluorescence microscope system coupled to a CCD camera. For the viral antibodies assayed here, the limit of detection (LOD) for the analyte concentration in our microchips was established to be 3-5 times lower than that obtained on commercial microwell plates using a fiftieth of the sample volume and less than a third of the incubation time. Our analyses further show that when compared to the end-point ELISA format, the kinetic mode of this assay yields an improvement in the LOD by over an order of magnitude in microfluidic devices. This benefit is primarily realized as the observed variation in the background fluorescence (signal at the start of the enzyme reaction period) was significantly larger than that in the rate of signal generation upon repeating these assays in different microchannels/microchips. Because the kinetic ELISA results depend only on the latter quantity, the noise level in them was substantially lower compared to that in its end-point counterpart in which the absolute fluorescence measurements are of greater significance. While a similar benefit was also recorded through implementation of kinetic ELISAs on the microwell platform, the improvement in LOD registered in that system was not as significant as was observed in the case of microfluidic assays.

No MeSH data available.


Related in: MedlinePlus