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This manuscript focuses on the production of plasma standards for the construction of plasma standard curves, as used in colorimetric assays. Whilst there are different ways of producing plasma standards, the presented method, whilst being

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A General Method for the Construction of Plasma Standards for Standard Curves as used in Colorimetric Assays
H. Edwards, Charles Sturt University
Abstract
This manuscript focuses on the production of plasma standards for the construction of plasma standard curves, as used in colorimetric assays. Whilst there are different ways of producing plasma standards, the presented method, whilst being mathematically exact, is time and cost effective, as it is practically easy to perform and requires minimal amounts of plasma and equipment. The concept here presented can be applied to similar problems. Keywords: Plasma; Standard curve; Colorimetric assay; Monetary and practical efficacy
Introduction
Standard curves are employed to determine the concentrations of analytes in fluids through comparison against a standard range of known concentration. Aqueous standard curves are easy to construct employing serial dilutions. Plasma standard curves however face a challenge if the analyte of interest naturally occurs in plasma. Another challenge occurs as assays require small volumes of plasma, whilst looking at low concentrations of analytes. To avoid needing large volumes of plasma or evaporation and resuspension procedures, these challenges are overcome with a mathematically exact method which to perform only requires pipetting. Whilst general protocols on setting up colorimetric assays are available from the internet (i.e. http://www.ruf.rice.edu/~bioslabs/methods/protein/protcurve.html) a solution to the identified challenge is not, and is therefore here focused on.
Method
Let us assume we want to monitor the concentration of an analyte which naturally occurs in plasma, over time, associated to treatment or other influences. As a first step we determine the analyte concentration in control plasma with a quantitative method which requires no standard comparison - such as chromatography assays. For studies which include a large amount of samples, these methods however may be too time and cost intensive. Therefore we use this plasma of now known analyte concentration to create our standard curve for a less time and cost intensive assay alike a colorimetric assay. For a plasma colorimetric assay, an agent may have to be employed to precipitate plasma proteins. Therefore the present method relies on a dilution of a given volume of the analyte-containing fluid of interest, with a given volume of agent. Alternatively, to follow the present protocol for other purposes, a given volume of water could be used instead of the agent, as long as such is consistently done for the standards which produce the standard curve, as well as for the samples of the analysis. For illustrative purposes let us assume a 10 fold dilution of 350
μ
L plasma to 3150
μ
L agent. Let us assume we determined the concentration of our analyte via chromatography as 40 mg/L to be the baseline concentration in the control plasma. Hence, the colorimetric assay will yield us an absorbance value associated to a plasma concentration of 40mg/L. We would like to have plasmas of known higher concentrations to produce a standard curve, such that we can determine analyte concentrations from absorbance values associated to known concentrations. Producing plasmas of higher concentrations by adding the analyte to plasma, would either require evaporation and resuspension procedures, or large volumes of plasma. However adding the required additional amount of analyte
within
the agent, is equivalent to employing plasmas of known higher concentrations. This is what we will therefore do. We will use an excel spreadsheet to do the calculations for us. In the first column (A) we note our desired plasma concentration range, i.e. ranging from 40 mg/L to 5000 mg/L of analyte. In the second column (B) we calculate the corresponding analyte quantity within 350
μ
L of plasma as
= /∗.
[1] In the third column (C) we calculate the difference between this desired analyte quantity and the analyte quantity in the 350
μ
L of the 40 mg/L control plasma - which we now know is 0.014 mg/350
μ
L - as
=−.
[2] This is the analyte quantity that we have to add
within
the 3150
μ
L of agent, to our 350
μ
L of our control plasma. For this purpose, we need to create a superstock of a known concentration of our analyte within our agent. This analyte-agent superstock can for example be produced in such a way, that 3150
μ
L of it exactly adds the right amount of analyte to the standard of highest concentration. Alternatively it can be produced at a higher concentration, whilst it has to be produced at at least this concentration. To produce the analyte-agent superstock, we calculate the concentration this superstock at least needs to be, to contain the quantity of analyte that needs to be added for the highest standard within 3150
μ
L. The quantity of analyte that needs to be added with 3150
μ
L of agent for the standard of highest concentration can i.e. be read from our table (see table 1) as 1.736 mg. If we want to i.e. create 100 mL analyte-agent superstock for convenience we therefore need to add
. . =
[3]
. ∗ . =.
[4] 55.1 mg analyte to 100 mL agent. This can be easily done by solving the analyte at a high concentration in laboratory water such that adding a small quantity of the analyte solution to the agent to make up 100 mL causes no significant dilution of the agent. For example a 5.51 mL of a 1000mg/100mL analyte solution could be added to 94.49 mL agent. To reduce the dilution of the agent, 0.551 mL of a 1000mg/10mL analyte solution could be added to 99.449 mL of agent. As we know from column C that we have to add 1.736 mg analyte for our highest standard and we produced our analyte-agent superstock
to yield this within 35 μ
L; in the fourth column (D) we calculate how many
μ
L of this analyte-agent superstock we have to add to our 350
μ
L plasma as
=. ∗
[5] In the fifth column (E), we calculate the difference that we have to make up for with our usual analyte-free agent as
= −
[6]
We pipette 350
μ
L of our control plasma for each standard and pipette the amount of analyte-containing and analyte-free agent as calculated by excel in columns D and E, to form the 1/10 dilution. Please consider table 1 to follow these 5 steps as displayed in columns A-E: Table 1: Calculations for the constructions of the plasma standard curve
A: Plasma conc. (mg/L) B: Plasma conc.
(mg/350
μ
L) C: Analyte Difference
(mg) D: Agent-analyte Superstock
(
μ
L) E: Agent
(
μ
L) Formula
A/1000*0.35 B-0.014 C/1.736*3150 3150-D
40
0.014 0 0 3150
100
0.035 0.021 38.10483871 3111.895161
150
0.0525 0.0385 69.85887097 3080.141129
200
0.07 0.056 101.6129032 3048.387097
300
0.105 0.091 165.1209677 2984.879032
400
0.14 0.126 228.6290323 2921.370968
500
0.175 0.161 292.1370968 2857.862903
600
0.21 0.196 355.6451613 2794.354839
700
0.245 0.231 419.1532258 2730.846774
800
0.28 0.266 482.6612903 2667.33871
900
0.315 0.301 546.1693548 2603.830645
1000
0.35 0.336 609.6774194 2540.322581
1100
0.385 0.371 673.1854839 2476.814516
1200
0.42 0.406 736.6935484 2413.306452
1300
0.455 0.441 800.2016129 2349.798387
1400
0.49 0.476 863.7096774 2286.290323
1500
0.525 0.511 927.2177419 2222.782258
2000
0.7 0.686 1244.758065 1905.241935
2500
0.875 0.861 1562.298387 1587.701613
3000
1.05 1.036 1879.83871 1270.16129
3500
1.225 1.211 2197.379032 952.6209677
4000
1.4 1.386 2514.919355 635.0806452
4500
1.575 1.561 2832.459677 317.5403226
5000
1.75 1.736 3150 0 More or less standards can be included to produce a curve as desired. The standards are then treated as appropriate with vortexing and centrifugation to use the supernatant for colorimetric reaction. When reading the absorbance values for the pipetted standards and blotting them against the known concentrations of the standards, a curve similar to the following should be obtained:
Diagram 1: Standard Curve A linear trendline can be chosen as long as absorbance and analyte concentration form a linear relationship in the linear range of the assay, if such is sufficient for the purpose. The equation of this trendline is then displayed on the graph and can be used to calculate concentration values from absorbance values by rearrangement of the equation. Higher or lower data points may have to be omitted depending on the individual assay to yield a best fitting curve. A meaningful standard comparison is established then, when the samples are treated the same way as the standards which produce the curve, and consistency is minded in every aspect. Such includes that the 1/10 dilution will be adhered to for the treatment of the plasma samples with the plain agent, or that the colorimetric reagent is added consistently at the same volume and same concentration. Footnote: When employing the presented method to produce the standard curve displayed in diagram 1, triplicates revealed a 1-2% pipetting error when aiming for speed.
Conclusions
The presented method for the preparation of plasma standards is fast and easy to follow, whilst requiring minimal amounts of plasma and equipment.
Acknowledgement
I would like to thank Dr. Scott Edwards, for trusting me with this aspect of his project, which made me think up and successfully apply the presented method. I also would like to thank him for financially supporting the open access publication of this manuscript.
y = 0.3025x + 158.140200400600800100012000500100015002000250030003500
A b s o r b a n c e
Plasma Analyte concentration in mg/L
Plasma Standard Curve

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