ELISA: Spectrophotometric Determination of Unknown Bovine Serum Albumin

Ramirez, Alyza Joy M.

Abstract

ELISA or Enzyme-Linked Immunosorbent Assay is a biochemical technique that is used for detecting
and quantifying antibodies or antigens. In reading the colorimetric reaction, an equipment called
spectrophotometer is used to measure the absorbance of a solution as light of a specified wavelength is
passed through it. In this study, Bovine Serum Albumin or BSA which is a serum albumin protein
isolated from cows was used, specifically in the determination of its unknown concentration.

Serial dilutions of 6 different concentrations (50 mg/mL, 25 mg/mL, 12.5 mg/mL, 6.25 mg/mL, 3.125
mg/mL & 1.5625 mg/mL) of BSA sample is performed, together with one sample containing the
unknown BSA concentration and another sample containing water (H 20) which served as the negative
control. Thereafter, triplicate samples were delicately transferred using a micropipette into an ELISA
microplate and each of it received 180 µL of Biuret reagent. After 5 minutes of storage and incubation,
absorbance of each sample was read by placing the microplate in the Biotek ELx800 ELISA reader. By
further calculations of the average absorbance of each samples for the wavelengths of 450nm, 490nm and
630nm using the MS Excel file in the computer as well as by plotting the calculated absorbance and
concentration values of each known samples in a graph, the R 2 value for the wavelength of 630nm is used
in comparison since it has the highest value. The result gathered after computing the value of x using the
y−b
equation x = was 25.42 or 25, so the researcher arrived at a conclusion that 25 mg/mL, more or
m
less is the concentration of the unknown BSA sample.

Introduction

ELISA or Enzyme-Linked Immunosorbent Assay is a biochemical technique that is used for detecting
and quantifying antibodies or antigens against viruses, bacteria and other materials [ CITATION Ali19 \l
13321 ]. ELISA is typically performed in 96-well polystyrene plates. In this technique, the polystyrene
plate is used to bind antigen or antibody and a corresponding conjugate linked to an enzyme is attached.
The addition of the substrate generates a colorimetric reaction that must be read by an equipment called
spectrophotometer (Conceincao, et.al, 2015).

Spectrophotometry is one of the most widely used techniques, due to the simplicity of its procedures and
its speed, precision and accuracy (Conceincao, et.al, 2015). It measures the absorption or light
transmission, which can help in determining the concentration of a sample substance. In line with this, a
spectrophotometer is used as the device which measures the absorbance of a solution as light of a
specified wavelength is passed through it [CITATION unk17 \l 13321 ].

Bovine Serum Albumin (also known as BSA or ‘Fraction V’) is serum albumin protein isolated from
cows. It is commonly used by researchers as a blocking agent to prevent non-specific binding of antigens
and antibodies to the microtiter well [ CITATION Stu12 \l 13321 ] and often used as a protein concentration
standard in protein experiments as well as in other biochemical applications. (G-Biosciences, 2019).
In this laboratory activity, you will learn about how the concentration of an unknown BSA sample was
determined using the ELISA reader through spectrophotometer, followed by plotting the absorbance and
concentration of the known BSA on a graph to create a standard curve. Thereafter, the determination of
y−b
the unknown concentration of BSA was finalized by using the equation x = .
m

Methodology

Preparation of Bovine Serum Albumin (BSA)

A total of eight tubes were prepared and labelled as 1, 2, 3, 4, 5, 6, 7 and 8. By using a micropipette, the
first six tubes were carefully placed with 100 µL of H 2O as well as the 8 th tube that will serve as the
negative control. The 7th tube will serve as the one that contains the unknown BSA concentration.
Through series of dilution, different concentrations of BSA is prepared. Starting from tube 1, 100 µL of
100% concentration of BSA is delicately diluted resulting to 50 mg/mL BSA solution. Thereafter, 100µL
of solution from the tube 1 is transferred to tube 2 obtaining a 25 mg/ml BSA solution; then from the
previous tube 2, 100µL from its solution is transferred into tube 3 obtaining 12.5 mg/mL of BSA solution;
another 100µL solution from tube 3 is transferred into tube 4 obtaining 6.25 mg/mL BSA solution; from
tube 5 and 6, the same process is done resulting with 3.125 mg/mL and 1.5625 mg/mL of BSA solution.
To simplify, while each of the first 5 tubes contain 100µL of BSA solution with different concentrations,
tube 6 contains 200 µL.

ELISA Microplate

Each of the prepared tubes with various concentrations of BSA is transferred to the ELISA microplate in
a triplicate manner from left to right well at the top going to bottom. Using a micropipette, triplicate
samples from tube 1 with 50mg/mL BSA is transferred into Row A. Same process is done to the rest of
the row, where 25 mg/mL from tube 2 is transferred to Row B, 12.5 mg/mL from tube 3 is transferred to
Row C, 6.25 mg/mL from tube 4 is transferred to Row D, 3.125 mg/mL from tube 5 is transferred to Row
E, 1.5625 mg/mL from tube 6 is transferred to Row F, unknown BSA concentration from tube 7 is
transferred to Row G and 100 µL of H 2O which is the negative control from tube 8 is transferred to Row
H.

Biuret Reagent

All of the used well are pipetted with 180 µL of Biuret reagent. After 5 minutes of storing and
incubation, there is an indication of the presence of proteins due to the change in color of each samples.

ELISA Microplate Reader

Absorbance of the samples for the specific wavelengths of 450nm, 490nm and 630nm is read by placing
the microplate in the Biotek ELx800 ELISA reader. Followed by computing the average of all the
concentrations for each of the given wavelengths after accessing the MS Excel file on the computer.
Results

After the procedure was completely done, the following results were gathered:

Table 1. Raw Data Showing the Absorbance of Each Samples of Known Concentrations

  10 11 12
0.246 0.257 0.244 450
A 0.414 0.428 0.418 490
0.368 0.38 0.353 630
0.221 0.153 0.207 450
B 0.343 0.188 0.304 490
0.35 0.25 0.324 630
0.17 0.176 0.174 450
C 0.229 0.241 0.243 490
0.273 0.277 0.277 630
0.182 0.154 0.138 450
D 0.21 0.194 0.176 490
0.257 0.25 0.22 630
0.324 0.144 0.15 450
E 0.32 0.163 0.163 490
0.302 0.222 0.225 630
0.197 0.135 0.138 450
F 0.204 0.146 0.151 490
0.263 0.215 0.218 630
0.207 0.192 0.19 450
G 0.282 0.257 0.272 490
0.318 0.292 0.302 630
0.15 0.138 0.129 450
H 0.157 0.15 0.135 490
0.231 0.218 0.21 630

Table 2. Average Absorbance of each Concentration for 450nm, 490nm and 630nm Wavelengths.

Concentration 450nm 490nm 630nm

A 50 0.249 0.42 0.367
B 25 0.1936667 0.2783333 0.308
C 12.5 0.1733333 0.2376667 0.2756667
D 6.25 0.158 0.1933333 0.2423333
E 3.125 0.206 0.2153333 0.2496667
F 1.56 0.1566667 0.167 0.232
H 0 0.139 0.1473333 0.2196667
 Absorbance (nm) Graph Showing Absorbance for each Concentration
0.6
450
0.4
f(x) Linear
f(x) =
= 0.01
0 x +x0.23
+ 0.17
0.2 R² = 0.96 (450)
R² ==0.97
f(x) 0 x + 0.16
R² = 0.71 490
0
Linear
0 10 20 30 40 50 (490) 60
Concentration (mg/mL)

Figure 1. Graph Showing the Absorbance for each Concentration

In figure 1 above, it shows that the absorbance in 630 has the highest R 2 value. Thus, the absorbance of
the unknown sample in 630 will be the one to use.

Conclusion

After plotting the measured absorbance and concentration in a graph, R 2 value of each wavelengths
namely: 450nm, 490nm, and 630nm were obtained. 630nm, the wavelength with the highest R 2 value of
0.973 is used together with its y value which is 0.302. From the equation y = mx+b, the equation of x =
y−b
is derived where y = 0.302, b = 0.2308 and m = 0.0025, to determine the concentration of the
m
unknown BSA sample which is represented by x. The calculated value of x is 25.42 or 25, so the
researcher arrived at a conclusion that 25 mg/mL, more or less is the concentration of the unknown BSA
sample.

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