FUNDAMENTAL MEDICAL SCIENCE I

FINAL REPORT (PROTEOMIC)

ANGELINE TANCHERLA
01071170034
GROUP A2-2

Universitas Pelita Harapan

Mochtar Riady Institute for Nanotechnology
Faculty of Medicine
2017
 ABSTRACT

Proteomics is the large-scale study of proteomes. Proteomes refer to a set of

proteins produced in an organism, system, or biological context [1]. Proteins are
important macromolecules in our body, so it is also important for us to study about
proteins in order to improve health of mankind. Up until now, proteomics explores
many different fields, such as protein-protein interaction studies, protein functions,
protein modifications, and protein localization studies. Most of the time, proteomics
corresponds with genomics. Genomics is the study of gene in order to make analysis
about its products (proteins), whereas proteomics is the study of functionally
modified protein produced by gene.

An example of proteomics is immunoassay. The principle of immunoassay is

to detect and quantitate the analyte by using the antibody-antigen reaction. In this
experiment, immunoassay is applied to conduct ELISA (enzyme-linked
immunosorbent assay) and HBsAg rapid test. Through ELISA, we aim to detect and
quantitate human AFP antigen in plasma samples. Through HBsAg rapid test, we
aim to detect Hepatitis B surface antigen by using HBsAg test card.

Aside from proteomics, we also studied about blood glucose. We measured

blood glucose level in plasma samples using the o-toluidine method. It is important
for us to measure our blood glucose so that we can maintain it at a normal level and
avoid having hyperglycemia (high level of blood glucose) or even hypoglycemia (low
level of blood glucose) that can cause many health problems.

After doing the experiments, we successfully achieved the aims. We found out
that our samples are healthy, because they do not contain HBsAg and have normal
amount of AFP, which are 0.697 ng/ml for sample A2 and 0.842 ng/ml for sample C2.
The blood glucose levels are also in the normal range, wherein sample A2 has
concentration of 89.64 mg/dl and sample C2 has concentration of 91.85 mg/dl.
I. INTRODUCTION

Proteins are macromolecules that are needed by our body in order to function
well. They are made up of amino acids linked by peptide bonds. We can obtain
protein through foods from animal products such as meat, poultry, fish, and also from
plants like nuts, beans and peas. Proteins are responsible for building, and repairing
or replacing worn out body tissues. Some proteins also act as enzymes, hormones,
receptors, signaling proteins and antibodies.

Antibodies, also known as immunoglobulins, are proteins that are in charge of

immunity. They can detect and fight foreign materials (antigens) in the bodies.
Antigen entering the body will activate the immune system to produce specific
antibodies. Specific antibodies will bind to specific antigens and inactivate them. This
property is applied to carry out immunoassays. Immunoassays are bioanalytical
methods to detect and quantitate analyte by using the reaction of an antigen and an
antibody [2].

An example of immunoassay is ELISA or EIA. ELISA (enzyme-linked

immunosorbent assay) or EIA (enzyme immunoassay) is a plate-based assay
technique to detect and quantify substances such as proteins, antibodies and
hormones [3]. In a sandwiched ELISA, capture antibody is attached on a solid
surface. The antigen added into the plate will bind to the capture antibody, and then
followed by detection antibody that is linked to an enzyme. This makes the antigen
trapped in between two antibodies. In the detection step, substrates were added to
react with enzymes and establish blue color. Lastly, stop solution is added to stop
the reaction, which will turn the color into yellow. The concentration of antigen can be
quantitated by measuring the absorbance, and then comparing it with a standard
curve. In this experiment, ELISA is used to detect and quantitate the concentration of
AFP in plasma samples. AFP or alpha feto protein is a glycoprotein normally
produced during fetal and neonatal development [4]. However, it decreases as
humans grow older. Normally in adults, only small amounts (about 0-20 ng/ml) can
be detected in serum or plasma. Increased amount of AFP may indicate liver
disorder or pregnancy.
 Another example of immunoassay is HBsAg rapid test. It is a qualitative,
lateral flow immunoassay for the detection of Hepatitis B surface antigen in serum or
plasma that indicates Hepatitis B virus infection [5]. Colloidal gold conjugated
monoclonal antibodies reactive to HBsAg are dried onto a nitrocellulose membrane
test strip. When the sample is added, it migrates by capillary action through the strip.
HBsAg that is present in the sample will bind with the gold conjugated antibodies
forming particles. These particles will continue to migrate along the strip until they
arrive at test region (T) where they will be captured by anti-HBsAg antibodies
immobilized there [6]. This will create a visible purplish red line. The appearance of
the colored line in the test region indicates a positive result, while the absence of it
indicates a negative result. To serve as procedural control, a colored line will always
appear in the control region, no matter what the result is.

Blood sugar, or blood glucose, is the sugar that is present in our blood. Sugar
is important to generate energy for our body to do work. We obtain sugar from the
food we eat. The obtained sugar will be broken down into glucose in our body. Our
blood will then carry glucose throughout our body to be utilized into energy.
However, too much blood glucose could bring threat to health, such as diabetes,
kidney and nerve damage. Therefore, it is important to maintain our blood sugar
level in normal range. For people without diabetes, a fasting blood sugar should be
under 108 mg/dl. Postprandial sugars taken two hours after meals should normally
be less than 140 mg/dl [7]. In this experiment, blood sugar is measured using the o-
toluidine method. It reacts in hot glacial acetic acid with glucose and produces blue-
green condensation product, which will be measured colorimetrically at λ max 630
nm. The absorbance and concentrations of standards are used to make a standard
curve that will be used to quantify the samples' blood sugar level.

To conduct proteomic experiments like immunoassays, or blood sugar level

measurement, we need to use human plasma. Plasma is clear, yellowish fluid that
carries the red blood cells, white blood cells, and platelets. It contains more than
90% of water, 7-8% of proteins and clotting factors, and small amounts of salts,
glucose and lipids. The immunoassays require antibodies and the blood sugar test
requires glucose. Therefore, plasma is an important substance that can be used for
us to carry out the tests, because it contains all the substances we need.
II. MATERIALS AND METHODS

To conduct a HBsAg Rapid Test, HBsAg rapid test cards were prepared.
Firstly, all reagents and specimens were brought to room temperature. The test
cards were removed from the foil pouch and were placed on clean dry surface. Then,
100 µl of the two samples (A2 and C2 plasma samples from Anastasia and Christine's
blood) and one positive control were each dispensed into respective sample wells on
the test cards. At 15 minutes, the results were interpreted.

To prepare for ELISA, desired numbers of coated wells in the holder were
fixed. Then, 20µl of standards (with AFP concentration of 0, 5, 20, 50 ng/ml),
samples (A2 and C2), and control were each dispensed into appropriate wells. 100 µl
of zero buffers were dispensed into each well and mixed thoroughly for 30 seconds.
The mixtures were incubated at room temperature (18-25ºC) for 30 minutes. After 30
minutes, the incubation mixture was removed by flicking the plate into a waste
container. The microtiter wells were rinsed and flicked 5 times with distilled water.
The wells were sharply struck on paper towel to remove all residual water droplets.
150µl of Enzyme Conjugate Reagent were dispensed into each well and mixed
gently for 5 seconds. The mixture was incubated at room temperature for 30
minutes. The incubation mixture was removed by flicking the plate contents into
waste container. The microtiter wells were rinsed and flicked 5 times with distilled
water. The wells were struck sharply on paper towel to remove residual water
droplets. 100µl of TMB Reagent were dispensed into each well and mixed gently for
30 seconds. The mixture was incubated at room temperature for 20 minutes. To stop
the reaction, 100µl of Stop Solution were added to each well and mixed gently for 30
seconds. Ensuring the changing of blue color to yellow color is important. Then,
optical density at 450 nm was read using a microtiter reader within 15 minutes. The
results were inputted into Microsoft Excel using a computer. A standard curve was
made to determine the AFP concentration in our samples.

To measure blood glucose level from blood serum, 9 clean dry test tubes
were prepared and were covered with aluminium foils. The test tubes were labeled
for 4 standards (with concentration of 50 mg/dl, 100 mg/dl, 150 mg/dl, and 200
mg/dl), 4 samples (A1, C1, A2, and C2) and 1 blank. 50 µl of standards and plasma
samples were added into each test tubes. 1 ml of o-toluidine reagent (1% o-toluidine
in glacial acetic acid) was added into each tubes. Additional 50 µl of o-toluidine
reagent were added into the blank tube. The tubes were placed in a boiling water
bath for 10 minutes. After that, the test tubes were removed from the water bath and
cooled under tap water. The absorbance of the mixtures were read at λ max 630 nm.
A standard curve was made in Microsoft Excel based on the measured absorbance
and known concentrations of standards. The equation of the curve was used to
calculate the blood glucose level in our samples.
III. RESULTS

A. ELISA

Absorbance (at 450 nm)

Original Subtracted by Blank

Blank 0.05 -

Standard with concentration of 0 ng/ml 0.07 0.07-0.05 = 0.02

Standard with concentration of 5 ng/ml 0.27 0.27-0.05 = 0.22

Standard with concentration of 20 ng/ml 0.785 0.785-0.05 = 0.735

Standard with concentration of 50 ng/ml 1.637 1.637-0.05 = 1.587

Table 1 : ELISA Absorbance Value of Blank and Standards (at 450nm)

Absorbance (at 450 nm)

Subtracted by Blank
Original
Calculation Mean

1st Measurement 0.12 0.12-0.05 = 0.07

Sample A2 0.0785
2nd Measurement 0.137 0.137-0.05 = 0.087

1st Measurement 0.109 0.109-0.05 = 0.059

Sample C2 0.083
2nd Measurement 0.157 0.157-0.05 = 0.107

Table 2 : ELISA Absorbance Value of Samples (at 450 nm)

 Figure 1 : ELISA Standard Curve in Ms. Excel

Calculation of Samples' AFP Concentration :

y = 32.123x - 1.8247 and R² = 0.99647

where, y = AFP Concentration

x = Absorbance
R² = Coefficient of Determination

Therefore,

AFP
Concentration = 32.123x − 1.8247 = 32.123(0.0785) − 1.8247 = 0.697 ng/ml
of A2 Sample

AFP
Concentration = 32.123x − 1.8247 = 32.123(0.083) − 1.8247 = 0.842 ng/ml
of C2 Sample
 S20

Legend
A2 : A2 Sample
C2 : C2 Sample
S5
S0 : 0 ng/ml Standard
C2 S5 : 5 ng/ml Standard
S0 S20: 20ng/ml Standard
A2

Figure 2 : ELISA AFP Graph (samples included)

B.HBsAg

S T C
Description
+ : Positive Control
A : Sample A2
C : Sample C2
S : Sample Well
T : Test Region
C : Control Region

Figure 3 : HBsAg rapid test cards

C. Blood Sugar Quantitation

Absorbance (at 630 nm)

Original Subtracted by Blank

Blank 0.059 -

Standard with concentration of 50 mg/dl 0.138 0.138-0.059 = 0.079

Standard with concentration of 100 mg/dl 0.232 0.232-0.059 = 0.173

Standard with concentration of 150 mg/dl 0.335 0.335-0.059 = 0.276

Standard with concentration of 200 mg/dl 0.402 0.402-0.059 = 0.343

Sample A1 0.243 0.243-0.059 = 0.184

Sample A2* 0.213 0.213-0.059 = 0.154

Sample C1 0.209 0.209-0.059 = 0.15

Sample C2* 0.217 0.217-0.059 = 0.158

Table 3 : Absorbance Value of Blank, Standards and Samples (at 630 nm)

* Only sample A2 and C2 were analysed by our subgroup (2nd subgroup), whereas sample A1 and C1
belong to the other subgroup
 Figure 4 : BSL Standard Curve in Ms. Excel

Calculation of Samples' AFP Concentration :

y = 554.73x + 4.207 and R² = 0.99297

where, y = Blood Glucose Concentration

x = Absorbance
R² = Coefficient of Determination

Therefore,

Blood Glucose
Concentration = 554.73x + 4.207 = 554.73(0.154) + 4.207 = 89.64 mg/dl
of A2 Sample

Blood Glucose
Concentration = 554.73x + 4.207 = 554.73(0.158) + 4.207 = 91.85 mg/dl
of C2 Sample
IV. DISCUSSION

The ELISA experiment is done to measure the concentration of AFP in our

plasma samples. It doesn't require any radioisotopes or a radiation-counting
apparatus. We used immobilized monoclonal antibodies, which are the anti-AFP
antibodies that will specifically bind to AFP antigens. We also added enzyme-linked
detection antibody to trap the immobilized antigen. Substances aside from the
'sandwiched' AFP will be washed out, leaving only the AFP in between the
antibodies. This makes ELISA effective, because it specifically traps the antigen
(AFP) needed in this experiment.

During ELISA experiment, we made a standard curve based on the measured

absorbance of the standards. Using Ms. Excel, absorbance of the standards (X-axis)
are plotted against the known concentrations of the standards (Y-axis). The equation
derived from the curve is y = 32.123x - 1.8247, with R² = 0.99647. Coefficient of
determination (R2) is a measure that assesses the ability of a model to predict or
explain an outcome in the linear regression setting [8]. In general, a high R2 value
indicates that the model is a good fit for the data. Our R2 value is 0.99647, which is
very close to 1. This indicates that our curve fits the data well. Now, using the
equation of the standard curve, we calculated the samples' AFP concentration.
Sample A2 has an AFP concentration of 0.697 ng/ml, while sample C2 has an AFP
concentration of 0.842 ng/ml. Normally, human plasma contains less than 20
ng/ml of AFP. Therefore, we can conclude that our samples are healthy and have
normal concentration of AFP.

In contrast to ELISA that can be used to detect and quantify concentrations,

HBsAg test can only be used to detect substances present in plasma or serum. The
result of HBsAg test can be observed from Figure 3. The first test card, which is the
positive control, presents purplish red test bands in both control and test regions.
The result is positive and this indicates that the positive control contains HBsAg.
From the remaining test cards, which are samples A2 and C2, we can see that the
purplish red bands appear only in the control regions. The results are negative,
which means that both of our samples do not contain HBsAg. The appearance of
purplish red test band in control region is normal and is used to show that the test is
done correctly.

In blood sugar quantitation, we used the o-toluidine method. It is simple,

efficient, low-cost and valid to quantitate blood glucose level. The additional
advantage is that it can be conducted directly on serum or plasma without
deproteinization. It only requires orthotoluidine (o-toluidine). O-toluidine, an aromatic
amine, reacts in acetic acid with the terminal aldehyde group of glucose and
generates colored condensation product that will be measured colorimetrically. The
final test color has good stability. However, o-toluidine method is not widely used
anymore because it is believed to be carcinogenic [9].

Similar to ELISA, we also made a standard curve based on the standards'

absorbance and concentration. Using Ms. Excel, absorbance of the standards (X-
axis) are plotted against the known concentrations of the standards (Y-axis). The
equation derived from the curve is y = 554.73x + 4.207, with R² = 0.99297. The R²
value is more than 0.95, which indicates that the standard curve is a good fit for our
data. Using the equation, we calculated our samples' blood sugar level. Sample A2
has a concentration of 89.64 mg/dl and sample C2 has a concentration of 91.85
mg/dl. Normal blood sugar should be less than 108 mg/dl in fasting state, or less
than 140mg/dl in 2 hours postprandial state. Therefore, we can conclude that both of
our samples have normal blood sugar level.
V. REFERENCES

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