FUNDAMENTAL MEDICAL SCIENCE I

FINAL REPORT (PROTEOMIC)

DEVINA
07120110064
GROUP A1-3

Universitas Pelita Harapan

Mochtar Riady Institute for Nanotechnology
Faculty of Medicine
2011

ABSTRACT

Proteomic experiment was involved protein so we used plasma which contain it

to be tested. Protein have four distinct of structures which were primary structure,
secondary structure, tertiary structure, and quaternary structure. It have several function
such as enzyme, structural, and body defense (antigen versus antibody).Immunoassay
was the chemical reaction to detect specific substrate, the analyte in blood/ body fluid
sample. It can be done by using ELISA and HBsAg Rapid Test.
The first experiment was using ELISA to detect the concentration of antigen
properties (with direct technique), antibody (with indirect technique), and primarily
protein (with both technique). Second experiment was by doing HBsAg experiment for
checking part of component virus Hepatitis B with a test pack. The differences between
ELISA and Rapid Test were the template. ELISA was using microwells to checking the
concentration. Beside, rapid test using test pack and it was a qualitative immunoassay
by checking the presence.
There were also third experiment looking for the concentration of blood glucose
in case it was in normal levels or not. In this experiment, the blood glucose
concentration was determined based on visual observation in the comparison with the
standard series which has been reacted with O-toluidine.

I. INTRODUCTION

Proteins are very important molecules in our cells. They are involved in virtually
all cell functions. Each protein within the body has a specific function. Some proteins
are involved in structural support, while others are involved in bodily movement, or in
defense against germs.
Proteins vary in structure as well as function. They are constructed from a set of
20 amino acids and have distinct three-dimensional shapes. Below is a list of several
types of proteins and their functions. Antibodies, contractile, enzymes, hormonal,

structural, storage, and transporter were some of protein functions (Regina Bailey,
2011).
A reaction that occurs when an antigen combines with a corresponding antibody
to produce an immune complex. A substance that induces the immune system to form a
corresponding antibody is called an immunogen. All immunogens are also antigens
because they react with corresponding antibodies; however, an antigen may not be able
to induce the formation of an antibody and therefore may not be an immunogen. For
instance, lipids and all low-molecular-weight substances are not immunogenic.
An assay that quantifies antigen or antibody by immunochemical means. The
antigen can be a relatively simple substance such as a drug, or a complex one such as
a protein or a virus (McGraw, 2011).
It is very important that blood sugar levels are kept as close to normal as
possible. For most people with diabetes, a healthy range is between 90 and 130 mg/dl
before meals and less than 180 mg/dl at one to two hours after a meal (see chart
below). A doctor or health care provider can tell a person with diabetes about how and
when to test blood sugar. It is helpful to keep a record of blood sugar readings several
times during the day.

Target blood glucose levels for

most people who have diabetes

(mg/dl)

Before meals

90 to 130

1 to 2 hours after the start of a meal

less than 180

Hypoglycemia (low blood glucose)

70 or below

We could have referred instead to the serum proteome but chose plasma
because it is in a sense the larger, parent collection from which other related samples
are derived (N. Leigh Anderson and Norman G. Anderson,2002). We were able to
identify many proteins present in the moderate- to low-abundance range in the plasma
proteome. It can also reflect differences in blood glucose levels over time, as well as the

individual effects of biological factors that influence cellular glucose transport and the
nonenzymatic protein glycation/ deglycation cycle.
HBsAg Hepatitis B Surface Antigen Rapid Test Device (Whole
Blood/Serum/Plasma) is a qualitative, solid phase, two-site sandwich immunoassay for
the detection of HBsAg in whole blood, serum or plasma. The membrane is pre-coated
with anti-HBsAg antibodies on the test line region of the Device. During testing, the
whole blood, serum or plasma specimen reacts with the particle coated with anti-HBsAg
antibodies. HBsAG stands for hepatitis B surface antigen. If it is found, along with other
specific antibodies, it means the person has a hepatitis B infection (Charles Daniel,
2008).
The purpose of an ELISA is to determine if a particular protein is present in a
sample and if so, how much. There are two main variations on this method: you can
determine how much antibody is in a sample, or you can determine how much protein is
bound by an antibody. The distinction is whether you are trying to quantify an antibody
or some other protein. In this example, we will use an ELISA to determine how much of
a particular antibody is present in an individuals blood (Davidson, 2002).
A blood glucose test measures the amount of a type of sugar, called glucose, in
your blood. Glucose comes from carbohydrate. Several different types of blood glucose
tests are used. Fasting blood sugar (FBS) measures blood glucose after you have not
eaten for at least 8 hours. 2-hour postprandial blood sugar measures blood glucose
exactly 2 hours after you start eating a meal. Random blood sugar (RBS) measures
blood glucose regardless of when you last ate (Health Wise, 2009).

II. MATERIALS AND METHOD

In the experiment of HBsAg rapid test, we brought all the reagents and specimen
to room temperature and used the test pack by removed it from foil pouch and placed
on clean dry surface. 100uL of the specimen or control were dispensed by using pipette
into the sample well on the card. The result was interpreted after 15 minutes.
In ELISA (Enzyme-Linked Immunosorbent Assay), first the 20uL of standard,
specimens, and controls were dispensed into appropriate wells, then 100 uL of zero
buffers were also dispensed into each well and they were all mixed before had to
incubated at room temperature for 30 minutes. after that, the incubation mixture were
removed and the microtiter wells were rinsed and flicked 5 times with distilled water.
The need to be striked sharply onto absorbent paper to removed all residual water
droplets. 150 uL of enzyme conjugate reagent was dispensed into each well and gently
mixed for 5 seconds, then incubated at room temperature for 30 minutes.
Against, the incubation mixture was removed by flicking plates contents into
waste container and the microtiter wells was also rinsed and flicked 5 times with distilled
water. The wells were sharply striked before 100uL of TMB reagent was dispensed into
each well and mixed for 5 seconds then incubated at room temperature for 20 minutes.
The reaction was stopped by adding 100 uL stop solution to each well, and mixed for 30
seconds until or until the blue color changes to yellow color completely. The optical
density was read at 450 nm with a microtiter reader within 15 minutes.
We also determined colorimetric of blood sugar lever by provided 200mg/dl of
glucose standard and prepared concentration glucose standard point and 2 ml of 1% Otoluidine was added and mixed then incubated for 10 minutes in boiling water bath.
Absorbance had to be measured at 630nm and 5 tubes was prepared to be filled by
concentration, glucose standard, and water.
Tube 1 filled with 50 concentration, 0.25ml of glucose standard, and 0.75 water.
Tube 2 filled with 80 concentration, 0.4ml of glucose standard, and 0.6 ml of water. Tube
3 filled with 100 concentration, 0.5ml of glucose standard, and 0.5ml of water. Tube 4
filled with 150 concentration, 0.6ml of glucose standard, and 0.4 of water. Last tube was
blank and only filled with 1ml of water.

Blood glucose had to be measured before blood from vein was drawn and
transferred to centrifuge tube. Serum need to being obtained by centrifugation of blood
for 10 minutes. glucose concentration had to be determined in the provided serum
sample of patient using O-toluidine method. In dry test tube, 0.1ml of distilled water/
standard glucose/ serum then 2 ml of toluidine reagent was added and mixed in each
tube. The tube have to covered with aluminium foil and put in boiling water bath for 10
minutes. after that, test tubes had to being cooled under tap water and the absorbance
ready to read at 1 max 630nm. The concentration had to be calculated of BSC in
provided blood samples using absorbance reading of standard glucose.

III. RESULT

A. ELISA

R2

= 0.8379

S1

= (0.058 X 0.188) : 2
= 0.123

S2

= (0.130 X 0.064) : 2
= 0.097

= ax + c

= concentration

= absorbance

y1

= 209.7(0.123) 29.589
= - 3.7957

y2

= 209.7(0.097) 29.589
= - 9.2481

B. HBsAg Rapid Test

Left

(A) = Our sample (negative)

Right (B) = Sample from a donor whom known had positive Hepatitis B acute
C. Blood Sugar Quantitation

S1
S2
S3

Concentration (mg/dL)
50
80
100

A1
0.164
0.331
0.446

A2
0.166
0.331
0.448

Average
0.165
0.331
0.447

S4
BLAN

150

0.656
0.287

0.658
0.289

0.657
0.288

K
Y

= 0.004x 0.065

R2

= 0.993

= 0.004x 0.065

0.288 = 0.004x 0.065

0.353 = 0.004x
x

= 88.25 mg/dL

IV. DISCUSSION

The principle of ELISA is to detection of antibody-antigen complex that involves

enzyme reaction. ELISA has direct technique and indirect technique. In direct ELISA,
capture antibody has been coding in solid phase for checking the serum wether it was
exist or not and reacted with secondary HRPO. After they were bend, the TMB
substrate was added. Direct ELISA was use to checking the antigen when indirect
ELISA was for checking the antibody which antigen was coding first. But On ELISA
experiment, we found that our curved wasnt acceptable because it seems abnormal
that it should be. We thought it might happen because our group error due mixed the
sample so we cant get any conclusion to determined the health range. When we trying
to make the curve, it seems unusual. It wasnt straight as it should, but looks bend. So
we only inserted three samples of data to get better curve and conclusion. And its
appeared as in the result. And because of the error we made, we cant get any
conclusion about the healthy range which was known was less than 8.5 ng/ml.
On HBsAg Rapid Test we have had negative result of hepatitis-B acute.
We determined it because only one purplish red test band appears in the control region.
Because if it was positive, in addition of the control purplish red test band, a distinct
purplish red test band also appears in the test region. And if neither test band nor

control band appears, the specimen should be tested again using a new test card
because it was invalid. We Interpret the result based on the band appearance because
one strip means the person is negative for Hepatitis B. This is because while the serum
moves through capillary action and goes to the reaction site, no antigen was found with
the antibody. At test site, there is no binding of the antigen with antibody to the antiHBsAg neither there was a dye substrate to colored the polyclonal of anti-HBsAg. At the
control zone, all the antibodies bind with the anti-antibodies (anti-anti HBsAg) because
there was an anti-mouse Ab, and the enzyme there can work.
We also did experiment for looking of blood sugar quantitation and based on our
result, we had agreed that it was normal. Our curved also acceptable with our final
result that have been interpreted by it colored which was observed and compared to the
standard series as its reacted with the O-toluidine based on visual. More glucose, more
reaction with O-toluidine more color created. O-toluidine reacts in hot glacial acetic acid
with the terminaldehyde group of glucose to produce a blu-green colored condensation
product that can be measured colorimetrically at lamda maximum 630nm in this
method. Thats why the highest rate concentration of blood glucose has the most dense
colored. The concentration of normal blood glucose was 4.4 - 6.1 mmol/L (82-110
mg/dL). After having meal was 7.8 mmol/L (140mg/dL). Both of our sample was taken
from the same person which means had same blood glucose concentration. Based on
our result, the concentration of blood glucose was 88.25mg/dL so it was a normal levels
of blood glucose.

V. REFERENCES

Regina Bailey. Protein Functions. About Biology. 2011.

http://biology.about.com/od/molecularbiology/a/aa101904a.htm/2011_November21
McGraw Hill.Immunoassay. Science and Technology.2011.
Department of Health. The Importance of Controlling Blood Sugar.2010. Available from;
URL:
www.health.ny.gov/.../controlling_blood_sugar_importance.htm/2011_November_22
Qibin Zhang, Ning Tang, Athena A. Schepmoes, Lawrence S. Phillips, Richard D.
Smith,and Thomas O. Metz. . Proteomic Profiling of Nonenzymatically Glycated Proteins
in Human Plasma and Erythrocyte Membranes. Available from; URL:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731429/2011_November_22
N. Leigh Anderson and Norman G. Anderson. The Human Plasma Proteome. History,
Character, and Diagnostic Prospects;2002.

Charles Daniel. HBsAg. Hepatitis. 2008. Available from; URL:

http://hepatitis.about.com/od/ghi/g/HBsAG.htm/2011_November_23
Hepatitis B virus surface Antigen Rapid Test Device. FirstVue HBsAg Rapid Test . 2011.
Available from; URL: http://www.hbsagrapidtest.com//2011_November_23
Blood Glucose. Diabetes Health Center. 2009. Available from; URL:
http://diabetes.webmd.com/blood-glucose/2011_November_26