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FUNDAMENTAL MEDICAL SCIENCE I

FINAL REPORT (PROTEOMIC)

DEVINA

07120110064

GROUP A1-3

Univ!"i#$" P%i#$ &$!$'$n

M*#$! Ri$+, In"#i## .! N$n#*n%/,

F$%#, . M+iin

2011

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ASTRACT

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 !"I#$ and %&s$g 'apid est.

he first experiment was using !"I#$ to detect the concentration of antigen

properties (with direct technique), antibody (with indirect technique), and primarily

protein (with both technique). #econd experiment was by doing %&s$g experiment for 

checing part of component virus %epatitis & with a test pac. he differences between

!"I#$ and 'apid est were the template. !"I#$ was using microwells to checing the

concentration. &eside, rapid test using test pac and it was a qualitative immunoassay

by checing the presence.

here were also third experiment looing 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 *+toluidine.

I INTRODUCTION

Proteins are very important molecules in our cells. hey are involved in virtually

all cell functions. !ach protein within the body has a specific function. #ome 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. hey are constructed from a set of

- amino acids and have distinct three+dimensional shapes. &elow is a list of several

types of proteins and their functions. $ntibodies, contractile, enzymes, hormonal,

http://biology.about.com/library/weekly/aa050301a.htmhttp://biology.about.com/library/weekly/aa050301a.htm

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structural, storage, and transporter were some of protein functions ('egina &ailey,

-).

 $ reaction that occurs when an antigen combines with a corresponding antibody

to produce an immune complex. $ substance that induces the immune system to form a

corresponding antibody is called an immunogen. $ll 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. 0or

instance, lipids and all low+molecular+weight substances are not immunogenic.

 $n assay that quantifies antigen or antibody by immunochemical means. he

antigen can be a relatively simple substance such as a drug, or a complex one such as

a protein or a virus (1c2raw, -).

It is very important that blood sugar levels are ept as close to normal as

possible. 0or most people with diabetes, a healthy range is between 3- and 4- mgdl

before meals and less than 5- mgdl at one to two hours after a meal (see chart

below). $ doctor or health care provider can tell a person with diabetes about how and

when to test blood sugar. It is helpful to eep a record of blood sugar readings several

times during the day.

T$!/# %+ /%" %v%" .!

"# ''% * *$v +i$#" (/5+%)

&efore meals 3- to 4-

to hours after the start of a meal less than 5-

%ypoglycemia (low blood glucose) 6- or below

7e could have referred instead to the 8serum9 proteome but chose plasma

because it is in a sense the larger, parent collection from which other related samples

are derived (:. "eigh $nderson and :orman 2. $nderson,--). 7e 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

http://www.answers.com/topic/antigenhttp://www.answers.com/topic/antibodyhttp://www.answers.com/topic/lipidhttp://www.answers.com/topic/immunogenichttp://www.answers.com/topic/antigenhttp://www.answers.com/topic/antigenhttp://www.answers.com/topic/antibodyhttp://www.answers.com/topic/antigenhttp://www.answers.com/topic/antibodyhttp://www.answers.com/topic/lipidhttp://www.answers.com/topic/immunogenichttp://www.answers.com/topic/antigenhttp://www.answers.com/topic/antibody

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individual effects of biological factors that influence cellular glucose transport and the

nonenzymatic protein glycation deglycation cycle.

%&s$g %epatitis & #urface $ntigen 'apid est ;evice (7hole

&lood#erumPlasma) is a qualitative, solid phase, two+site sandwich immunoassay for

the detection of %&s$g in whole blood, serum or plasma. he membrane is pre+coated

with anti+%&s$g antibodies on the test line region of the ;evice. ;uring testing, the

whole blood, serum or plasma specimen reacts with the particle coated with anti+%&s$g

antibodies. %&s$2 stands for hepatitis & surface antigen. If it is found, along with other

specific antibodies, it means the person has a hepatitis & infection (

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In the experiment of %&s$g rapid test, we brought all the reagents and specimen

to room temperature and used the test pac by removed it from foil pouch and placed

on clean dry surface. --u" of the specimen or control were dispensed by using pipette

into the sample well on the card. he result was interpreted after > minutes.

In !"I#$ (!nzyme+"ined Immunosorbent $ssay), first the -u" of standard,

specimens, and controls were dispensed into appropriate wells, then -- u" of zero

buffers were also dispensed into each well and they were all mixed before had to

incubated at room temperature for 4- minutes. after that, the incubation mixture were

removed and the microtiter wells were rinsed and fliced > times with distilled water.

he need to be stried sharply onto absorbent paper to removed all residual water

droplets. >- u" of enzyme con?ugate reagent was dispensed into each well and gently

mixed for > seconds, then incubated at room temperature for 4- minutes.

 $gainst, the incubation mixture was removed by flicing plates contents into

waste container and the microtiter wells was also rinsed and fliced > times with distilled

water. he wells were sharply stried before --u" of 1& reagent was dispensed into

each well and mixed for > seconds then incubated at room temperature for - minutes.

he reaction was stopped by adding -- u" stop solution to each well, and mixed for 4-seconds until or until the blue color changes to yellow color completely. he optical

density was read at @>- nm with a microtiter reader within > minutes.

7e also determined colorimetric of blood sugar lever by provided --mgdl of

glucose standard and prepared concentration glucose standard point and ml of A *+

toluidine was added and mixed then incubated for - minutes in boiling water bath.

 $bsorbance had to be measured at B4-nm and > tubes was prepared to be filled by

concentration, glucose standard, and water.

ube filled with >- concentration, -.>ml of glucose standard, and -.6> water.

ube filled with 5- concentration, -.@ml of glucose standard, and -.B ml of water. ube

4 filled with -- concentration, -.>ml of glucose standard, and -.>ml of water. ube @

filled with >- concentration, -.Bml of glucose standard, and -.@ of water. "ast tube was

blan and only filled with ml of water.

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&lood glucose had to be measured before blood from vein was drawn and

transferred to centrifuge tube. #erum need to being obtained by centrifugation of blood

for - minutes. glucose concentration had to be determined in the provided serum

sample of patient using *+toluidine method. In dry test tube, -.ml of distilled water

standard glucose serum then ml of toluidine reagent was added and mixed in each

tube. he tube have to covered with aluminium foil and put in boiling water bath for -

minutes. after that, test tubes had to being cooled under tap water and the absorbance

ready to read at max B4-nm. he concentration had to be calculated of < in

provided blood samples using absorbance reading of standard glucose.

III RESULT

A ELISA

'  C -.5463

# C (-.->5 D -.55) =

C -.4

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# C (-.4- D -.-B@) =

C -.-36

y C ax E c

y C concentration

x C absorbance

y C -3.6(-.4) F 3.>53

C + 4.63>6

y C -3.6(-.-36) F 3.>53

C + 3.@5

&"A/ R$'i+ T"#

"eft ($) C *ur sample (negative)

'ight (&) C #ample from a donor whom nown had positive %epatitis & acute

C %+ S/$! $n#i#$#in

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#@ >- -.B>B -.B>5 -.B>6

&"$:

G

-.56 -.53 -.55

H C -.--@x F -.-B>'  C -.334

y C -.--@x F -.-B>

-.55 C -.--@x F -.-B>

-.4>4 C -.--@x

x C 55.> mgd"

IV DISCUSSION

he principle of !"I#$ is to detection of antibody+antigen complex that involves

enzyme reaction. !"I#$ has direct technique and indirect technique. In direct !"I#$,

capture antibody has been coding in solid phase for checing the serum wether it was

exist or not and reacted with secondary %'P*. $fter they were bend, the 1&

substrate was added. ;irect !"I#$ was use to checing the antigen when indirect

!"I#$ was for checing the antibody which antigen was coding first. &ut *n !"I#$

experiment, we found that our curved wasnt acceptable because it seems abnormal

that it should be. 7e thought it might happen because our group error due mixed the

sample so we cant get any conclusion to determined the health range. 7hen we trying

to mae the curve, it seems unusual. It wasnt straight as it should, but loos bend. #o

we only inserted three samples of data to get better curve and conclusion. $nd its

appeared as in the result. $nd because of the error we made, we cant get anyconclusion about the healthy range which was nown was less than 5.> ngml.

*n %&s$g 'apid est we have had negative result of hepatitis+& acute.

7e determined it because only one purplish red test band appears in the control region.

&ecause 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. $nd if neither test band nor

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control band appears, the specimen should be tested again using a new test card

because it was invalid. 7e Interpret the result based on the band appearance because

one strip means the person is negative for %epatitis &. his is because while the serum

moves through capillary action and goes to the reaction site, no antigen was found with

the antibody. $t test site, there is no binding of the antigen with antibody to the anti+

%&s$g neither there was a dye substrate to colored the polyclonal of anti+%&s$g. $t the

control zone, all the antibodies bind with the anti+antibodies (anti+anti %&s$g) because

there was an anti+mouse $b, and the enzyme there can wor.

7e also did experiment for looing of blood sugar quantitation and based on our

result, we had agreed that it was normal. *ur 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 *+toluidine based on visual. 1ore glucose, more

reaction with *+toluidine more color created. *+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 B4-nm in this

method. hats why the highest rate concentration of blood glucose has the most dense

colored. he concentration of normal blood glucose was @.@ + B. mmol" (5+-

mgd"). $fter having meal was 6.5 mmol" (@-mgd"). &oth of our sample was taen

from the same person which means had same blood glucose concentration. &ased onour result, the concentration of blood glucose was 55.>mgd" so it was a normal levels

of blood glucose.

V REFERENCES

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'egina &ailey. Protein 0unctions. $bout &iology. -.

http=biology.about.comodmolecularbiologyaaa-3-@a.htm-J:ovember

1c2raw %ill.Immunoassay. #cience and echnology.-.

;epartment of %ealth. he Importance of