Biomolecular Analysis

Techniques in Disease
Diagnostic
Yudit Oktanella

Veterinary Medicine Faculty

Universitas Brawijaya
Indonesia
 Please check out:
https://fkh.ub.ac.id/
Biomolecular Analysis Techniques

Definition Benefit? Application

Molecular biology - the branch of biology that deals with the structure and function
of the macromolecules (e.g. proteins and nucleic acids) essential to life.

Molecular biology analysis is needed to make it easier to understand various

molecular mechanisms including gene identification, protein expression and
exploration.

--- Techniques - methods specifically used to study these molecular mechanisms

 Basic Techniques of
Molecular Biological Basic Molecular
Analysis for Nucleic Acids Analysis Techniques
for Proteins
Basic Molecular Analysis Techniques for
hormone, antibody and enzymes

Protein
Quantitative
separation
Proteins

Spectrofotometer SDS PAGE

Isoelectric
ELISA Focussing

High-performance liquid chromatography

(HPLC)/ Thin layer chromatography (TLC)

Elektroforesis 2
Dimensi (2-D)
Teknik Dasar Analisis Biologi Molekuler Untuk Asam Nukleat
Hibridisasi Dengan
Hibridisasi dengan Menggunakan Hibridisasi In Situ
Probe Asam Nukleat Matriks Padat – dot
dalam Bentuk Larutan blot, RFLP

single-strand Mikroarray
conformational
polymorphism,
SSCP

Polymerase chain reaction

The enzyme-linked immunosorbent assay (ELISA) is a plate/well-based assay
technique designed to detect and quantify peptides, proteins, antibodies
and hormones.
- with the principle of antigen-antibody binding

Detection is carried out by assessing the activity of the conjugate enzyme

through incubation with the substrate to produce a measurable product.

Non
Kompetitif
Kompetitif
 Coating/Capture
Basic
components for Plate
ELISA Blocking

Probing/Detection

Signal
Measurement
 ELISA DIRECT

INDIRECT SANDWICH
DIRECT ELISA
 • Ag binding in the solid phase.
Sample (antigen) dissolved in
buffer - put in well - incubated at
1 37⁰C overnight

• Blocking – with several types of

blocking reagents: bovine serum
albumin (BSA), ovalbumin (OA),
2 gelatin, skim milk.

• Substrate addition. Substrate:

triggers a color development
reaction through an enzyme-
3 catalyzed reaction.

• Reading - the intensity of the color

formed is read at a certain
4 wavelength - microplate reader
 Direct elisa
Advantage Disadvantage

This ELISA examination is faster than Antigen immobilization is less specific, causing background
noise when compared to indirect ELISA. Mostly because all
other types of ELISA, with a technique the proteins in the sample, including the target protein will
with fewer stages bind to the plate.

Less flexible because each protein

Has fewer errors because it uses less
reagents and requires fewer steps
target requires a specific conjugated
primary antibody

There is no signal
amplification which can
reduce the sensitivity of the
assay
Direct elisa
INDIRECT ELISA
• - Indirect ELISA technique to measure the presence of antibodies/IgG in
the sample
• The indirect technique has the characteristic that the antigen does not
attach directly to the antibody detector, that's why it is called indirect
elisa. The antigen will bind to other antibodies first, then it will bind to
antibodies that have been labeled with enzymes.
• Indirect Elisa also uses a specific antigen (monoclonal) as well as a signal-
specific secondary antibody to detect the presence of the desired
antibody in the sample being tested.
Indirect elisa
Sandwich elisa
SANDWICH ELISA
The Sandwich ELISA is the most common type of ELISA test to
be performed. This ELISA test requires two specific antibodies
for different epitopes of the antigen. The two antibodies are
usually referred to as a matched antibody pair. One of the
antibodies will bind to the surface of the plate and be used as
capture antibody to facilitate immobilization of the antigen.
Other antibodies will conjugate and facilitate detection of the
antigen.
 • The plate used has been prepared with specific antibodies (non-specific binding
1 sites are blocked)

• The sample containing the antigen sought is placed on the plate.

2 • excess antigen that does not react, then the plate is washed

• The addition of primary antibody to bind to the antigen specifically.

• Enzyme-labeled secondary antibody is added to bind to the primary antibody.
3 • If the antibody-enzyme conjugate does not bind, then the plate is washed

• The substrate is added so that it can be converted by the enzyme into a color
precipitate whose intensity can be measured
• Stop solution is given
4 • Reading - the intensity of the color formed is read at a certain wavelength -
microplate reader
Competitive elisa
ELISA APPLICATION:

Enzyme :
Hormone SOD,
quantification Gluthatione
peroxidase,
Katalase, etc

ANTIGEN,
Antibody titer
Sitokin,
post
Growth factor,
vaccination
MDA
Hormonal detection using
ELISA
Infetility Testosterone,
in
male/fe Gonadotropin
male Hormone – FSH/LH

Folicular cyst- Cow

testosterone disease
propionate

Estrone sulphate,
Conception Progesterone,
state
PMSG, etc
FSH concentration in Rat’s serum
Raw data/wavelength 450

1 2 3 4 5 6 7 8 9 10 11 12
0.206 0.25 0.227 0.171 0.235 0.275 0.282 0.255 0.001 0.001 0.06 0.049
A
0.295 0.258 0.262 0.248 0.209 0.261 0.166 0.193 0.001 0.001 0.118 0.088
B
0.286 0.261 0.263 0.252 0.39 0.229 0.402 0.296 0.001 0.001 0.21 0.216
C
0.443 0.465 0.594 0.203 0.565 0.03 0.001 0.001 0.001 0.001 0.377 0.426
D
0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.785 0.663
E
0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001
F
0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001
G
0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001
H
No Standard Standard X (concentration Y (Abs 450 nm) Concetration ng/L
1 2 ng/L)
1 0.06 0.049 75 0.0545 128.83
2 0.118 0.088 150 0.103 209.67
3 0.21 0.216 300 0.213 393
4 0.377 0.426 600 0.4015 707.17
5 0.785 0.663 1200 0.724 1244.67
 DNA ANALYSIS
Summary of DNA methylation analysis methods.

Source: https://www.researchgate.net/publication/232810210_Deciphering_the_Epigenetic_Code_An_Overview_of_DNA_Methylation_Analysis_Methods
STEP TO STEP FOR PCR
DNA
extraction • Total DNA / genome

Identify
Target Gen
• Primer

Amplification • Amplikon
Gene - PCR

• Elektroforesis DNA - SDS PAGE

Result/ horizontal : Identifikasi kesesuaian
amplicon gen target dengan amplifikasi
• Sekuensing Gen
 AMPLIFICATION SET UP
Primer Urutan Oligonukleotida
Forward 5’-GCAAATCAGAAGTGATAGA-3’
Reverse 5’-CCAAGCCAAACGTATGAGTT - 3
Primer Urutan Oligonukleotida
Forward 5’-GCAAATCAGAAGTGATAGA-3’
Reverse 5’-CCAAGCCAAACGTATGAGTT - 3

Steps and duration Temp

Pre denaturation 3 menit 94OC
Denaturation 30 detik 94OC
Annealing 30 detik 59OC
Elongation 1 menit 72OC
Post elongation 7 menit 72OC
Hasil Elektroforesis PCR dengan Target Amplifikasi 290 bp Pada Gel Agarose
Konsentrasi 2%
PCR
product
PCR RFLP

Figure 2. Electrophoresis visualization of genomic extracts on 1,8 %

agarose gel
Note: M = DNA Ladder 1Kb, 1-13 = sample code
Note: M = DNA Ladder 100bp, 1-13 = PCR sample product.

Figure 3 Electrophoresis visualization of genomic extracts on 1,8 % agarose gel

Note: M = DNA Ladder 1Kb, 1-13 = sample code