CURRICULUM VITAE

Nama : Dr. Budi Santosa, MSi.Med

Pekerjaan : Dosen
Unit Kerja : Universitas Muhammadiyah Semarang
Program Studi Analis Kesehatan
1. 1990 : AAK Surakarta
Pendidikan :
2. 2000 : FKM UNDIP
3. 2009 : Biomedik UNDIP
4. 2013 : Kedokteran/Kesehatan
UNDIP

PATELKI (Ketua DPW JATENG : 2013-

Organisasi : 2017)
 IMMUNOLOGI ASSAY:
Multiplex Immunoassay
Budi Santosa
Seminar Ilmiah Rakernas PATELKI
Swiss Bell Hotel SKA Pekanbaru 22 Mei 2015
 An immunoassay is a test that uses
antibody and antigen complexes as a
means of generating a measurable
result.
 Immuno & assay

Immuno refers to an immune response that causes

the body to generate antibodies,
and
assay refers to a test. Thus, an immunoassay is a test
that utilizes immunocomplexing when antibodies and
antigens are brought together
 Immunoassays are different from other
types of laboratory tests, such as
colorimetric tests, because they use
antibody:antigen complexes to generate
a signal that can be measured.
Immunoassay: Antibodies, Antigens
and Analytes Defined

An antibody is a protein that is produced by

the body in response to an invading
(foreign) substance.

Antibodies are produced as part of the

bodys immune response to protect itself.
 Antibodies

Antibodies possess high

a) specificity
and
b) affinity
for a specific antigen. It is the specific binding of an
antibody to an antigen that allows the detection of analytes
by a variety of immunoassay methods.
 Structure of Antibodies

Antibodies (Ab) are a type of protein called

immunoglobulins.
The most common one is immunoglobulin G
(IgG).
IgG is a protein composed of two main
structural and functional regions
Struktur Antibodi
 An antigen

An antigen is the substance that the body is

trying to fight off (eliminate or reduce) by
mounting an immune response. Some
immunoassays test for antigens directly. For
example, the drug is the antigen that binds to
the antibody.
 An analyte

An analyte is anything measured by a

laboratory test. In immunoassay testing, the
analyte may be either an antibody, or an
antigen.
 Immunoassays

Immunoassays utilize one or more select

antibodies to detect analytes of interest.
The analytes being measured may be those that
are:
a) naturally present in the body (such as a
thyroid hormone),
b) the body produces but are not typically
present (such as a cancer antigen),
c) do not naturally occur in the body (such as an
abused drug).
 IMMUNOASSAY

ELISA
MULTIPLEX
-
TEST
What is an ELISA?
Enzyme-linked immunosorbent assay
Name suggests three components
Antibody
Allows for specific detection of analyte of
interest
Solid phase (sorbent)
Allows one to wash away all the material that is
not specifically captured
Enzymatic amplification
Allows you to turn a little capture into a visible
color change that can be quantified using an
absorbance plate reader
What is it used for?

Measure antibody levels (allergies,

vaccines)
Detect viruses (hepatitis, HIV, venereal
diseases)
Detect hormonal changes (pregnancy)
Detect circulatory inflammatory markers
(cytokines)
Advantages

Sensitivity Relative sensitivities of tests (approx)

Quantitative
Usual operating range
[Ab] or [Ag]

Reproducible precipitation
10 g/ml - 1 mg/ml
immunoelectrophoresis
Kit format double/radial diffusion

immunofluorescence 0.1 - 10 g/ml

ELISA (colour) 0.1 - 10 ng/ml

(chemiluminescence) 0.01 - 10 ng/ml

radioimmunoassay 0.01 - 10 ng/ml

 Type ELISA

Different Types
Indirect
Sandwich
Competitive
Can Be Used To Detect Both Antibody
and Antigen
Very Sensitive, pg/mL (Ex. 10 pg/mL)
 Indirect ELISA
A substrate for this enzyme is then added.
Often, this substrate changes colour upon
reaction with the enzyme. The colour change
shows that secondary antibody has bound to
primary antibody, which strongly implies that
the donor has had an immune reaction to the
test antigen.

The higher the concentration of the primary

antibody that was present in the serum, the
stronger the colour change. Often a
spectrometer is used to give quantitative
values for colour strength
Indirect ELISA
Sandwich ELISA
Sandwich ELISA
The ELISA plate is coated with
Antibody to detect specific antigen
Sandwich ELISA
Competitive ELISA

Less is more. More antigen in your sample

will mean more antibody competed away,
which will lead to less signal
 One step competitive format
In the one step competitive format (see Figure) both the
labeled antigen reagent (Ag*) and the unlabeled specimen (or
test sample analyte) compete for a limited amount of antibody.
Two step competitive format
a. Indirect ILISA RESUME

b. Sanwich ILISA

c. Competitive ILISA
Antibody Steps
 Result of Elisa reader

Std 1 Std 2 Dcs PGE2 LPS LPS + -5 -6 -7 -8 Neg Ctrl

6.125 0.331 0.275 0.099 0.094 0.315 0.168 0.268 0.289 0.319 0.098
3.0625 0.183 0.18 0.1 0.095 0.31 0.172 0.268 0.285 0.297 0.095
1.53125 0.155 0.136 0.106 0.099 0.286 0.179 0.263 0.263 0.266 0.104
0.765625 0.139 0.13 0.105 0.105 0.322 0.205 0.278 0.298 0.279 0.102
0.382813 0.127 0.12 0.111 0.106 0.324 0.204 0.309 0.353 0.292 0.12
0.191406 0.118 0.112 0.112 0.12 0.31 0.204 0.326 0.308 0.324 0.108
0.116 0.11 0.045 0.042 0.052 0.052 0.053 0.051 0.042 0.042
0.123 0.123 0.044 0.052 0.051 0.052 0.054 0.052 0.052 0.053
Data Analysis

Sample Standard Curve

0.5
0.45
0.4
Aborbance (490 nm)

0.35
0.3
0.25
0.2
0.15
0.1
absorbance y = -0.0583Ln(x) + 0.3858
0.05
Log. (absorbance) R2 = 0.9919
0
0.1 1 10 100
Concentration (ug/mL)
Type of Assay
 MULTIPLEX
IMUNOASSAY
What is Multiplex Assay?

Laxman B, Morris DS, Yu

J et al. (2008)).
Multiplex Assay Desaign
Principles of Bead-Based Multiplex
Immunoassays
 Bead-based multiplex immunoassays

Assay Principle

Microspheres are dyed to create Microspheres are coated with

100 distinct color capture antibody
Sample is added to
microspheres and analite
is capture
Fluorescent tagged
detection antibody is added
Laser detect both bead
dyes and tagged
detection antibody
 A magnet capture
analyte and hold
Dual Laser:
bead magnetic
1 klasification
bead
2. Derived Two dioda
sgnal to illuminate the
proportion beads
with analyte
1. LED for detection
2. Derived sgnal to
proportion with analyte
Schematic representation of a sandwich-based Bio-Plex assay workflow.
Schematic illustration of the multiplex bead-based competitive
immunoassay
Scheme of magnetic microsphere immunoassay
Benefits Include:
Simultaneous detection of up to 100 analytes
Get results in 3-4 hours
Low sample volume requirement saves precious
sample
Better sensitivity and dynamic range compared to
ELISA
Easy-to-use software for instrument control data
visualization and data management
Flexible ordering options: preconfigured ready-
to-use kits or create-your-own custom assays
World class support from Bio-Rad, the first
company to partner with Luminex to deliver
xMAP assays, instruments, and software
1 Allergy Testing 8 Genotyping
2 Autoimmune 9 Infectious Disease
3 Cancer Markers 10 Isotyping
4 Cardiac Markers 11 Metabolic Markers
5 Cytokine 12 Tissue Typing
6 EndocrineACTH (h), 13 Kinase Phosphorylated
Adiponectin Protein
(h,m), Amylin (m) (rt)
(h), CPeptide
(h), Calcitonin (h), CRF
Linco
7 Gene Expression 14 Transcription Factors-
Nuclear Receptors
The challenges of multiplex
immunoassays in clinical settings:
1.Biomarker validation;
2.Standardisation of immunoassay design and
quality control (calibration and
quantification);
3.Availability, stability, specificity and cross
reactivity of reagents;
4. Assay automation and the use of validated
algorithms for transformation of raw data
into diagnostic results
Impact using Multiplex Testing

Efficiency workflow and staffing

Limiting hands on time
Single operator
Shorter Turn around time
Multiplex : All target 1 6 h
Singleplex
Bacterial : 2-3 days
Viral : EIA 1-2 h single plex 5-8
Parasite : 3 5 days
Preventive drug resistance
Cost still a big challenge
Value of shorter hospitalization
Earlier initiate therapy
Avoiding unnecessary treatment viral infection