TEKNIK IMUNOLOGI

Oleh
Evy Diah Woelansari, S.Si, M.Kes
Immunoassay

 Detection methods to localize antigen or

proteins either in cells, tissue section or
soluble proteins
 Using labelled antibodies as spesific reagent
through antigen-antibody interaction that are
visualized by a marker such as fluorescent dye,
enzyme or colloidal gold
Principle of Immunoassay
 Labels in immunoassay

 Radioactive label
 Nonradioactive label
– Colloidal gold
– Virus particle
– Ferritin
– Fluorescent
– Enzyme
 Immunoassay techniques

Type of Antigen Immunodetection techniques

Tissue •Immunohistochemistry (IHC)

•Immunofluorecence assay (IFA)

Soluble •ELISA
•Immunoblotting

Cell •Immunocytochemistry (ICC)

•FACS
•IFA
 Techniques at DNA, RNA and protein level
PCR Western blot
RT-PCR Western blot
Sequencing ELISA
FISH ELISA
DISH IHC
etc Amino acid seq.
FACS analysis

RNA Immune
DNA Protein
(message) Response
(database) (action)
(action)

Activity
Presence of gene
gene/genome “activity” Host Reaction
expression “Immune status”
“ dosis” “activity”
Key component in immunoassay
 Basic method in immunoassay

 Direct
 Indirect
Direct method
Indirect method
 Antibody is adaptive immune component with 2 specific
characteristics:
1.Recognize specific antigen
2.Precipitate antigen that binds to it
 How to measure result of immunodetection method?
1. substrate precipitation
1.ELISA
Intensity of color spectrum spectrophotometer
Semi quantitative (indirect ELISA), quantitative (sandwich
ELISA)

2. IHC
Staining of specific surface antigen

3. Immunoblotdetection
Presence of specific protein band
 How to measure result of immunodetection method?
2. light excitation

3. Fluorescence
microscope:
Specific antibody labelled
with fluoresenceprobe
Antibody testing limitations

 Difficulties in interpretation

 Limitations - ‘window period’

 antibodies appear within 3-4 weeks

 Direct detection – HIV p24 antigen or DNA/RNA (NAT) –
pre-antibody
 Combo test = earlier detection

 Primary infection + therapy = delayed antibody

response
ELISA
(enzyme-linked immunosorbentassay)
 A biochemical technique used mainly in immunology
to detect the presence of an antibody or an antigen
in a sample, by using solid phase matrix (ELISA-
plate) for reaction to occurs, and colorimetric (OD) as
the read-outs
 Systems:
 1.Direct detect presence of antigen
 2.Indirect : determine serum antibody conc.
 3.Sandwich : determine antigen conc.
 ELISA
Measures : presence of
antigen Antibody concentration
Antigen concentration

Qualitative Semi Quantitative Quantitative

Westernblot--‐Immunoblot
 Westernblot--‐Immunoblot
 Antigen is immobilized on nitrocellulose
membrane.

 Preparation of immobilized antigen consist of :

• protein separation (SDS--‐PAGE)
• Protein immobilization (Western--‐blot)

 Immunodetection (immunobloting)
Reaction on Immunobloting
Western Blot

 Expensive – $ 80 - 100
 technically more difficult
 visual interpretation
 lack standardisation
 - performance
 - interpretation
 - indeterminate reactions –
resolution of ??
 ‘Gold Standard’ for confirmation
Electrophoretic mobility-shift assays
(EMSA)
 are techniques to detect the association of two
molecules, by running them (electrophoresis) through
a nondenaturing, polyacrylamide gel.
 Bound molecules have different characteristics (size
and/or charge) from unbound molecules and therefore
their mobility will be shifted in the gel.
 The technique can be used to determine (for
example) whether a transcription factor is associated
with a DNA segment.
 If a complex of a transcription factor and a DNA probe
then the identity of the factor can be determined by
adding antibody against it into the mixture
Definition
Flowcytometry

 a technique wich cells/ events passed individually

through a beam of laser light, amplified and
converted to digital signal and can be plotted to form
a scattergram.
 Cells or other particles can be analyzed using
fluorescence technique resulted after previously
fluorochrome labeled
What is flowcytometry?
 Cyto = sel
 Metry = measurement
 Mengukur bermacam –macam karakteristik tiap
satu sel
 Hitungan tiap sel antara 500-4000 sel/detik
Clinical Function
 CD4/CD8 cell count
 Subset Lympocyte
 Phenotyping Leukemia
 CD 34 cell count
 DNA
 Circle cell analyze
Basics of Flowcytometry

 Cells in suspension
 Flow in line through
 an illuminated volume where they
 Scatter light and emit fluorescence
 That is collected, filtered and
 Converted to digital values
 That are stored in a computer
Fluorescence-activated cell sorter (FACS)

 is an instrument that carries out flow cytometry on a

mixed population of cells.
 Basic instruments analyze the cells and can
quantitate the proportions and phenotype of each
subpopulation.
 A sorter is also able to separate the cells into
different subpopulations so that they can be used in
subsequent experiments.
 The parameters for the sorting (size, fluorescence
intensity, etc.) a
A cytometer Needs a Combined System of

 Fluidics
To introduce and focus the cells for interrogation
 Optics
To generate and collect the light signals
 Electronics
To convert the optical signals to proportional
electronic signals and digitize them for computer
analysis
Perbedaan flow cytometry dgn FACS (Fluorecence
activated sorter)
 Flow cytometry
1. Alat yg dpt mengukur berbagai karakteristik sel dlm
wkt cepat secara stimulan
2. Merupakan pemeriksaan yg paling baik untuk
limfosit T CD4+ dan limfosit T CD8+
 FACS
1. Memisahkan sel berdasarkan subtipe dan epitop
2. Menggunakan berlabel fluorokrom dalam
mengidentifikasi antigen
Fluidics -Hydrodynamic focussing
 Sample (cells in suspension)
 Cells need to be focussedin liquid stream
 Under the right conditions the sample fluid will not
mix with the sheath fluid
Optics -Light Source
 Lasers
 Single wavelength
 Argon (488 nm)
 Helium-Neon (633 nm)

 Intersection point
Laser focussed on the same point where cells are
focused in liquid stream
Forward Scatter (FSC)

 Related to cell surface area

 Intensity of FSC is proportional to the size of a cell
 Diffracted laser light at low angles(1–10)
 Along the axis that the laser light is traveling
Side Scatter (SSC)
 SSC is related to cell granularity and complexity
 Intensity of SSC is proportional to the shape and
optical homogeneity of a cell
 Diffracted laser light, detected at high angles (90)
from the laser beam
Fluorescence

 Excitation Fluorochrome absorbs energy from the

laser light
 Emission Fluorochrome releases absorbed energy
by emitting photons of longer wavelenghth
Detector types
 Two common detector types:
 Photodiode
 Used for strong signals (FSC, SSC)

 Photomultiplier tube (PMT)

 Used for weak signals
 More sensitive than photodiode
Procedures of CD4 absolute measurement
 PRINCIPLE
 Tritest3-color reagents use a time-saving lyse/no-
wash method for direct immunoflorescencestaining
of human peripheral blood specimen.
 When whole blood is added to TruCOUNT Tubes
containing TriTES Treagent, the fluorochrome-
labelledantibodies binds specifically to antigens on
the surface of lymphocytes. The FACSCaliburflow
cytometerdetects three fluorescent colors as well as
forward scatter and side scatter.
Absolute Counts
 Multiset software perform flow cytometricdata
acquisition and automated analysis.
 A Trucount bead region is automatically
identified and the number of events within this
region is determined.
 Persiapan sampel
 3ml darah vena dalam tabung vakum K3EDTA
 Stabil < 30 jam suhu ruang

 Pengiriman Sampel
 Dipertahankan dalam suhu kamar
 Dimasukkan dalam styroform

 Penolakan sampel
 Hemolisis atau beku
 > 48 jam
Cara kerja
 Menggunakan tabung BD truCOUNT
 20 l reagen BD tritest CD3/CD4/.CD8
 50 l whole blood
 Vortex
 Inkubasi 15 menit
 Suhu kamar, tempat gelap
 450 l lysing solution
 Vortex
 Inkubasi 15 menit
 Suhu kamar
 Baca dg cytometer FACS Calibur
Tanpa Trucount
 Reagen 10 l
 Sampel 50 l
 DL : WBC, limfosit
DNA analisis
What about simple assays?
HIV Determine test
• Detect HIV-1 & HIV-2
• Cannot differentiate
• Procedural control – anti Human
IgG
• Whole blood or serum/plasma
• Widely available
• No additional reagents required
• Room temperature storage
• 15 minutes to result
 BioRad HIV-1/2 Multispot
• Detects HIV-1 and HIV-2
• Will differentiate 1 and 2
• Procedural control – anti-Hu IgG
• Serum / plasma only
• Additional reagents (included)
• Requires refrigerated storage
• ‘Immunoconcentration’ principle
• 15 minutes to result
 Aims in Developing HIV Testing
Strategies
• To arrive at the correct sero-diagnosis
• To minimise total testing; thus cost
• Minimise samples classed as indeterminate
or dual reactors
• Detect HIV-1 negative but HIV-2 positive
• Follow likely seroconverters (HIV-1 or -2)
Diagnostic Testing for SARS-CoV 2
Terimakasih