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Immunoassay

 Detection methods to localize antigen or

proteins either in cells, tissue section or
soluble proteins
 Using labelled antibodies as spesific reagent
TEKNIK IMUNOLOGI through antigen-antibody interaction that are
visualized by a marker such as fluorescent dye,
enzyme or colloidal gold
Oleh
Evy Diah Woelansari, S.Si, M.Kes

Principle of Immunoassay

Labels in immunoassay Immunoassay techniques

Type of Antigen Immunodetection techniques

 Radioactive label
 Nonradioactive label
Tissue •Immunohistochemistry (IHC)
– Colloidal gold •Immunofluorecence assay (IFA)
– Virus particle
Soluble •ELISA
– Ferritin •Immunoblotting
– Fluorescent
Cell •Immunocytochemistry (ICC)
– Enzyme •FACS
•IFA

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Techniques at DNA, RNA and protein level Key component in immunoassay

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”

Basic method in immunoassay Direct method

 Direct
 Indirect

 Antibody is adaptive immune component with 2 specific

Indirect method characteristics:
1.Recognize specific antigen
2.Precipitate antigen that binds to it

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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 Antibody testing limitations

3. Fluorescence  Difficulties in interpretation

microscope:
Specific antibody labelled  Limitations - ‘window period’
with fluoresenceprobe
 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.

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ELISA
Measures : presence of
antigen Antibody concentration
Antigen concentration

Qualitative Semi Quantitative Quantitative

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

 Preparation of immobilized antigen consist of :

• protein separation based on molecular weight
(MW) (SDS--‐PAGE)
• Protein immobilization (Western--‐blot)

 Immunodetection (immunobloting)

Reaction on Immunobloting

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Westernblot-Immunoblot Western Blot

 Expensive – $ 80 - 100
 technically more difficult
 visual interpretation
 lack standardisation
 - performance
 - interpretation
 - indeterminate reactions –
resolution of ??
 ‘Gold Standard’ for confirmation

Immunoblotting
Blocking buffer 37°C1 hr

3x wasing with PBS Tween

Sampel serum in bloking buffer (1:100) RT 1 hr

3x wasing with PBS Tween

Rabbit  human IgG-HRP in dilution buffer (1:1000), RT 1 hr

3x wasing with PBS Tween

Substrate/ chromogen (chloronaphtol)

Stop with water

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