Serology Test for Infectious Disesase:What & When

Umi S. Intansari

Departemen Patologi Klinik & Kedokteran Laboratorium

Fakultas Kedokteran

Kesehatan Masyarakat & Keperawatan

PERTANYAAN SEPUTAR DIAGNOSTIK LAB

TERKAIT INFEKSI

Pemeriksaan apa yang bisa dilakukan? Parameter apa saja?

Kapan sebaiknya mulai diperiksa darah ?

Bagaimana interpretasi hasil lab serologi?

Apa pemeriksaan lab untuk diagnosis pasti ?

Bagaimana memantau perjalanan penyakit denganpemeriksaan lab ?

The choice of diagnostic method depends on:

3

• clinical diagnosis

• epidemiological survey,

• Research, vaccine development

Purpose of the tests

laboratory facilities

technical expertise available

costs

the time of sample collection

Laboratory diagnostic method

4

Ease of use

Confidence

DIRECT METHODS INDIRECT METHODS

Virus Isolation Genome

detection

Antigen

detection

Serology

IgM

Serology

IgG

TDR Cit. J. Cardosa

• The branch of laboratory medicine

• scientific study of blood serum

• In practice refers to the diagnostic identification of Ab (& Ag)

Serology

• Antigen and antibody reactions in vitro

• Detections and quantization of antigens and antibodies.

Serological test

Majority Diagnostic tests are serological test

Keith chaitoff, 2004 abbot diagnostic division

Antigen

+Antibody

+ Indicator system

/ detector

Immuno-serology techniqueWhat

Antigen antibody reactions (serological tests)

Precipitation reactions

Agglutination reactions

Virus neutralization test

Immuno fluorescence test

Radioimmuno assay

Enzyme- linked immunosorbent assay(ELISA

Immunoelectroblot eg., westren blot

Immunochromatographic tes

Flow cytometry, etc

Immunoassay

• Precipitation

• Agglutination

Unlabelled

immunoassay

• RIA, ELISA, CLIA, FIA

• Lateral flow Chromatography

• Flow cytometry

Labeled

immunoassay

Immuno-serological testing

10

Figure A-10

Different Ab bind to distinct epitopes on Ag

Affinity?

Avidity?

Antigen-Antibody interaction

Antibody isotypes

IgM is the first to produced before isotypeswitching

After maturation B cell will express different set of isotypes depend on effector site:

low affinity but compensate by pentameric form

Figure A-9

12

Precipitin curve

Agglutination

13

Antibody can agglutinate to Ag on the surface of a large particle (bacteria, latex)

Hemaglutination if Ag on the surface RBC

• Widal test, C-Reactive Protein,

• ASTO, TPHA

Clinical application:

Agglutination

Haemagglutination Inhibition assay(Neutralization test)

15

Neutralization

Test

Labelled Immunoassay

17

Basic parameters:

• Solid phase 1 reactant

• Separation bound & free reagent

• Color development enzyme

Label:

• Enzyme: Enzyme-linked immunosorbent assay

• Chemiluminescense molc

• Radioactive molc

• Fluorescense molc

20

Figure A-20

Flow cytometry

Wicking Material

Colloidal Gold Pad

• Flavivirus specific MAb

conjugated to gold colloid

• Dengue 1-4 Recombinant Antigens

Add 10µL of blood or serum

Membrane with Immobilised

Antibodies

• IgM capture line

• IgG capture line

• Control Line

Blood Separation Device

Absorbent Pad

Backing Sheet

IgM Capture Line

IgG Capture Line

Control Line

Add 2 drops of running

buffer

Rapid Assay

Cassette EnclosureRelease of Serum

Components

Release of Assay

Reagents

Antibody Complexing

15 min

Processing Center

Supply Center

Matrix Cell Hopper

System Control Center

Sampling

Center

Automation

Interpretation

• Qualitative:

• Positive/ Negative

• Optical density, Index value : ODs/Odst, Cutoff

• Quantitative: analyte consentrationunit/mL

Results

Method & performance

Course & history of disease

24

Interpretation of Ag Detection

Recent infection

• HBsAg : acute/ chronic infection/ carrier

• HBeAg : replication marker, infectious

Depend on the marker:

Qualitative/ quantitative

25

Interpretation of Ab Detection

•IgM : recent•IgG : recent/ past/secondary paired sera?•Total

Recent/ Past infection :

• Anti HBs: protective• Anti HCV & anti HIV: diagnostic

Depend on the marker:

• high • low

Avidity

26

Innate versus Adaptive Mechanisms

When

• Seroconversion:

• development of detectable Ab in serum

Seroconversion

• Seroreversion:

• The opposite of seroconversion

• When test is no longer detect Ag/Abin patient’s sera

Seroreversion

Primary versus Secondary Response

IMMUNE RESPONSE

Primary Antibody response:

• First exposure to ag

• IgM appears 3-5 days,

increased and then drops

over a few weeks-months

• IgG detectable 1-2 weeks ,

increased and decreases

over a period of time

Secondary Antibody response/ Anamnestic response:

• After reexposure to ag

• Antibody production increases rapidly

• IgG increase in 2 – 3 days and

increases higher levels than primary

response and remains detectable for

months or years

Antibody Titer• Antibody titre is the

concentration of antibodies against a particular antigen

• Serology test is usually done using micro well plate.

• So that the test sample can be done in a very small sample.

Results Titers provide more valuable information when tested in parallel with a previous titer specimen.

A comparison of the current specimen’s results and previous specimen’s current results should be made.

A change in titer of 2 or more tubes is considered to be significant.

Early Diagnosis Late Diagnosis

days-4 0 4 6 14-21 >50

Hematology Testing

Molecular Detection (RT-PCR)

Virus Isolation

Viral Antigen Detection

Neutralisation Test

Hemaglutination Inhibition

Serology Methods IgG / IgM

By Elisa or Rapid Tests

Timing is critical in the Use of Dengue Diagnostics

Course of dengue infection and timings of diagnosis

Interpretation of Den Diagnostic tests

No single diagnostic assay can diagnose all dengue patients at the various times they may present with symptoms.

WHO, 2011

Interpretasi

35

Respon

Antibodi

Interval

S1-S2

Titer S2 Interpretasi

4X 7 hr 1:1280 Infeksi akut, primer

4X Random 1:2560 Infeksi akut, sekunder

4X < 7 hr 1:1280 Infeksi akut, primer/sekunder

tetap Apapun >1:2560 Infeksi sekunder, baru terjadi

tetap 7 hr 1:1280 Bukan dengue

tetap Spesimen

tunggal

1:1280 Tak dpt diinterpretasi

HIV Time Line

Acute Hepatitis B Virus Infection with Recovery

Typical Serologic Course

IgM anti-HBc

HBsAg

0 4 8 12 16 20 24 28 32 36 52 100

Weeks after Exposure

Titer

HBeAg anti-HBe

Symptoms

anti-HBs

Total anti-HBc

HBV DNA

Window

Period

38

Primary Infection

IgM: day 5 after onset rise for 1-3 weeks, up to 60 days.

IgG: day 10-14 after onset of fever and persists for life.

Secondary Infection

• IgM: Produced at low or undetectable levels

• IgG: Rising rapidly 1-2 days after onset of symptoms

I had high fever last night.

So I go to a hospital this morning.Dr. said it is not dengue fever.

Because they tested Dengue IgG/IgM.

Case 1 *for example

SymptomNS1 Ag

DAY -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10 11 12

AntibodyBite

Ag/A

b level

Day

NS1 Ag

IgM

IgG

Case 2 *for example

DAY -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10 11 12

AntibodyBite

I had high fever from 1 week ago.

So I just took a aspirin.

But I could not get over, so I visit

hospital this morning.

Dr. said it is not dengue fever.

Because they tested Dengue NS1 Ag.

SymptomNS1 Ag

Ag/A

ble

vel

Day

NS1 Ag

IgM

IgG

1 2 3 5 7 10 12