Diagnosa

Lab.Mikrobiologi
Penyakit Demam Berdarah
dengue (DHF)

TIK
MAMPU

MENJELASKAN CARA
MENEGAKKAN DIAGNOSIS PENYAKIT
DHF
MAMPU MENJELASKAN CARA
PEMERIKSAAN DHF DENGAN CARA
SEROLOGI TES
MAMPU MENGINTERPRETASIKAN HASIL
PEMERIKSAAN SEROLOGIS TES DENGAN
TEKNIK ELISA (DENGUE RAPID TEST)

Penyebab DHF
Virus

dengue
Genus Flavivirus
4 serotipe: Den-1, Den-2, Den-3 dan
Den-4
Punya 3 protein struktural: envelope,
premembran, core dan 7 protein
nonstruktural

Penyebaran
Nyamuk

aedes aegypti: adesi virus

netralisasi
gangguan
pembekuan darah
kebocoran
pembuluh darah

Diagnosis Dengue virus

Mikroskopik:

mikroskop elektron
Makroskopik (in vivo)
Serologis tes (elisa, PCR, aglutinasi,
presipitasi dll)
Dengue IgG/IgM Test
C

T1

T2

S
1 tetes serum
+ 2 tetes
buffer

 Interpretasi

hasil:
1. C positif: kit yg digunakan valid
2. T1 positif: IgM thd antigen envelope
positif
3. T2 positif: IgG thd antigen envelope
positif
4. Ti dan T2 negatif: tdk ada antibodi thd
antigen envelope
5. C, T1 positif, T2 negatif: apa artinya?
6. C, T2 positif, T1 negatif: apa artinya?
7. C, T1, T2 positif: apa artinya?

Langkah2 ELISA
Sampel yang diukur
Kit (ready for use)
Titrasi antigen dan antibodi (optimasi konsentrasi/
checkerboard)
kurva standard
Coating, pelapisan pada media (mis: microplate 96 wells)
Coating solution

Blocking, menghindari reaksi non spesifik

Blocking solution

Washing, setiap langkah

Washing solution

Antibodi pertama
Antibodi kedua yang telah dilabel/melabel dengan enzim
Substrat
Substrat solution

Stop reagent: 1N NaOH

Stage One
The antigen is added to the well and attaches itself to
the bottom and sides of the well which is coated with a
special substance. Incubated at 40C overnight or 370C for
1 hr

Any excess antigen is washed out the

antigen which is adsorbed onto the
well surface stays in the well.
Detergent is used for the wash.

Stage Two
Neutralisation - a milk protein is added to fill the gaps on
the well. It is then incubated for an hour at room
temperature. Incubated at RT for 1-11/2 hr

Any excess milk protein is washed out

using a detergent

Stage Three
Antibody is added and attaches only to the antigen, not to
the neutral milk protein. Not all the sites get taken up and
it important that there is more antigen than antibody. This
is because it is the level of antibodies that is being
measured. All the antibodies in the sample must be
attached to an antigen to get an accurate measurement. At
this point effectively the antibodies are invisible.
Incubated at RT for 1 hr
Any excess antibody is washed out, leaving
only the antibodies attached to the
antigens.

Stage Four
Conjugate is added and binds to the antibodies.
At this point there is a biological sandwich.
The conjugate contains an enzyme which indicates
the presence of the antibody.
Incubated at RT for 1 hr
Any excess conjugate is washed out
leaving the enzyme markers attached to
the antibodies.

Stage Five
Substrate is added. This part is a chemical reaction. the
substrate reacts with the conjugate and forms a colour
which you can see. By using a colourimeter and measuring
the optical density of the solution, the technician is able
to determine the number of antibody sites available.
Incubated at RT for 30.
A substrate reacts with the conjugate to
form a colour

Stage Six
The reaction is stopped with sulfuric acid, nitric
acid. Acid stops the activity of the enzyme. The
timing of this step is critical. If you leave one
reaction longer than another, the colour may be
more intense because the reaction has had more
time to proceed. The process is useful only if each
step and concentration of reactants is standardized.

Pemeriksaan IgM, IgG

Dengue

stick IgM, IgG

DOT BLOTT

Reaksi serologis
Semikuantitatif
Deteksi spesifitas reaksi antigen dan
antibodi
Dapat menentukan imunogenisitas antigen
Dapat membedakan tinggi rendahnya
antibodi yang direaksikan

Membran NC + Ag + Abs primer

Washing sol
Membran NC + Ag + Abs primer + Abs sekunder
Washing sol

Membran NC + Ag + Abs primer + Abs sekunder + substrat

Stop
Ada noda: +

Tak ada noda: -