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PENGUJIAN BIOASSAY DENGAN

METODE ELISA (Enzyme Linked


Immunosorbent Assay )

Mata kuliah Analisis Mikrobiologi dan


Bioassay
Hanafi, MSi AKA BOGOR

Definitions
Antibodies

(
immunoglobulins)
are
gamma globulin proteins that are found in
blood and are used by the immune system
to identify and neutralize foreign objects

The

antibody recognises and bind to the


antigenic determinant region of the antigen

Specific

antibodies are produced by


injecting an antigen into a mammal, such
as a mouse.

Definitions- cont

Antigens
Suatu bahan biokimia yang berada di dalam
tubuh akan menstimulasi produksi antibody
Is present naturally in the body like hormones
Is not present in the body in normal condition like
drugs

Immunoassay
Teknik
pengujian
yang
dilakukan
dengan
menggunakan
ikatan
antara antigen
dan
antibody
yang
sesuai
agar
teridentifikasi
kuantitas specific antigen or antibody in a sample

Definitions- cont
Analyte

Sampel yang dianalisis menggunakan


immunoasssays. Analit bisa berupa
Antibody or Antigen

ELISA technique
Yaitu
cara
pengujian
bioassay
menggunakan uji immunology untuk
mendeteksi keberadaan antibody or an
antigen in a sample.
The

technique is divided into


1- Competitive ELISA
2- Sandwich ELISA (also called direct ELISA)
Indirect ELISA- 3
Non Competitive-4

Competitive ELISA

Antigen berlabel dan antigen sampel berkompetisi


mengikat antibodi. Banyaknya antigen sampel
yang terikat antibodi diukur dari pembentukan
warna

:Tahapan Uji
Antibodi dilapiskan pada mikroplat
Penambahan hapten-enzim konjugat dan larutan
contoh
Terjadi kompetisi analit contoh dan enzim untuk
mengikat antibodi
Penambahan Substrat pembentuk warna

KOMPETISI LANGSUNG (SANDWICH)


Analit sampel (antigen) ditempelkan pada plat
antibodi. Setelah pencucian matriks sampel,
antigen sampel ditempelkan antigen
berlabel). Banyaknya antigen sampel yang
dapat mengikat antibodi ditentukan dengan
penambahan pereaksi pembentuk warna

Sandwich ELISA

Indirect ELISA
Analit sampel dan antigen berlabel
tidak
langsung
berkompetisi
mengikat antibodi, banyaknya analit
yang berikatan dengan antibodi,
menunjukkan
banyaknya
antigen
berlabel.
Pada metode ini yang
terukur
adalah
jumlah
antigen
berlabel
yang terwarnai oleh
.pereaksi

Indirect ELISA

An example of an ELISA experiment


Before

starting the work read kit


instruction carefully

1-

The 96 well plate is labeled


carefully and the first wells are used
to draw the standard curve

An example of an ELISA experiment-Cont


The

sample is added to plate in


duplicate or triplicate and then the
mean result is calculated

The

quality control sample which is


provided with the kit is treated as the
test samples

Results
After

reading the results the standard


curve is drawn were the
concentration is blotted on the X-axis
and the absorbance on the Y-axis

Absorptio
n nm

Concentration ng/ml

Results-cont
The

standards concentrations is
specified on the x-axis and the
reading of each standard is specified
on the y-axis and the standard curve
is drawn