Rifki Febriansah

Dasar Teori
Metode uji imunologi berdasarkan reaksi spesifik
antara antigen dan antibodi.
 
Tujuan
Memahami prinsip kerja ELISA, mengetahui
konsentrasi antigen atau antibodi dalam sampel.

Menentukan suatu sampel negatif atau positif

mengidap penyakit Hepatitis berdasarkan teknik
ELISA.
Prinsip kerja
Reaksi spesifik antara antigen dan antibodi dengan
menggunakan enzim sebagai penanda reaksi
(indikator)
3 tipe elisa
Direct ELISA = mendeteksi antigen dengan cara mengikat
antigen dengan antibodi yang telah dilabel dengan enzim.
 Butuh: antibodi yang khas untuk antigen yang dideteksi.

Indirect ELISA = Enzim diikatkan pada antibodi sekunder

yang berikatan dengan antibodi primer.

Sandwich ELISA = dicirikan oleh antibodi penangkap

antigen yang diikatkan pada fase padat
 Antibodi penangkap  dalam wells. Lalu Ag dari darah atau
urine  + dlm wells. => Ag berikatan dg Ab penangkap.
Prosedur
100 uL sampel (well E1, dst)
100 uL Kontrol - (well A1, B1, dan C1)
100 uL kontrol + (well D1)

Perlakuan:
Tutup dg kertas seal, homogenkan (goyang plate perlahan)
Inkubasi 37 C (60’) pd inkubator
Keluarkan plate, cuci plate 4x (dg washer instrument)
+ 100uL substrat (@wells ke semua sampel/kontrol)
Inkubasi lagi (30’) = diamkan, hindari cahaya.
+100 uL H2SO4 2N @wells (isi sampel/kontrol)
Baca hasil
Analisis
Nilai cut off diperoleh dari :
hasil rata-rata kontrol negatif + 0,05
(cut off = NCx + 0,05)

Sampel dg Abs < cut off  non-reactive HbsAg.

Sampel dg Abs ≥ cut off  initial reactive HbsAg.

initial reactive
 ditest ulang dlm duplicate/duplo
dg serum yang sama dan dalam waktu yang sama.

Jika hasil running duplo:

keduanya non-reactive  sampel non-reactive

Jika hasil running duplo:

salah satu/keduanya memberikan hasil reactive 
pengujian sampel dilanjutkan dg tes hepanostika
HbsAg Uni-Form II Confirmatory
 Syarat kontrol negatif (-)
Nilai kontrol negatif harus < 0,200.

Jika nilainya > 0,200 jangan digunakan  ulangi

pemeriksaan.

Nilai kontrol negatif (NC) harus masuk range antara

0,6 ≤ NCx ≤ 1,4 x kontrol negatif

*Assay dinyatakan valid jika nilai

kontrol positif – kontrol negatif ≥ 0,400 (PC-NC ≥ 0,400)
 ELISA:
Enzyme-Linked ImmunoSorbent Assay

96-well
microtiter Small volumes
plate
Use less of
Run multiple reagents
tests on one
plate

Can use bound Ab to quantitate soluble antigen

Can use bound antigen to quantitate serum Ab

 Steps in the ELISA

Partially purified,
protein HBsAg
antigens pre-coated
onto an ELISA plate

ELISA plate wells already coated with protein HBsAg

 Steps in the ELISA

Primary (1) antibody

(“body fluid” sample)

Patient serum added which contains antibodies to

protein HBsAg
 Steps in the ELISA

e e

Secondary (2) antibody

e e e (conjugated to horse
e e e radish peroxidase)
Variable domain of 2
antibody binds constant
domain of
1 antibody
Antisera added which contains anti-human
secondary antibody-enzyme conjugate
Steps in the ELISA

e e

e Secondary (2) antibody

e
Variable domain of 2
antibody binds constant
domain of 1 antibody

Unbound antibodies washed away

 Steps in the ELISA

Chromagen or substrate added

(tetramethylbenzidine or TMB)

Addition of chromagenic substrate results in color reaction

Color intensity increases with concentration (titer) of antibodies
Darker color = higher titer
 Steps in the ELISA

Primary (1) antibody

NEGATIVE REACTION:
There are no antibodies present that bind HBsAg
antigen
 Steps in the ELISA

NEGATIVE REACTION:
All primary antibodies washed away
 Steps in the ELISA

e e

e e e Secondary (2) antibody

e e e

NEGATIVE REACTION:
There are no primary antibodies for the
secondary antibodies to bind
 Steps in the ELISA

NEGATIVE REACTION:
All secondary antibodies washed away
 Steps in the ELISA

Substrate added

NEGATIVE REACTION:
There is no enzyme to carry out the color reaction
NO COLOR CHANGE
 Color Reaction

Enzymatic reaction:
Alkaline phosphatase Colorless
BCIP Phosphate + indolyl
BCIP = 5-bromo-4-chloro-3-indolyl phosphate

Chemical reaction:
Indolyl + NBT Blue color
NBT = nitroblue tetrazolium
 Scoring ELISA
• Compare color to positive and negative
controls
• Indicate the range of dark blue
• The larger the range of color, the more
concentrated the antibodies
• Highest concentration of antibodies in
those samples, highest HBsAg expression

Back
Referensi
Akin, H. M. 2006. Virologi tumbuhan. Kanisius,
Yogyakarta: 191 hlm.
Anonim, 2010, Mikro Elisa, Biomerieux, Jakarta.
Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G.
Seidman, J.A. Smith & K. Struhl. 2003. Current Protocols in
Molecular Biology. John Wiley & Sons, Inc., USA: xii + 4384
hlm.
Ravichandra, N. G. 2010. Methods and techniques in plant
nematology. PHI Learning Private Limited, New Delhi: 616
hlm
Terima Kasih
0,3518 0.0757 0.0513 0.1833 0,1106 0,094 0,3187 0,5438

0,1363 0,0674 0,0838 0,738 0,1299 0,5685