ANTIBODI POLIKLONAL

DAN MONOKLONAL

ANA INDRAYATI
FAKULTAS FARMASI
UNIVERSITAS SETIABUDI SURAKARTA

anabintisuryanto@gmail.com
08156266891
The first line of defense
ANTIBODI
 SISTEM PERTAHANAN TUBUH ATAU SISTEM IMUN
 MERUPAKAN GLIKOPROYEIN YANG DIHASILKAN OLEH SEL B
UNTUK MELAWAN BENDA ASING ’NON-SELF’ ATAU ANTIGEN
 DISEBUT JUGA IMUNOGLOBULIN (Ig) atau SERUM PROTEIN
GLOBULIN
 KEMAMPUAN TUBUH MENGHASILKAN ANTIBODI-
MENUNJUKKAN SEBERAPA TINGGI SISTEM
 ANTIBODI DIHASILKAN 10-14 HARI SETELAH TERPAPAR
ANTIGEN
 The antibodies are transported through the blood and the lymph
to the pathogen invasion site
 The body contains millions of different B cells, each able to
respond to one specific antigen
SEL PENGHASIL ANTIBODI

TIDAK TERDAPAT BUTIRAN SITOPLASMA

BUTIRAN (diwarnai dengan
SITOPLASMA Noda Wright)

Nilai normal leukosit adalah pada kisaran

4.000-10.500 sel/mm³ pada dewasa dan 5.000-
Sel penghasil antibodi
15.500 sel/mm³ pada anak-anak
 STRUKTUR ANTIBODI

• Antibodi mempunyai struktur seperti huruf Y

• Terdiri atas dua rantai polipeptida: rantai
berukuran besar (ranti berat) da berukuran
kecil (rantai ringan)
• Kedua rantai dihubungkan oleh ikatan disulfida
ANTIBODI DAN ANTIGEN

 Antibody: imunoglobulin
 Antigen: imunogen
 The strength of binding
between the antibody
and an antigen at a single
binding site is known as
the antibody’s affinity for
the antigen
 EPITOP DAN PARATOP

 PARATOP: sisi pada antibodi tempat epitop berikatan

 EPITOP: sisi pada antigen tempat paratop berikatan
BAGAIMANA ANTIBODI BEKERJA?

AGLUTINASI

NETRALISASI

FIKSASI KOMPLEMEN

FAGOSITOSIS
ANTIBODI POLIKLONAL DAN
MONOKLONAL
 Antibodi poliklonal:
 antibodi yang
mengenali banyak
epitop dari antigen
 Antibodi
monoklonal:
 antibodi yang
hanya mengenali
satu epitop dari
antigen
 ANTIBODI MONOKLONAL
 ANTIBODI YANG HANYA MENGENALI SATU
ANTIGEN (SPESIFIK)
 ANTIBODI HOMOGEN, SPESIFISITAS SAMA
 BERASAL DARI SATU (MONO) SEL HIBRIDOMA
 MONO: SATU
 KLON: SEKELOMPOK SEL YANG BERASAL DARI SATU
SEL, SECARA GENETIK SAMA
 TEKNOLOGI HIBRIDOMA UNTUK
PRODUKSI MAb
 Imunisasi mencit dengan antigen X, antibodi terhadap antigen X
dihasilkan oleh sel B
 Isolasi dan seleksi sel B penghasil antibodi tehadap X dari limfa
mencit
 Penggabungan sel B dengan sel mieloma (sel kanker sumsum tulang)
pada kultur sel dengan PEG (polietilen glikol) atau elektro fusi
 Sel B: umurnya pendek-penghasil antibodi spesifik

 Sel mieloma: imortal-dapat dibiakkan terus menerus

 PABRIK PRODUKSI ANTIBODI YANG TIDAK ADA HABISNYA

 Penggabungan antara sel B dengan sel mieloma menghasilkan sel

hibrid (hibridoma)
 SELEKSI SEL HIBRIDOMA
 Sel memiliki dua jalur dalam sintesis nukleotida yaitu jalur de
novo dan jalur salvage (non-esensial/penyelamatan)
 Sel B
 HGPRT positif (HGPRT: hypoxanthine phosphoribosyl
transferase) merupakan enzim untuk sintesis basa
purin/DNA jalur salvage). HGPRT mengkatalis pembentukan
nukleotida purin dari ribosa, hipoxantin, dan guanin.
 Jalur de novo dan salvage on
 Sel mieloma
 HGPRT negatif ->mutasi
 Jalur de novo on
 Jalur salvage off
Sintesis nukleotida dari
prekursor metaboliknya: Sintesis nukleotida dari
asam amino, ribosa-5- daur ulang basa bebas
fosfat, CO2, dan unit satu atau nukleosida dari
karbon pemecahan asam nukleat.
 Medium HAT (hipoxantine, aminopterin, timidin)
 Aminopterin (obat) menghambat sintesis DNA jalur de novo
 sel mensintesis nukleotida melalui jalur salvage
 Sel B: mati (umurnya pendek)
 Sel mieloma: mati (HGPRT negatif sehingga jalur salvage off)
 Sel hibridoma: hidup (HGPRT positif sehingga jalur salvage on)
 Sel hibridoma yang terbentuk ini akan terus menerus tumbuh
secara in vitro dan mensekresikan antibodi monoklonal
 Berikutnya, penting untuk
 Pada saat dilakukan fusi sel B dengan sel mieloma didapatkan
6x106 sel hibridoma yang berbeda
 Skrining sel hibridoma yang menghasilkan antibodi yang
diinginkan
 Skrining menggunakan metode ELISA
SELEKSI
 Problem…..
ANTIBODI MONOKLONAL Many patients develop immune response to
monoclonal antibodies produced in mice, as
GENERASI BARU these are foreign proteins

HAMA (human anti-mouse

antibody)-> membuang bagian
antigen dari bagian tikus
tersebut, dengan membuat
antibodi chimeric dan
humanized mAB.
• Menurunkan tingkat imunogenitas secara
signifikan (80% )
• Waktu paruh di serum lebih lama (14-23
hari dibandingkan dengan 30-40 jam)->
frekuensi pemberian bisa dikurangi
 APLIKASI Mab
• DIAGNOSIS
• TERAPI
ANTIBODI POLIKLONAL UNTUK DETEKSI

 ANTIBODI POLIKLONAL: CAMPURAN ANTIBODI

 MENGENALI LEBIH DARI SATU MACAM EPITOP

 KURANG SPESIFIK

 ANTIBODI MONOKLONAL: SPESIFIK

TERAPI KANKER
Monoclonal antibodies work in
different ways and some work in more
than one way. They may:
• Trigger the immune system to
attack cancer cells
• Block molecules that stop the
immune system working
(checkpoint inhibitors)
• Block signals telling cancer cells to
divide
• Carry cancer drugs or radiation to
cancer cells
Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy;
ADCC: antibody dependent cell-mediated cytotoxicity; CDC: complement-dependent
cytotoxicity; MAb: monoclonal antibody; scFv, single-chain Fv fragment
Therapeutic mAbs and their derivatives can affect target cell function and
viability via several different mechanisms. For oncology indications where
directed tumor cell lysis is the desired endpoint, mAbs can be used to engage
cytotoxic or phagocytic effector cells (ADCC or ADCP, respectively), to drive pro-
apoptotic signaling via ligation of death receptors (PCD), or to activate complement
cascades (CDC). An antibody–drug conjugate (ADC) consists of a mAb directed
against a tumor cell antigen coupled to a small cytotoxic molecule, resulting in a highly
specific and targeted chemotherapeutic agent.
TRASTUZUMAB (HERCEPTIN): kanker payudara

 Pada sel kanker payudara,

terjadi overekspresi HER2.
 Protein ini berperan dalam
tranduksi sinyal untuk proliferasi
sel
 Semakin banyak HER2 maka
proliferasi sel meningkat
 mAb menghambat EGF
(epidermal growth factor
berikatan dengan reseptor-nya
sehingga ekspresi HER2
dihambat.
 Proliferasi sel yang sangat
cepat tidak terjadi
 Rituximab
 The first monoclonal antibody approved for the treatment of cancer, rituximab is a
chimeric IgG1 monoclonal antibody targeting the CD20 antigen expressed on the
surface of B cells.
 Rituximab's mechanism of action includes the induction of apoptosis, inhibition of cell
growth, activation of complement-mediated cell lysis, and induction of ADCC.
 Rituximab was approved by the FDA in 1997 for the treatment of low-grade or
follicular lymphoma, relapsed, or refractory CD20-positive non-Hodgkin's
lymphoma.
 Recently, the FDA granted approval for use of rituximab as first-line treatment for
patients with diffuse large B-cell, CD20-positive, non-Hodgkin's lymphoma in
combination with CHOP (cyclophosphamide, doxorubicin HCl, vincristine [Oncovin],
prednisone) or other anthracycline-based chemotherapy. Rituximab has also been
approved to treat rheumatoid arthritis.
 Side effects observed with rituximab include acute infusional reactions, ranging from
fever, chills, nausea, vomiting, rash, pruritis, and angioedema, to bronchospasm,
dyspnea, and hypotension
 Alemtuzumab
 Alemtuzumab (Campath) is a humanized IgG1 rat monoclonal
antibody targeting the CD52 antigen. CD52 is expressed at
high levels on most lymphocytes, natural killer cells, monocytes,
and macrophages.
 Alemtuzumab depletes normal and malignant lymphocytes by
causing cell lysis through a variety of mechanisms, including
antibody-dependent cellular cytotoxicity (ADCC), complement-
dependent cytolysis (CDC), and apoptosis.[26-29]
 Alemtuzumab was approved by the FDA in 2001 for the
treatment of B-cell chronic lymphocytic leukemia (CLL)
There are three mechanisms by which alemtuzumab mediates immune cell depletion: antibody-
dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and apoptosis. In vitro
experiments showed that antibody-dependent cell-mediated cytotoxicity occurs at much lower
concentrations of alemtuzumab (0.01 mg/mL) than the concentration required for complement-
dependent cytotoxicity and apoptosis.The cellular CD52 epitope recognized by alemtuzumab is the C-
terminal peptide and is part of the glycophosphatidylinositol anchor. Binding of the antibody to the
epitope promotes deposition of activated complement molecules and facilitates cell-mediated killing
 ELISA

Horseradish peroxidase (HRP)

Tetramethylbenzidine (TMB): chromogenic substrate
Direct fluorescent-antibody tests are used to
identify specific microorganisms (antigens)

Indirect fluorescent-antibody tests are used to

demonstrate the presence of antibodies against
a specific antigen in serum
Direct fluorescent antibody technique
1. obtain human samples and antibody conjugated with a fluorophore
2. fix the samples to a microscope slide. Prior to fixation, it may be
necessary to homogenize the sample so that it can be pipetted
easily.
3. fix the sample to the slide surface through air-drying, formaldehyde,
or another method. Microscope slides with small wells are ideal,
since a small aliquot of human sample can be placed inside the well.
4. add antibody solution to the sample and incubate at a specified
temperature and humidity.
5. wash the slide thoroughly to remove any excess antibody, as this
"loose" antibody will increase background fluorescence.
6. observe under regular fluorescent or confocal microscopy.
The antibodies to HIV-1
envelope glycoproteins
gp120 and gp41 labeled
with radioisotopes 213Bi
and 188Re selectively killed
chronically HIV-1-infected
human T cells and acutely
HIV-1-infected human
peripheral blood
mononuclear cells
(hPBMCs) in vitro.
1. Obtain sample for testing and commercially available kit; several kits are
listed in Figure 3. Follow kit instructions exactly. Figure 3 gives an
overview of the ELISA process, and although each kit listed detects both
antigen and antibody, they may differ in the exact method or reagents
used for HIV detection.
2. Monoclonal antibodies to p24, the capsid protein of HIV, are immobilized
on a solid surface. Also immobilized is an antigen cocktail, with
recombinant antigens representing both HIV-1 and HIV-2 strains. Also
contained within the testing device are unbound labeled polyclonal anti-
p24 antibodies, and unbound labeled antigen.
3. An aliquot of the blood sample (often diluted) is added to the testing
device. If HIV antigen is present, it will bind the anti-p24 antibodies.
Similarly, if HIV antibodies are present, they will bind the labeled antigen.
Wash excess label antibody and antigen from the testing device and then
quantify antigen/antibody complexes by their respective labels.
 DIAGNOSIS HIV
 The test of HIV infection is based on detecting the presence of
HIV antibody in the patient’s blood serum
TES KEHAMILAN
RAPID TEST-MALARIA
RAPID TEST-DENGEU

The construction of the strip with a novel design; (B) one-way

automatic blood separation device.
The novel dengue IgG/IgM rapid test strip in a
plastic cassette
RAPID TEST-HBV
PLASMA VS SERUM
Terima kasih