vaksin

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1 IMUNISASI IMUNISASI Usaha pencegahan suatu penyakit Usaha pencegahan suatu penyakit dengan pemberian vaksin dengan pemberian vaksin (imunitas aktif) (imunitas aktif) atau antisera (imunitas pasif). atau antisera (imunitas pasif).

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IMUNISASIIMUNISASI

Usaha pencegahan suatu penyakit Usaha pencegahan suatu penyakit

dengan pemberian vaksin dengan pemberian vaksin

(imunitas aktif)(imunitas aktif)

atau antisera (imunitas pasif).atau antisera (imunitas pasif).

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• ANTISERA• Serum yang telah mengandung

antibodi• Contoh :• ATS (Anti Tetanus Serum)• ABU (Anti Bisa Ular)

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• Vaksinasi• Suatu imunoterapi anti-mikrobial,

melibatkan pengaktifan sistem imun untuk merespon agen penyebab infeksi.

• Apakah perlu imunisasi pada bayi?• Bayi yang baru lahir sudah

mendapat antibodi dari ibunya usia 6 bulan

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• Setelah usia 6 bulan, bayi memerlukan imunisasi

• Tetapi tidak harus diimunisasi• Persetujuan orang tua• Memberi penjelasan vaksin yang

akan diberikan

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Vaksin adalah preparat antigenik yang Vaksin adalah preparat antigenik yang digunakan untuk meningkatkan imunitas digunakan untuk meningkatkan imunitas

terhadap suatu penyakit.terhadap suatu penyakit.Istilah vaksin berasal dari bahasa latin, Istilah vaksin berasal dari bahasa latin,

vacca=sapi.vacca=sapi.Istilah ini untuk memberi penghargaan Istilah ini untuk memberi penghargaan

pada Edward Jenner yang meneliti tentang pada Edward Jenner yang meneliti tentang cowpox. Pasteur menemukan vaksin cowpox. Pasteur menemukan vaksin

terhadap smallpox.terhadap smallpox.Proses distribusi dan pemberiannya Proses distribusi dan pemberiannya

disebut vaksinasi.disebut vaksinasi.

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• Vaksin dapat untuk profilaktik (untuk pencegahan atau memperbaiki efek dari infeksi oleh patogen) atau terapeutik (untuk pengobatan, misal terhadap kanker).

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• Tipe vaksin • Vaksin dapat berupa virus atau

bakteri hidup, yang dilemahkan.• Atau dapat berupa organisme yang

dimatikan atau diinaktifkan atau produk murni yang berasal dari bagian atau fragmen organisme.

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• Ada 4 tipe vaksin tradisional :• 1. Inaktivasi : dibuat dari mikroorganisme

virulen yang dimatikan dengan bahan kimia atau pemanasan.

• Bahan kimia yang digunakan : formalin atau fenol

• Vaksin tipe ini menimbulkan respon imun yang tidak komplit atau yang pendek waktunya dan memerlukan penggunaan secara boster.

• Contoh : vaksin flu, kolera, bubonic plague dan hepatitis A.

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• 2. Mikroorganisme hidup yang dikurangi virulensinya,

adalah : mikroorganisme hidup yang dikultivasi dibawah

kondisi yang tidak enak bagi sifat virulennya. Misal : virus polio dikultur (ditumbuhkan dalam telur ayam yang sudah dibuahi) Helicobacter pylori, hidup di lambung.

Memproduksi urease nyeri. Bakteri ini dikultur berkali-kali (20-50 X),

fimbrie nya akan hilang bakteri gundul• Vaksin tipe ini menimbulkan respon imun lebih

panjang dan lebih banyak digunakan untuk orang dewasa

sehat.• Contoh : yellow fever, measles, rubella, dan mumps

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• 3. Toksoid, tipe ini dibuat dari senyawa toksik dari mikroorganisme yang diinaktifkan.

• Senyawa tersebut pada keadaan tertentu lebih dapat menimbulkan penyakit dibanding dengan mikroorganisme itu sendiri.

• Contoh : tetanus, dipteri• 4. Sub unit, berupa fragmen dari

mikroorganisme, yang dapat menimbulkan respon imun.

• Misal : vili• Contoh : vaksin HBV, dibuat dari protein

permukaan virus

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VIRION

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Eukaryote versus prokaryote

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• Vaksin tuberkulosis hidup bukan merupakan strain TB yang dapat menyebabkan penyakit menular, tetapi dari strain BCG.

• Di USA, vaksin ini jarang digunakan.

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• Beberapa vaksin inovatif (modern) yang sedang dikembangkan dan juga dalam penggunaan :

• 1. Konjugat - bakteri tertentu mempunyai • lapisan luar berupa polisakarida yang sifat imunogeniknya lemah. Dengan melekatkan polisakarida pada protein (misal toksin), maka sistem imun dapat mengenali polisakarida tersebut sebagai antigen protein• Contoh : Vaksin Haemophilus influenzae tipe B

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• 2. Vektor rekombinan- dengan mengkombinasi sifat fisiologi satu mikroorganisme dengan DNA dari mikroorganisme yang lain.

• Imunitas dapat dibentuk terhadap penyakit yang mempunyai proses infeksi yang komplek

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• 3. Vaksin DNA- Akhir-akhir ini, tipe baru dari vaksin dikembangkan dari DNA agen infeksi.

• Transformasi DNA bakteri atau virus kedalam sel manusia atau binatang.

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• Karena sel tersebut dapat hidup lama, jika ada patogen yang biasanya mengekspresikan protein tersebut masuk kedalam hospes, mereka akan diserang dengan segera oleh sistem imun.

• Salah satu keuntungan vaksin DNA adalah sangat mudah diproduksi dan disimpan.

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• 4. Vaksin sintetik

• 5. Vaksin sub unit• Yang bisa menimbulkan respon imun,

bagian yang muncul di permukaan (epitop). Terdiri dari 10-15 asam amino, yang

digunakan sebagai vaksin.

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• Di USA, the Advisory Committee on Immunization Practices, memberikan rekomendasi vaksinasi rutin pada anak-anak terhadap hepatitis A, hepatitis B, polio, mumps, measles, rubella, diptheria, pertussis, tetanus, chicken pox, rotavirus, influenza, meningitis dan pneumonia.

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• Banyaknya jumlah vaksin dan boster yang direkomendasikan (sampai dengan 24 injeksi pada umur 2 tahun) telah menjadi masalah untuk pencapaian yang penuh. Vaksin kombinasi telah dipasarkan (misal vaksin Prevnar dan ProQuad), dengan tujuan untuk pencegahan terhadap bermacam-macam penyakit.

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• Vaksin yang direkomendasikan untuk pemberian ulang seumur hidup :

measles, tetanus, influenza, dan pneumonia.

Seringkali wanita hamil diskrining untuk terus resisten terhadap rubella.

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• Vaksin yang direkomendasikan untuk usia tua : pneumonia dan influenza, yang lebih mematikan untuk golongan tersebut.

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• Kontroversi Vaksin• Sejumlah vaksin, mengandung thimerosal,

(pengawet) yang dimetabolisme menjadi etilmerkuri. Contoh : vaksin influenza, DTP (diphtheria, tetanus dan pertussis).

• Sejak tahun 1997, penggunaan thimerosal sudah banyak berkurang.

• Di USA telah ditetapkan hukum yang melarang penggunaan thimerosal dalam vaksin untuk anak-anak.

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• Penelitian tentang thimerosal :• Laporan akhir tahun 1990, vaksin MMR

(Measles, Mumps, Rubella) dan hepatitis B, menimbulkan efek samping autisme.

• Laporan pada tahun 2004-2005, banyaknya penderita mumps (gondok) pada anak-anak dan orang tua.

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• Efektifitas yang rendah dari pemberian vaksin • 1. Pada hospes yang tak mampu membentuk antibodi, seperti AIDS, yang diserang sel Tnya• 2. Pada hospes yang menerima obat yang dapat menurunkan sistem imun, misal : golongan obat steroid, juga propanolol• 3. Pada hospes yang menderita kelainan genetik• Tidak mempunyai ADE (Adenin Deaminase), sehingga sel T terganggu, menderita seperti AIDS atau lebih dikenal SCID (Severe Complex Immuno Deficiency)• 4. Penerima transplantasi, sering diberi obat imunosupresi

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• Efektifitas atau penampilan vaksin, tergantung pada beberapa faktor :

• 1. Penyakitnya (untuk beberapa vaksinasi penyakit tertentu, hasilnya lebih baik daripada penyakit lainnya)

• 2. Strain dari vaksin• 3. Skedul waktu pemberian• 4. Individual• 5. Etnik atau genetik

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• Efikasi Vaksin• Vaksin tidak menjamin perlindungan yang

komplet, masih ada kemungkinan, orang tersebut terkena penyakit.

• Bisa disebabkan : • 1. Sistem imun hospes. • 2. Adanya imunitas yang direndahkan

(diabetes, penggunaan steroid, infeksi HIV) • 3. Sistem imun hospes tidak mempunyai sel

B • Contoh : Polio 90-100% Tetanus >90% Diphteria 87-96%

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• Bahkan apabila hospes mengembangkan antibodi, sistem imun manusia tidak sempurna. Beberapa germ dapat bermutasi (virus penyebab demam dan influenza), dan dalam beberapa kasus sistem imun masih belum mampu untuk memerangi infeksi.

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• Vaksin yang dapat meningkatkan resiko terjadinya diabetes melitus

• 1. Pertussis• 2. Hemophilus• 3. Hepatitis B• 4. Tuberculosis (BCG)• Bisa berasal dari hospes atau dari

vaksinnya• Hospes, karena kelainan genetik• Vaksin , karena bahan pengawetnya

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• Efek Samping• Beberapa penyakit autoimun, seperti

Acute disseminated encephalomyelitis, Guillain-Barré syndrome, Transverse myelitis and multiple sclerosis diketahui ada hubungannya dengan vaksin.

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• Produksi vaksin• Melalui beberapa tahap• 1. Kultivasi secara in vivo dan invitro• Tak semua agen infeksi bisa dikultivasi in

vitro. • Contoh : Mycobacterium leprae• Hanya bisa hidup di hewan trenggiling,

jika di luar tubuh manusia• Virus MMR bisa dikultivasi in vivo di

dalam embrio dengan kultur jaringan atau

• sel• Vibrio cholerae dapat dikultivasi in vitro

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• Pemanenan dan pemurnian Bisa dilakukan secara sentrifugasi

dan kromatografi• Formulasi Untuk menjaga konsistensi vaksin,

terhadap temperatur, maka ditambahkan gliserin, agar kalau didinginkan tidak membeku

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• Pengawet• Bisa digunakan antibiotik, tetapi bisa

menimbulkan resistensi Thimerosal, digunakan dalam jumlah

sangat kecil, sekitar 0,00125%• Elektrolit dan bufer, untuk menjaga pH

larutan vaksin• Adjuvan, suatu material yang dapat

meningkatkan respon imun Syarat : tidak imunogenik, inert,

mudah didegradasi, tidak pirogenik, tentunya dapat meningkatkan imunogenik suatu antigen

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• Vaksin yang ideal • Dapat digunakan untuk semua umur• Proteksi seumur hidup, hanya sekali

pemberian• Mudah diberikan, jika mungkin per

oral, karena injeksi bisa menimbulkan trauma pada anak-anak.

• Tidak ada efek samping• Stabil • Tersedia dalam jumlah besar• Murah

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• Adjuvan • Senyawa peningkat respon imun.• Contoh :• Adjuvants in common use: • 1. Garam Aluminium • 2. Liposomes and

Immunostimulating complexes (ISCOMS)

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• 3. Complete Freunds adjuvant is an emulsion of Mycobacteria, oil and water

• Too toxic for man • Induces a good cell mediated  immune response. • 4. Incomplete Freund's adjuvant as above,

but without Mycobacteria. • 5. Muramyl di-peptide • Derived from Mycobacterial cell wall. • 6. Cytokines • IL-2, IL-12 and Interferon-gamma.

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• Imunoterapi yang lain• Kanker• Imunoterapi BCG

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• Imunoterapi topikal menggunakan krim (imiquimod) untuk kutil, jerawat, basal cell cancer, squamous cell cancer, cutaneous lymphoma, dan superficial malignant melanoma.

• Imunoterapi injeksi menggunakan antigen mumps, candida atau trichophytin untuk mengobati kutil.

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• Imunoterapi dengan sel dendritik• Tujuannya untuk mengaktifkan respon

sitotoksik terhadap suatu antigen.• Sel dendritik (sel APC), diambil dari

pasien, dicampur dengan antigen ataupun dengan vektor virus. Sel dendritik yang sudah aktif, dikembalikan lagi ke pasien; sel tersebut akan menyajikan antigen ke sel limfosit efektor (sel TCD4+, sel TCD8+, dan sel dendritik, juga sel B).

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• Imunoterapi adoptif menggunakan sel T• Terapi ini menggunakan respon sitotoksik

oleh sel T untuk menyerang kanker. Sel T dari pasien yang mempunyai reaktivitas secara alami maupun genetik pada pasien kanker, ditingkatkan aktivitasnya, kemudian dipindahkan lagi ke pasien tersebut.

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• Tumor pasien dipanen, dan ditingkatkan aktivitasnya secara in vitro dengan IL-2, anti-CD3 dan kadar tinggi

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• Imunosupresi• Menekan respon imun abnormal

dalam penyakit autoimun atau usaha untuk mengurangi respon imun normal untuk mencegah penolakan terhadap sel atau organ yang ditransplantasikan.

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• Imunoterapi juga digunakan untuk mengobati alergi.

• Obat alergi, antihistamin atau kortikosteroid hanya mengobati simtom penyakit alergi, sedangkan imunoterapi dapat memodifikasi secara alami penyebab penyakit alergi, dengan mereduksi sensitivitas terhadap alergen.

• Imunoterapi tidak efektif untuk semua orang.

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• Perbedaan ASI dengan susu formula• ASI mengandung antibodi, susu

formula tidak• ASI mengandung laktalbumin, susu

formula tidak• Walau ada usaha, sapi memproduksi

laktalbumin dengan rekayasa genetik, tetapi efikasinya masih kalah dibanding ASI

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• Western blot• The western blot (alternatively,

immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

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• There are now many reagent companies that specialize in providing antibodies (both monoclonal and polyclonal antibodies) against many thousands of different proteins. Commercial antibodies can be expensive, although the unbound antibody can be reused between experiments. This method is used in the fields of molecular biology, biochemistry, immunogenetics and other molecular biology disciplines.

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• Polyvinylidene Fluoride, or PVDF is a highly non-reactive and pure thermoplastic fluoropolymer. It is also known as KYNAR, HYLAR or SYGEF.

• PVDF is a specialty plastic material in the fluoropolymer family; it is used generally in applications requiring the highest purity, strength, and resistance to solvents, acids, bases and heat and low smoke generation during a fire event. Compared to other fluoropolymers, it has an easier melt process because of its relatively low melting point of around 177°C.

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• It has a relatively low density (1.78) and low cost compared to the other fluoropolymers. It is available as piping products, sheet, tubing, films, plate and an insulator for premium wire. It can be injected, molded or welded and is commonly used in the chemical, semiconductor, medical and defense industries, as well as in lithium ion batteries.

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• Other related techniques include using antibodies to detect proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA).

• The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blotting.

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• Steps in a Western blot• Tissue preparation• Samples may be taken from whole tissue or

from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only.

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• Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes.

• A combination of biochemical and mechanical techniques – including various types of filtration and centrifugation – can be used to separate different cell compartments and organelles.

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• Gel electrophoresis• Immunoblot (Western blot) analysis of

proteins separated by SDS-PAGE gradientgel electrophoresis.

• The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel.

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• By far the most common type of gel electrophoresis employs polyacrylamide gels and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS polyacrylamide gel electrophoresis) maintains polypeptides in a denatured state once they have been treated with strong reducing agents to remove secondary and tertiary structure (e.g. disulfide bonds [S-S] to sulfhydryl groups [SH and SH]) and thus allows separation of proteins by their molecular weight.

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• Sampled proteins become covered in the negatively charged SDS and move to the positively charged electrode through the acrylamide mesh of the gel. Smaller proteins migrate faster through this mesh and the proteins are thus separated according to size (usually measured in kilo Daltons, kDa). The concentration of acrylamide determines the resolution of the gel - the greater the acrylamide concentration the better the resolution of lower molecular weight proteins. The lower the acrylamide concentration the better the resolution of higher molecular weight proteins. Proteins travel only in one dimension along the gel for most blots.

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• Samples are loaded into wells in the gel. One lane is usually reserved for a marker or ladder, a commercially available mixture of proteins having defined molecular weights, typically stained so as to form visible, coloured bands. When voltage is applied along the gel, proteins migrate into it at different speeds. These different rates of advancement (different electrophoretic mobilities) separate into bands within each lane.

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• Samples are loaded into wells in the gel. One lane is usually reserved for a marker or ladder, a commercially available mixture of proteins having defined molecular weights, typically stained so as to form visible, coloured bands. When voltage is applied along the gel, proteins migrate into it at different speeds. These different rates of advancement (different electrophoretic mobilities) separate into bands within each lane.

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• Transfer• In order to make the proteins accessible

to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of tissue papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it.

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• Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this "blotting" process, the proteins are exposed on a thin surface layer for detection (see below).

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• Both varieties of membrane are chosen for their non-specific protein binding properties (i.e. binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings.

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• The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie or Ponceau S dyes. Coomassie is the more sensitive of the two, although Ponceau S's water solubility makes it easier to subsequently destain and probe the membrane as described below.

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• Blocking• Since the membrane has been chosen for its

ability to bind protein, and both antibodies and the target are proteins, steps must be taken to prevent interactions between the membrane and the antibody used for detection of the target protein. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically Bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive), with a minute percentage of detergent such as Tween 20.

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• The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein. This reduces "noise" in the final product of the Western blot, leading to clearer results, and eliminates false positives.

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• Detection• During the detection process the

membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colourimetric reaction and produces a colour. For a variety of reasons, this traditionally takes place in a two-step process, although there are now one-step detection methods available for certain applications.

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• After blocking, a dilute solution of primary antibody (generally between 0.5 and 5 micrograms/ml) is incubated with the membrane under gentle agitation. Typically, the solution is composed of buffered saline solution with a small percentage of detergent, and sometimes with powdered milk or BSA. The antibody solution and the membrane can be sealed and incubated together for anywhere from 30 minutes to overnight. It can also be incubated at different temperatures, with warmer temperatures being associated with more binding, both specific (to the target protein, the "signal") and non-specific ("noise").

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• Two step• Primary antibody• Antibodies are generated when a

host species or immune cell culture is exposed to the protein of interest (or a part thereof). Normally, this is part of the immune response, whereas here they are harvested and used as sensitive and specific detection tools that bind the protein directly.

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• Secondary antibody• After rinsing the membrane to remove

unbound primary antibody, the membrane is exposed to another antibody, directed at a species-specific portion of the primary antibody. This is known as a secondary antibody, and due to its targeting properties, tends to be referred to as "anti-mouse," "anti-goat," etc. Antibodies come from animal sources (or animal sourced hybridoma cultures); an anti-mouse secondary will bind to just about any mouse-sourced primary antibody.

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• This allows some cost savings by allowing an entire lab to share a single source of mass-produced antibody, and provides far more consistent results. The secondary antibody is usually linked to biotin or to a reporter enzyme such as alkaline phosphatase or horseradish peroxidase. This means that several secondary antibodies will bind to one primary antibody and enhance the signal.

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• Most commonly, a horseradish peroxidase-linked secondary is used in conjunction with a chemiluminescent agent, and the reaction product produces luminescence in proportion to the amount of protein. A sensitive sheet of photographic film is placed against the membrane, and exposure to the light from the reaction creates an image of the antibodies bound to the blot.

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• As with the ELISPOT and ELISA procedures, the enzyme can be provided with a substrate molecule that will be converted by the enzyme to a colored reaction product that will be visible on the membrane (see the figure below with blue bands).

• A third alternative is to use a radioactive label rather than an enzyme coupled to the secondary antibody, such as labeling an antibody-binding protein like Staphylococcus Protein A with a radioactive isotope of iodine. Since other methods are safer, quicker and cheaper this method is now rarely used.