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Steel RGD, Torrie JH. 1984. Prinsip dan Prosedur Statistika : Suatu Pendekatan Biometrik. Edisi ke 2. Jakarta. PT. Gramedia Pustaka Utama. Suartha IN. 1999. Preparasi Antibodi Anti-Idiotype sebagai Dasar Pembuatan Vaksin untuk Pencegahan Streptokokosis. Tesis. Program Pascasarjana. IPB. Bogor. Sun S, Mo W, Ji Y, Liu S. 2001. Preparation and Mass Spectrometric Study of Edd Yolk Antibody (IgY) Againts Rabies Virus. Rapid Commun. Mass Spedtrom. 15:708-712. Thanavala YM, Bond A, Tedder R, Hay FC, Roitt IM. 1985. Monoclonal Internal Image Anti-id iotypic Antibodies of Hepatitis B Surface Antigen. Immunology 55(2):197-204. Tizard IR. 1988. Pengantar Imunologi Veteriner. Edisi kedua. Partodiredjo M., penerjemah. Surabaya. Penerbit Universitas Airlangga, Vizcaino SMJ. 2004. How Many Types are Known? Course of Introduction to the Swine Immunology. http://www.sanidadanimal.info/inmun/elautor.htm [29-06-2004]. Vogel M, Miescher S, Kuhn S, Zurcher AW, Stadler MB, Ruf C, Effenberger F, Kricek F, Stadler BM. 2000. Mimicry of Human IgE Epitopes by AntiIdiotypic Antibodies. J. Mol. Biol. 298(5):729 -735. WHO. 2002. Rabies vaccines. In : Immunization, Vaccines and Biologicals. Weekly Epidemiological Record, 77:109-120. http://www.who.int/wer/pdf/2002/wer7714.pdf. [22-06-2004]. WHO. 2003. Discussion on WHO Requirements for Rabies Vaccine for Human Use : Potency Assay. Report. Worl Health Organization. Genewa. Wibawan IWT, Djannatun T, Halimah LS. 2003. Pengujian Teknik Koaglutinasi Tidak Langsung untuk Deteksi Penyakit Unggas. Laporan Hibah Bersaing XI 2003-2004. Wise DJ, Carter GR, Flores EF. 2005. Prevention of Viral Diseases, Vaccines and Antiviral Drugs. In: A Concise Review of Veterinary Virology. Carter GR, Wise DJ and Flores EF (Eds.). International veterinary Information Service, Ithaca, New York. www.ivis.org. [08-06-2005]. Wunner WH. 1991. The chemical composition and molecular structure of rabies viruses. In The Natural History of Rabies. Boca Raton, FL: CRC Press. Pp. 31 – 67. Zhou EM, Afshar A, Heckert RA, Nielsen K. 1994. Anti-Idiotypic Antibodies Generated by Sequential Immunization Detect the Share Idiotype on Antibodies to Pseudorabies Virus Antigens. J. Virol. Methods. 48:301-313. Zhou EM, Fischer MMD, Rector ES, Sehon AH, Kisil FT. 1991. A Murine Monoclonal Anti-Idiotypic Antibody Detects a Common Idiotope on Human, Mouse and Rabbit Antibodies to Allergen Lol p IV. Scand. J. Immunol. 34:307-316. Zhou EM, Huang W. 1995. Anti-Idiotypic Antibody as Potential Serodiagnostic Reagent for Detection of Bluetongue Virus Infection. J. Clin. Microbiol. 33(4): 850-854. Zhou EM, Lohman KL, Kennedy RC. 1990. Administration of Noninternal Imag e Monoclonal Anti-idiotypic Antibodies Induces Idiotype-Restricted Responses Spesific for Human Immunodeficiency Virus Envelope Glycoprotein Epitopes. Virology 174:9 -17.
63
LAMPIRAN
64
Prosedur kerja pada uji potensi serum anti rabies. A. Persiapan kerja. 1. Disiapkan 4 deret tabung reaksi, masing-masing 8 tabung reaksi berukuran 12 X 100 mm masing-masing untuk pengenceran serum uji, serum referensi, CVS dan netralisasi. 2. Serum Kuda Normal (SKN) 2% dalam akuades disiapkan di dalam gelas erlenmeyer.
B. Pengenceran serum uji dan serum referensi. 1. Serum uji dan serum referensi dibuat pengenceran seri sesuai skema berikut : Skema pengenceran serum uji dan serum referensi Enceran
: 1
SKN 2%(ml) : 4,5 Serum (ml) Prosedur
2
3
4
5
6
7
8
4,5
3,0
1,0
1,0
1,0
1,0
1,0
: 0,5 à 0,5 à 1,0 à 1,0 à 1,0 à 1,0 à 1,0 à 1,0 à buang : Ke dalam tabung 1ditambahkan 0,5 ml serum uji atau serum referensi, dihomogenkan. Kemudian sebanyak 0,5 ml larutan di tabung 1 dipindahkan ke tabung 2, dihomegenkan lagi. Selanjutnya 1,0 ml larutan di tabung 2 dipindahkan ke tabung 3 dan dihomogenkan lagi, demikian seterusnya dari 3 ke 4, 4 ke 5, 5 ke 6, 6 ke 7, hingga tabung 7 ke 8, sehingga didapatkan seri pengenceran serum sesuai dengan langkah 2. Untuk yang terakhir, 1 ml larutan dari tabung 8 dibuang (penyingkiran).
2. Pengenceran : 10-1
10-2
10 -2,6 10-2,9 10 -3,2 10-3,5 10 -3,8 10-4,1
3. Pengenceran yang akan digunakan dalam pengujian adalah dari tabung 4 (pengenceran 10-2,9) hingga tabung 8 (pengenceran 10-4,1)
65
C. Pengenceran CVS. 1. Serum uji dan serum referensi dibuat pengenceran seri sesuai skema berikut : Skema pengenceran serum uji dan serum referensi Enceran
: 1
SKN 2%(ml) : 9,7
2
3
4
5
6
7
8
4,5
4,5
4,5
18,0
4,5
4,5
4,5
Serum (ml)
: 0,3 à 0,5 à 0,5 à 0,5 à 2,0 à 0,5 à 0,5 à 0,5 à buang
Prosedur
: Ke dalam tabung 1 ditambahkan 0,3 ml suspensi CVS, dihomogenkan. Kemudian disentrifugasi 1500 rpm selama 15 menit. Sebanyak 0,5 ml supernatan di tabung 1 dipindahkan ke tabung 2, dihomegenkan lagi. Demikian seterusnya dari tabung 2 ke tabung 3 dan 3 ke 4. Selanjutnya 2,0 ml larutan di tabung 4 dipindahkan ke tabung 5 dan dihomogenkan lagi, kemudian 0,5 ml dari tabung 5 ke 6 dan dari 6 ke 7, sehingga didapatkan seri pengenceran CVS sesuai dengan langkah 2. Untuk yang terakhir, 0,5 ml larutan dari tabung 7 dibuang (penyingkiran).
2. Pengenceran : 10-2
10 -3
10-4
10 -5
10-6
10 -7
10-8
10 -9
3. Berdasarkan nilai potensi (LD 50) CVS, dipilih pengenceran CVS yang memenuhi syarat untuk netralisasi serum, yaitu yang potensinya antara 31,6 – 316 LD50/0,03 ml. Misalnya diketahui 1 LD50/0,03 ml CVS adalah 10-7,1, maka pengenceran CVS yang memenuhi syarat untuk netralisasi serum adalah pada pengenceran 10-5, karena pada pengenceran ini nilai potensi CVS adalah 10 -5/10 -7,1 = 10 -21 = 125,89 LD50/0,03 ml. 4. Dibuat sediaan CVS dengan pengenceran sesuai dengan nilai dalam langkah 3 sebanyak sesuai dengan kebutuhan untuk netralisasi serum.
66
Contoh perhitungan uji potensi serum kuda anti rabies (SAR). Tabel 7. End -point proteksi 50% populasi mencit dari SAR Pengenceran Jumlah hewan model
10 -2,9
10 -3,2,
10-3,5,
10-3,8
10-4,1.
Positif (ri)
5
5
0
1
0
Per pengenceran (ni)
5
5
5
4
5
Dari data pada Tabel 7, diperoleh nilai X0 = 3,2 dan d = 1,0. Dengan rumus Spearman – Karber : Log X50 X0 d ri ni
: : : :
=
- {X0 – (1/2)d + d ? [(ri)/ni]} ; dimana
Log10 pengenceran terendah dimana semua hewan model positif Log10 faktor pengenceran Jumlah hewan model yang positif Jumlah hewan model per pengenceran (setelah dikurangi yang mati selama 5 hari pertama).
Maka, Log X50
=
- {3,2
- ½(1,0) + 1,0 (5/5 + 0/5 + ¼ + 0/5)}
=
- {3,2 – ½ + 1,25}
=
- 3,95.
Diperoleh Nilai Efective Dose (ED)50 Serum Anti Rabies (SAR) : 10-3,95 Tabel 8. End -point proteksi 50% populasi mencit dari Serum Referensi 10-2,9
10-3,2,
10-3,5,
10 -3,8
10-4,1.
Positif (ri)
5
2
1
1
0
Jumlah hewan model (ni)
5
5
5
5
5
Pengenceran
Dari data pada Tabel 8, diperoleh nilai X0 = 2,9 dan d = 1,0. Dengan rumus dan cara perhitungan yang sama, diperoleh Nilai Efective Dose (ED)50 Serum Referensi : 10 -4 Nilai potensi Serum Anti Rabies (SAR) dihitung dengan rumus : P = {(ED50 Serum Uji) / (ED50 Serum Referensi) X Potensi Serum Referensi (IU/ml). Potensi SAR
=
10-3,95 / 10 -4 X 86,6 IU/ml
=
100,05 X 86,6 IU/ml
67
=
97,2 IU/ml.
Reagensia untuk kromatografi afinitas (Fast Protein Liquid Chromatography = FPLC). 1. Elution buffer 20 mM, terdiri atas : NaH2PO4
2,75 gram
Dilarutkan dalam 1000 ml akuades, dibuat pH 7,5.
2. Binding buffer K2SO 4 (Potasium sulfat) 0,5 M dalam 20 mM elution buffer. K2SO 4
43,67 gram
Dilarutkan dalam 500 ml elution buffer, pH7,5.
3. Clearing buffer Sebanyak 30 ml dipropanol dilarutkan dalam 70 ml elution buffer, pH 7,5.
4. Pelarut sampel K2SO 4
17,427 gram
Dilarutkan dalam 100 ml elution buffer, pH 7,5. Untuk melarutkan sampel, campurkan 1 bagian pelarut dengan 1 bagian sampel.
68
Reagensia untuk ELISA 1.. Coating buffer, terdiri atas : Na2CO3
1,06 gram
Na3HCO
0,84 gram
Dilarutkan dalam 100 ml akuades, dibuat dengan pH 9,6.
2. Phosphat Buffer Saline (PBS) stock (10 X konsentrasi), terdiri atas : NaCl
85 gram
KCl
2 gram
Na2HPO4
11,5 gram
KH 2PO4
2 gram
Dilarutkan dalam 1000 ml akuades. Sebelum dipakai, PBS diencerkan 10 kali dengan melarutkan 1 bagian PBS stock dengan 9 bagian akuades, pH diatur 7,2.
Untuk proses pencucian, digunakan PBST (PBS Tween) yang dibuat dari PBS ditambah dengan 0,05% Tween 20.
3. Citrat Phosphat Buffer, terdiri atas : C6H8O7 H2O
21,01 gram
Na2HPO4
14,2 gram
Masing-masing bahan dilarutkan ke dalam 500 ml akuades, kemudian dicampur hingga pH mencapai 4,2.
4. Substrat ABTS ABTS
286 mg,
Dilarutkan dalam 10 ml akuades. Sebanyak 200 µl ABTS yang sudah dilarutkan tadi dimasukkan ke dalam a0 ml buffer sitrat pH 4,2 yang telah dibubuhi 0,005% H2O2.
69
Perhitungan Berat Molekul Protein Berat molekul protein sampel dapat dihitung dari persamaan regresi antara mobilitas relatif protein marker (penanda protein) dengan logaritme dari berat molekul marker yang telah diketahui. Mobilitas relatif (Rf) protein dihitung dengan membandingkan jarak migrasi protein yang diukur dari garis awal separating gel sampai ujung pita protein dibandingkan dengan jarak migrasi tracking dye. Mobilitas relatif (Rf) dapat dirumuskan dengan :
Jarak migrasi protein Rf = Jarak migrasi tracking dye
Tabel 9. Berat molekul protein marker Jarak migrasi tracking dye 4.2 4.2 4.2 4.2 4.2
Jarak migrasi protein 0.2 0.4 0.6 0.9 1.3
Rf 0.047619 0.095238 0.142857 0.214286 0.309524
Log BM 5.342423 5.230449 5.064458 4.880814 4.724276
Kurva Standar HMW y = -2.4213x + 5.4405 R2 = 0.9826
5.4
Log BM
5.2 5 4.8 4.6 0
0.05
0.1
0.15
0.2
Rf
0.25
0.3
0.35
BM 220000 170000 116000 76000 53000
70
Rumus regresi : y = -2,4213x + 5,4405, selanjutnya digunakan untuk menghitung berat molekul protein sampel.
Tabel 10. Berat molekul protein pada serum ayam Jarak migrasi tracking dye 4.2 4.2 4.2
Jarak migrasi protein 0.3 0.8 1.3
Rf = x 0.071429 0.190476 0.309524
a -2.4213 -2.4213 -2.4213
b 5.4405 5.4405 5.4405
Log BM (y) 5.26755 4.9793 4.69105
BM (Dalton) 185160 95345 49096
Tabel 11. Rekapitulasi hasil pembacaan n ilai OD Ab 3 antibodi rabies pada serum kelinci yang diuji dengan metode ELISA. Minggu
Ulangan
I
1 2 3 Rata-rata 1 2 3 Rata-rata 1 2 3 Rata-rata 1 2 3 Rata-rata
II
III
IV
Nilai OD Ab 3 pada kelompok imunisasi Kontrol Ab 2 Vaksin 0,206 0,721 1,831 0,228 0,900 1,507 0,217 0,973 1,729 0,217 0,865 1,689 0,206 0,695 2,101 0,255 0,983 1,629 0,257 0,717 1,906 0,239 0,798 1,879 0,231 0,735 2,023 0,250 1,001 1,235 0,241 0,966 1,787 0,237 0,901 1,682 0,083 0,771 1,905 0,329 0,847 1,217 0,087 1,003 1,631 0,166 0,874 1,584
71
Tabel 12 : Hasil pembacaan ELISA OD serum kontrol (+) dan (- ) pemeriksaan serum kelinci kontrol pada panjang gelombang 414 nm Pengenceran Hasil pembacaan ELISA (OD) (IU) Serum kontrol (+) Serum kontrol (-) (+) – (-) I 0,685 0,510 0,393 0,368 0,331 0,297
1,0 0,5 0,25 0,125 0,06 0,03
II 0,683 0,508 0,397 0,368 0,329 0,299
rerata 0,684 0,509 0,395 0,368 0,330 0,298
I 0,050 0,046 0,044 0,035 0,032 0,029
II 0,046 0,046 0,045 0,037 0,031 0,031
rerata 0,048 0,046 0,045 0,036 0,032 0,030
0,696 0,463 0,350 0,344 0,298 0,268
Absorbance 414 nm (OD)
Kurva Standar Serum Kontrol y = 0.095Ln(x) + 0.5591
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0
0.5
1
1.5
IU/ml
Tabel 13 : Hasil pembacaan ELISA serum kelinci kontrol (diimunisasi dengan NaCl fisiologis) dibandingkan OD serum kontrol (+) dan (-) pada panjang gelombang 414 nm HASIL HASIL KETERANGAN KODE Minggu Ulangan (OD) (IU/ml) SAMPEL R1K1 1 1 0,206 0,024 Negatif R1K2 2 0,228 0,031 Negatif R1K3 3 0,217 0,027 Negatif Rerata 0,027 R2K1 2 1 0,206 0,024 Negatif R2K2 2 0,255 0,041 Negatif R2K3 3 0,257 0,042 Negatif Rerata 0,036 R3K1 3 1 0,231 0,032 Negatif R3K2 2 0,250 0,039 Negatif R3K3 3 0,241 0,035 Negatif Rerata 0,035 R4K1 4 1 0,083 0,007 Negatif R4K2 2 0,329 0,089 Negatif R4K3 3 0,087 0,007 Negatif Rerata 0,034
72
Tabel 14 : Hasil pembacaan ELISA OD serum kontrol (+) dan (-) pemeriksaan serum kelinci yang diimunisasi dengan Ab2 pada panjang gelombang 414 nm Pengenceran Hasil pembacaan ELISA (OD) (IU) Serum kontrol (+) Serum kontrol (-) (+) – (-) I 1,174 0,839 0,715 0,687 0,590
1,0 0,5 0,25 0,125 0,06
II 1,182 0,837 0,709 0,686 0,592
rerata 1,178 0,838 0,712 0,687 0,591
I 0,296 0,253 0,251 0,247 0,240
II 0,295 0,250 0,248 0,245 0,239
rerata 0,296 0,252 0,250 0,246 0,240
0,882 0,586 0,462 0,441 0,351
Kurva Standar Serum Kontrol
Absorbance 414 nm (OD)
y = 0.1717Ln(x) + 0.7838
1 0.8 0.6 0.4 0.2 0 0
0.5
1
1.5
IU/ml
Tabel 15: Hasil pembacaan ELISA serum kelinci yang diimunisasi dengan Ab 2 dibandingkan OD serum kontrol (+) dan (-) pada panjang gelombang 414 nm HASIL HASIL KETERANGAN KODE Minggu Ulangan (OD) (IU/ml) SAMPEL R1Y1 1 1 0,721 0,694 Protektif R1Y2 2 0,900 1,967 Protektif R1Y3 3 0,973 3,001 Protektif Rerata 1,887 Protektif R2Y1 2 1 0,695 0,596 Protektif R2Y2 2 0,983 3,190 Protektif R2Y3 3 0,717 0,678 Protektif Rerata 1,488 Protektif R3Y1 3 1 0,735 0,753 Protektif R3Y2 2 1,001 3,543 Protektif R3Y3 3 0,966 2,890 Protektif Rerata 2,395 Protektif R4Y1 4 1 0,771 0,928 Protektif R4Y2 2 0,847 1,445 Protektif R4Y3 3 1,003 3,585 Protektif Rerata 1,986 Protektif
73
Tabel 16 : Hasil pembacaan ELISA OD serum kontrol (+) dan (-) pemeriksaan serum kelinci yang diimunisasi dengan vaksin rabies pada panjang gelombang 414 nm Pengenceran Hasil pembacaan ELISA (OD) (IU) Serum kontrol (+) Serum kontrol (-) (+) – (-) I 0,684 0,509 0,394 0,365 0,330 0,297
1,0 0,5 0,25 0,125 0,06 0,003
II 0,684 0,508 0,395 0,370 0,329 0,298
rerata 0,684 0,509 0,395 0,368 0,330 0,298
I 0,050 0,048 0,045 0,240 0,233 0,030
II 0,045 0,044 0,245 0,232 0,230 0,029
rerata 0,048 0,046 0,045 0,036 0,032 0,030
0,696 0,463 0,350 0,344 0,298 0,268
Absorbance 414 nm (OD)
Kurva Standar Serum Kontrol y = 0.095Ln(x) + 0.5591
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0
0.5
1
1.5
IU/ml
Tabel 17 : Hasil pembacaan ELISA serum kelinci yang diimunisasi dengan vaksin rabies dibandingkan OD serum kontrol (+) dan (-) pada panjang gelombang 414 nm HASIL HASIL KETERANGAN KODE Minggu Ulangan (OD) (IU/ml) SAMPEL R1V1 1 1 1,831 3,389 Protektif R1V2 2 1,507 1,350 Protektif R1V3 3 1,729 2,536 Protektif Rerata 2,425 Protektif R2V1 2 1 2,101 7,295 Protektif R2V2 2 1,629 1,909 Protektif R2V3 3 1,906 4,193 Protektif Rerata 4,466 Protektif R3V1 3 1 2,023 4,944 Protektif R3V2 2 1,235 0,624 Protektif R3V3 3 1,787 2,990 Protektif Rerata 2,853 Protektif R4V1 4 1 1,905 4,181 Protektif R4V2 2 1,217 0,592 Protektif R4V3 3 1,631 1,920 Protektif Rerata 2,231 Protektif
74
Tabel 18. Kadar antibodi spesifik terhadap rabies (Ab3 ) serum kelinci yang diperiksa dengan metode ELISA. Minggu
Ulangan
I
1 2 3 Rerata 1 2 3 Rerata 1 2 3 Rerata 1 2 3 Rerata
II
III
IV
Kadar Ab 3 (IU/ml) pada kelompok imunisasi Kontrol Ab2 Vaksin 0,024 0,694 3,389 0,031 1,967 1,350 0,027 3,001 2,536 0,027 1,887 2,425 0,024 0,596 7,295 0,041 3,190 1,909 0,042 0,678 4,193 0,036 1,488 4,466 0,032 0,753 4,944 0,039 3,543 0,624 0,035 2,890 2,990 0,035 2,395 2,853 0,007 0,928 4,181 0,089 1,445 0,592 0,007 3,585 1,920 0,034 1,986 2,231
75
Tabel 19. Rekapitulasi kadar antibodi spesifik terhadap rabies (Ab 3) serum kelinci yang diperiksa dengan metode ELISA. KODE SAMPEL R1K1 R1K2 R1K3 R2K1 R2K2 R2K3 R3K1 R3K2 R3K3 R4K1 R4K2 R4K3 R1Y1 R1Y2 R1Y3 R2Y1 R2Y2 R2Y3 R3Y1 R3Y2 R3Y3 R4Y1 R4Y2 R4Y3 R1V1 R1V2 R1V3 R2V1 R2V2 R2V3 R3V1 R3V2 R3V3 R4V1 R4V2 R4V3
PERLAKUAN (Jenis imunogen) NaCl fisiologis (kontrol)
1
2
3
4
Antibodi Antiidiotipe (Ab2)
1
2
3
4
Vaksin Rabies
Ulangan
HASIL (IU)
Ratarata
KETERANG AN
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
0,024 0,031 0,027 0,024 0,041 0,042 0,032 0,039 0,035 0,007 0,089 0,007 0,694 1,967 3,001 0,596 3,190 0,678 0,753 3,543 2,890 0,928 1,445 3,585 3,389 1,350 2,536 7,295 1,909 4,193 4,944 0,624 2,990 4,181 0,592 1,920
0,027
Negatif Negatif Negatif Negatif Negatif Negatif Negatif Negatif Negatif Negatif Negatif Negatif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif Protektif
Minggu
1
2
3
4
0,036
0,035
0,034
1,887
1,488
2,395
1,986
2,425
4,466
2,853
2,231
