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Imunbaru2

  1. 1. Tutor Imunologi Putaran 1 <br />PEMERIKSAAN ANTIBODI ANTINUKLEARDENGAN METODE ELISA PADA PENDERITA SLE<br />Betty A Tambunan<br />Selasa, 1 Juli 2011<br />
  2. 2. MANIFES<br />BERMACAM-MACAM <br /> AUTOIMUN <br /> DISEASE<br />PENDAHULUAN<br />Systemic Lupus Erythematosus<br />
  3. 3. 3<br />KriteriaAMERICAN COLLEGE OF RHEUMATOLOGY<br />
  4. 4. AntibodiAntinuklear (ANA) <br /><ul><li> Autoantibodiyang melawankomponennuklear</li></ul>dansitoplasmasel<br /><ul><li> 4kategori: </li></ul>antiboditerhadapDNA<br />antibody terhadaphiston<br />antiboditerhadap protein nonhiston yang terikatpadaRNA<br />antiboditerhadap antigen nukleolus.<br />4<br />
  5. 5. PrinsipPemeriksaan ANA ELISA<br />5<br />
  6. 6. PrinsipPemeriksaan ANA ELISA<br />6<br />
  7. 7. Sampel Pemeriksaan ANA ELISA<br />Serum<br />Sampel yang terkontaminasi mikroba, mengandung partikel yang nyata, hemolisis dan lipemik tidak dapat digunakan untuk pemeriksaan ini.<br />Penyimpanan sampel : <br />Maksimal 48 jam pada suhu 2-80C<br />> 48 jam pada suhu -200C.<br />7<br />
  8. 8. Company name<br /><ul><li>Kontrolpositifrendah
  9. 9. Kontrolpositiftinggi</li></ul> Serum manusia yang mengandung antibodi terhadap antigen inti dan sitoplasma<br /><ul><li>Microwellplate ELISA
  10. 10. Kontrolnegatif ELISA serummanusia yang tidakmengandungantiboditerhadap antigen intidansitoplasma.</li></ul>Bahan dan Reagen Quanta Lite ANA ELISA <br />
  11. 11. GAMBAR BAHAN DAN REAGEN<br />9<br />Microwell Plate<br />ELISA High Positive Control<br />ELISA Low Positive Control<br />ELISA Negative Control <br />
  12. 12. Company name<br /><ul><li>HS HRP Ig G Conjugation,terdiridari bufferstabilisator protein.
  13. 13. TMB chromogen
  14. 14. HRP Stop Solution,berisi 0,344 M asam sulfur.
  15. 15. ANA Sample Diluent :Tris BufferedSaline, Tween 20, stabilisator protein.
  16. 16. HRP Wash Concentrate : TrisBufferedSalinedanTween 20</li></ul>Bahan dan Reagen Quanta Lite ANA ELISA <br />
  17. 17. GAMBAR BAHAN DAN REAGEN<br />11<br />TMB Chromogen<br />HRP Ig Conjugation<br />HRP Stop Solution<br />ANA Sample Diluent<br />HRP Wash Concentrate<br />
  18. 18. CHEMWELL<br />12<br />
  19. 19. Indirect ELISA<br />13<br />
  20. 20. Pemeriksaan Quanta Lite ANA ELISA dengan alat Chemwell<br />1. Sampeldanreagendiletakkan padasuhu<br />kamar(20-260C)<br />2. MengencerkanHRP Wash Concentrate<br /> (1:40)<br />3. Mengencerkansampel(1:41)<br />4. Menentukanletaksampeldankontrol<br />padamesinChemwell<br />14<br />
  21. 21. Prosedur Pemeriksaan ANA<br />Menempatkanmicrowellplateyang dibutuhkanpadaholder<br />Menambahkan100 mikroliterlarutan kontroldaninkubasipadasuhukamarselama 30 menit<br />Pencucian<br />Menambahkan 100 µLlarutan konjugat dan inkubasi selama 30 menit<br />15<br />
  22. 22. 9. Mengulangi langkah ke 7 untuk pencucian<br />10. Menambahkan 100 µL TMB kromogen pada masing- masing well dan inkubasi 30 menit<br />16<br />
  23. 23. 11. Menambahkan 100 µL HRP Stop Solution pada masing-masing well<br />12. Baca pada panjang gelombang 450 nm<br />17<br />
  24. 24. Quality Control<br />1. Semua kontrol pada ANA ELISA harus diperiksa bersama sampel untuk memastikan semua reagen dan prosedur baik<br />2. Semua kontrol tidak perlu diencerkan karena sudah diencerkan sebelumnya<br />3. Hasil tes dianggap valid jika :<br />OD kontrol (+) harus lebih tinggi dari OD kontrol rendah dan harus lebih tinggi dari OD kontrol (-)<br />18<br />
  25. 25. Perhitungan Hasil<br /> OD sampel <br /> Hasil = --------------------------------- x kontrol positif rendah <br /> OD kontrol positif rendah (unit)<br /> Dikatakan :<br /> Negatif bila kurang dari 20 unit.<br />Positif moderat bila nilai 20-60 unit.<br />Positif kuat bila nilai lebih dari 60 unit.<br />19<br />
  26. 26. Lembar hasil pemeriksaan <br />20<br />
  27. 27. TERIMA KASIH<br />21<br />
  28. 28. Epidemiologi<br />1kasus per 2500 orang<br />Lebih banyak pada perempuan (9:1), terutama pada perempuanusiasubur<br />Onset : dekadekeduaatauketigatetapidapat pula terjadi semua usia.<br />22<br />
  29. 29. Quality Control<br />23<br />OD : harus lebih dari 0,250 bila kurang dari 0,250 hasilnya dapat positif<br />OD high harus lebih dari 1<br />OD negatif harus kurang dari 0,15 <br />
  30. 30. 24<br />KriteriaAMERICAN COLLEGE OF RHEUMATOLOGY<br />
  31. 31. Prinsip teknik ELISA<br /><ul><li>Memasukkan antibodi pada serum ke dalam antigen yang sudah difiksasi pada penyangga padat.
  32. 32. Inkubasi dan cuci untuk menghilangkan antibodi yang berlebihan.
  33. 33. Menambahkan antibodi yang dikonjugasi dengan enzim. </li></ul>25<br />
  34. 34. Prinsip teknik ELISA<br /><ul><li>Aktivitas enzim ditentukan setelah ditambahkan substrat chromogenic spesifik
  35. 35. Intensitas reaksi warna yang terjadi sebanding dengan jumlah antibodi di dalam serum.</li></ul>26<br />
  36. 36. Keuntungan Teknik ELISA<br /><ul><li> Waktu pemeriksaan cepat : 2-4 jam.
  37. 37. Murah.
  38. 38. Dapat digunakan untuk pemeriksaan jumlah sampel yang besar. </li></ul>27<br />
  39. 39. 28<br />
  40. 40. Prinsip Capture ELISA<br />1. Antibodi Ig M pada serum diikatkan dengan antibodi IgM anti-human pada plat.<br />2.Mencuci plat dan menambahkan antigen dan dibiarkan agar berikatan dengan antibodi Ig M.<br />3. Setelah mencuci lagi kemudian menambahkan <br /> chromogen<br />29<br />
  41. 41. AntibodiAntinuklear (ANA) <br />Autoantibodiyang melawankomponennukleardansitoplasmasel<br />Dapatmerusakjaringansecaralangsungataupundalambentukendapankompleksimun<br />Tidakspesifikterhadap organ atauspesies.<br />4kategori: <br />antiboditerhadapDNA<br />antibody terhadaphiston<br />antiboditerhadap protein nonhiston yang terikatpadaRNA<br />antiboditerhadap antigen nukleolus<br />30<br />
  42. 42. Ruammalar<br />Eritematetap, datarataumeninggi, melebihitonjolanmalar, cenderungtidakmengenailipatannasolabialis.<br />Ruamdiskoid<br />Bercakeritematosamenonjoldenganskuamakeratosisdansumbatanfolikel; parutatrofidapatmunculpadalesi yang lebih lama.<br />31<br />
  43. 43. Double Antibody Sandwich ELISA<br />1. Bahan pemeriksaan yang mengandung antigen direaksikan dengan antibodi pertama yang terikat pada fase padat.<br />2. Selanjutnya ditambahkan antibodi kedua <br /> berlabel enzim.<br />3. Ditambahkan substrat dari enzim tersebut.<br />32<br />
  44. 44. False Negatif :<br />Kelebihan antigen sehingga antigen binding site telah terisi<br />Afinitas antibodi rendah, kadar antibodi yang berlabel suboptimal.<br />33<br />
  45. 45. False positif :<br /><ul><li> Pencucian yang tidak adekuat
  46. 46. Prevalensi dapat lebih tinggi pada beberapa penderita transplantasi dan penderita karsinoma dengan terapi antibodi monoklonal.</li></ul>34<br />
  47. 47. Persiapan Pemeriksaan ANA<br />Sampeldanreagendiletakkan padasuhukamar (20-260C).<br />Mengencerkan HRP Wash Concentrate (1:40) : mencampur 25 ml HRP Wash Concentrate dengan aqua destilata 975 ml.<br />Mengencerkansampel(1:41) dengan mencampur10 mikrolitersampeldengan 400 mikroliterpengencersampel ANA.<br />MenentukanletaksampeldankontrolpadamesinChemwell.<br />35<br />
  48. 48. Pencucian : mengisitiapsumuran 200-300 µL buffer HRP Wash yang telah diencerkankemudiandiaspirasi dan dibuang.Mengulangiurutankerjatersebutdua kali<br />Menambahkan 100 µLHS HRP Ig G konjugasi pada tiap-tiap sumuran dan inkubasi sumuran selama 30 menit.<br />36<br />
  49. 49. Prinsip ELISA indirect <br /><ul><li>Antigen pada permukaan penyangga padat dan menambahkan serum penderita.
  50. 50. Mendeteksi antibodi Ig M dengan menggunakan antibodi monoklonal anti-human Ig M yang dilabel enzim.
  51. 51. Menambahkan substrat cromogen. </li></ul>37<br />
  52. 52. Disease Autoantibody<br />Systemic Lupus Erythematosus<br />Rheumatoid Arthritis<br />SjogrensSyndrom<br />Systemic Sclerosis<br />Polymyositis/Dermatomyositis<br />Mixed Connective Tissue Disease<br />Wegener’s Granulomatosus<br />Anti-dsDNA, Anti-SM<br />RF (IgM), Anti-RA33, Anti-CCP<br />Anti-Ro(SS-A), Anti-La(SS-B)<br />Anti-Scl-70, Anti-centromere<br />Anti-Jo-1<br />Anti-U1-RMP<br />C-ANCA<br />
  53. 53.
  54. 54. Clinical Features of SLE<br />
  55. 55. QUANTA Lite TM ANA is an enzyme-linked immunosorbent Assay (ELISA) for the semi quantitative detection of anti-nuklear antibodies in human serum.The presence of ANA can be used in conjuntion with clinical findings and other laboratory test to aid in the diagnostic of rheumatic diseases such as systemic lupus erythematosus , Sjogren syndrom, scleroderma, and mixed connective tissue disease. <br />41<br />
  56. 56.  <br />QUANTA Lite® ANA<br /> <br />IVD-In Vitro Diagnostic Use CE markedTechnology: ELISACLIA Complexity: High<br />Description:<br />This assay is designed for the detection of anti-nuclear IgG antibodies (ANA) in human serum. The presence of these antibodies can aid in the diagnosis of rheumatic diseases such as systemic lupus erythematosus, Sjögren's Syndrome, scleroderma, and mixed connective tissue disease. <br />Antigen: native, recombinant, extracts from HEp-2 nuclei and nucleoli<br />Packaging: 1 X 96 wells<br />42<br />
  57. 57. QUANTA Lite TM ANA<br />Detects G59chromatin (dsDNA and histones), Sm/RNP, SS-A SS-B, Scl-70, centromere, and PCNA.<br />Shows cytoplasmic antigens such as Jo-1, mitochondria (M-2) and ribosomal-P protein.<br />
  58. 58. Hasil ANA Profile 1 Euroline, teskonfirmasiuntuk ANA (anti nuklearantibodi) yang positif<br /><ul><li>SS-A (Ro): positifkuat
  59. 59. Ro-52: positif
  60. 60. Ds-DNA: positiflemah
  61. 61. Nucleosomes: positif
  62. 62. Histones: positifkuat
  63. 63. Rib-P-Protein: positifkuat</li></ul>Dx Lab : SLE, 3 minggukemudianmeninggal<br />
  64. 64. Tes konfirmasi<br />ANA ELISA<br />45<br />
  65. 65. AUTOIMMUNE DISEASES (CMDT, 2011)Connective Tissue Diseases (CTD)1. Rheumatoid Arthritis 2. Systemic Lupus Erythematosus 3. Antiphospholipid Antibody Syndrome 4. Raynaud Phenomenon 5. Systemic Sclerosis ( Scleroderma ) 6. Idiopathic Inflammatory Myopathies (Polymyositis & Dermatomyositis) 7. Sjögren Syndrome 8. Wegner Granulomatosis<br />
  66. 66. RA = Rheumatoid arthritis SLE = Systemic lupus erythematosus<br />SjS = Sjögren’s syndrome DS = Diffuse scleroderma<br />LS = Limited scleroderma (CREST syndrome )<br />PD = Polymyositis / dermatomyositis WG = Wegener’s granulomatosis<br />
  67. 67. Radang sendi atau artritis reumatoid (bahasa Inggris: Rheumatoid Arthritis, RA) merupakan penyakit autoimun (penyakit yang terjadi pada saat tubuh diserang oleh sistem kekebalan tubuhnya sendiri) yang mengakibatkan peradangan dalam waktu lama pada sendi. Penyakit ini menyerang persendian, biasanya mengenai banyak sendi, yang ditandai dengan radang pada membran sinovial dan struktur-struktur sendi serta atrofi otot dan penipisan tulang. Berdasarkan studi, RA lebih banyak terjadi pada wanita dibandingkan pria dengan rasio kejadian 3 : 1.<br />Umumnya penyakit ini menyerang pada sendi-sendi bagian jari, pergelangan tangan, bahu, lutut, dan kaki. Pada penderita stadium lanjut akan membuat si penderita tidak dapat melakukan aktivitas sehari-hari dan kualitas hidupnya menurun. Gejala yang lain yaitu berupa demam, nafsu makan menurun, berat badan menurun, lemah dan kurang darah. <br />48<br />
  68. 68. Rheumatoid Arthritis<br />Abnormal antibodies can be found in the blood of people with rheumatoid arthritis. An antibody called "rheumatoid factor" can be found in 80% of patients with rheumatoid arthritis. Patients who are felt to have rheumatoid arthritis and do not have positive rheumatoid factor testing are referred as having "seronegative rheumatoid arthritis." Citrulline antibody (also referred to as anticitrulline antibody, anticycliccitrullinated peptide antibody, and anti-CCP) is present in most people with rheumatoid arthritis. It is useful in the diagnosis of rheumatoid arthritis when evaluating cases of unexplained joint inflammation. A test for citrulline antibodies is most helpful in looking for the cause of previously undiagnosed inflammatory arthritis when the traditional blood test for rheumatoid arthritis, rheumatoid factor, is not present. Citrulline antibodies have been felt to represent the earlier stages of rheumatoid arthritis in this setting. Another antibody called the "antinuclear antibody" (ANA) is also frequently found in people with <br />49<br />
  69. 69. Antiphospholipid syndrome (APS or APLS) or antiphospholipid antibody syndrome is a disorder of coagulation that causes blood clots (thrombosis) in both arteries and veins as well as pregnancy-related complications such as miscarriage, stillbirth, preterm delivery, or severe preeclampsia. The syndrome occurs due to the autoimmune production of antibodies against phospholipid (aPL), a cell membrane substance. In particular, the disease is characterised by antibodies against cardiolipin (anti-cardiolipin antibodies) and β2 glycoprotein I.<br />50<br />
  70. 70. Antiphospholipid antibody syndrome<br />Physicians use a combination of clinical symptoms (see above) and laboratory tests to diagnose APS. The common blood tests for antiphospholipid antibodies are as follows:<br />Anticardiolipin antibodies (IgG, IgM, and IgA)<br />Lupus anticoagulant – a panel of blood clotting tests that may include the dilute Russel Viper venom time (dRVVT), lupus aPTT, mixing studies, and hex phase phospholipid test, platelet neutralization procedure<br />Antibodies to b2-glycoprotein I (IgG, IgM, IgA)<br />Panels of tests for antibodies to phospholipids other than cardiolipin are available but have not undergone the rigorous international standardization efforts applied to anticardiolipin assays.  A number of experts in the field question the usefulness of these panels, which may be quite expensive.<br />51<br />
  71. 71. Scleroderma is a chronicsystemic autoimmune disease (primarily of the skin) characterized by fibrosis (orhardening), vascular alterations, and autoantibodies. <br />52<br />
  72. 72. Scleroderma<br />The diagnosis of the scleroderma syndrome is based on the finding of the clinical features of the illnesses. In addition, nearly all patients with scleroderma have blood tests that suggest autoimmunity, because antinuclear antibodies (ANAs) are usually detectable. A particular antibody, the anticentromere antibody, is found almost exclusively in the limited, or CREST, form of scleroderma. Anti-Scl 70 antibody (antitopoisomerase I antibody) is most often seen in patients with the diffuse form of scleroderma.<br />Other tests are used to evaluate the presence or extent of any internal disease. These may include upper and lower gastrointestinal tests to evaluate the bowels, chest X-rays, lung-function testing (pulmonary function test), and CAT scanning to examine the lungs, EKG and echocardiograms, and sometimes heart catheterization to evaluate the pressure in the arteries of the heart and lungs.<br />53<br />
  73. 73. Sjogren's syndrome is an autoimmune disease characterized by dryness of the mouth and eyes. Autoimmune diseases feature the abnormal production of extra antibodies in the blood that are directed against various tissues of the body. This particular autoimmune illness features inflammation in glands of the body that are responsible for producing tears and saliva. Inflammation of the glands that produce tears (lacrimal glands) leads to decreased water production for tears and dry eyes. Inflammation of the glands that produce the saliva in the mouth (salivary glands, including the parotid glands) leads to dry mouth and dry lips.<br />54<br />
  74. 74. Sjogren Syndrome<br />Patients with Sjogren's syndrome typically produce a variety of extra antibodies against body tissues (autoantibodies). These can be detected through blood testing and include antinuclear antibodies (ANA), which are present in nearly all patients. Typical antibodies that are found in most, but not all patients, are SS-A and SS-B antibodies (Sjogren's syndrome A and B antibodies), rheumatoid factor, thyroid antibodies, and others. Low red blood count (anemia) and abnormal blood levels of markers of inflammation (sedimentation rate) are seen.<br />55<br />
  75. 75. Wegener's granulomatosis (WG), more recently granulomatosis with polyangiitis, is a form of vasculitis(inflammation of blood vessels) that affects the nose, lungs, kidneys and other organs. Due to its end-organ damage, it is life-threatening and requires long-term immunosuppression.[1] Five-year survival is up to 87%, with some of the mortality due to toxicity of treatment. It is named after Dr. Friedrich Wegener, who described the disease in 1936.[2] In 2011, three professional bodies proposed a more descriptive name.[3]<br />Wegener's granulomatosis is part of a larger group of vasculitic syndromes, all of which feature an autoimmune attack by an abnormal type of circulating antibody termed ANCAs (antineutrophilcytoplasmic antibodies) against small and medium-size blood vessels. Apart from Wegener's, this category includes Churg-Strauss syndrome and microscopic polyangiitis.[1] Although Wegener's granulomatosis affects small and medium-sized vessels,[4] it is formally classified as one of the small vessel vasculitides in the Chapel Hill system.[5]<br />56<br />
  76. 76. Wegener Syndrome<br />Blood tests that detect the abnormal inflammation include the sedimentation rate (sed rate) and C-reactive protein. A more specific blood test used to diagnose and monitor Wegener's granulomatosis is the antineutrophilcytoplasmic antibody (ANCA test), which is commonly elevated when the disease is active.<br />The diagnosis of Wegener's granulomatosis is confirmed by detecting both abnormal cellular formations, called granulomas, and vasculitis in a biopsy of tissue involved with the inflammatory process. For examples, an open lung biopsy or a kidney biopsy are commonly used in making a diagnosis of Wegener's granulomatosis.<br />57<br />
  77. 77. Polymyositis (PM)is a type of chronic[1]inflammatory myopathy related to dermatomyositis and inclusion body myositis. Polymyositis means 'many muscle inflammation'.<br />58<br />
  78. 78. Polimyositis<br />Blood testing usually (but not always) reveals abnormally high levels of muscle enzymes, CPK or creatininephosphokinase, aldolase, SGOT, SGPT, and LDH. These enzymes are released into the blood by muscle that is being damaged by inflammation. They can also be used as measures of the activity of the inflammation. Other routine blood and urine tests can also look for internal organ abnormalities. Chest X-rays,mammograms,PAP smears, and other screening tests might be considered.<br />59<br />
  79. 79. Raynaud's phenomenon (RP) is a condition resulting in a particular series of discolorations of the fingers and/or the toes after exposure to changes in temperature (cold or hot) or emotional events.<br />60<br />
  80. 80. Raynaud phenomenon<br /><ul><li>In patients with the characteristic sequence of skin-color changes of the digits upon cold exposure, diagnosing RP is not difficult.</li></ul>61<br />Typically, patients with Raynaud's phenomenon that is a manifestation of a rheumatic disease have elevated blood sedimentation rates and antinuclear antibodies. Furthermore, capillary nail-fold abnormalities can frequently be found as described above.<br />
  81. 81. Prosedur Pemeriksaan dengan Mesin Chemwell<br />Pengenceran sampel (1:40)<br /> 400 µl sample diluent + 10 µl serum sampel<br /> dihomogenkan<br /> ambil 100 µl larutan<br /> (dalam tabung Eppendorf)<br />62<br />
  82. 82. Prosedur Pemeriksaan dengan Mesin Chemwell<br />Nyalakan program komputer<br />Klik ‘Start of Day’<br />63<br />
  83. 83. Prosedur Pemeriksaan dengan Mesin Chemwell<br />64<br />Menjalankan program komputer<br />Klik ‘Run’  klik ‘New Job’<br />
  84. 84. Prosedur Pemeriksaan dengan Mesin Chemwell<br />65<br />Memasukkan nomor sampel<br />Klik ‘OK’<br />
  85. 85. Prosedur Pemeriksaan dengan Mesin Chemwell<br />66<br />Menjalankan program Assay<br />Klik ‘Assay’<br />
  86. 86. Prosedur Pemeriksaan dengan Mesin Chemwell<br />67<br />Menyiapkan tempat reagen pada mesin<br />Pada layar komputer<br />Pada mesin Chemwell<br />
  87. 87. Prosedur Pemeriksaan dengan Mesin Chemwell<br />68<br />Menyiapkan tempat sampel pada mesin<br />Pada layar komputer<br />Pada mesin Chemwell<br />
  88. 88. Prosedur Pemeriksaan dengan Mesin Chemwell<br />69<br />Menyiapkan well pada mesin<br />Pada layar komputer<br />Pada mesin Chemwell<br />
  89. 89. Prosedur Pemeriksaan dengan Mesin Chemwell<br />70<br />Mengecek ulang tempat reagen, rak sampel dan well<br />
  90. 90. Prosedur Pemeriksaan dengan Mesin Chemwell<br />71<br />Mengecek ulang tempat reagen, rak sampel dan well<br />
  91. 91. Prosedur Pemeriksaan dengan Mesin Chemwell<br />72<br />Menjalankan proses pemeriksaan<br />Klik ‘RUN’  klik ‘Start”<br />
  92. 92. Prosedur Pemeriksaan dengan Mesin Chemwell<br />73<br />Hasil pemeriksaan<br />
  93. 93. 74<br />
  94. 94. 75<br />
  95. 95. 76<br />
  96. 96. 77<br />
  97. 97. 78<br />
  98. 98. HRP<br />79<br />

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