1. Pemeriksaan Immunoglobulin A (IgA) dengan metode Radial Immunodiffusion Ferdy,dr/ Endang Retnowati, dr, MS, SpPK(K) TUTOR IMUNOLOGI 1
2. 2 subklas: IgA1 dan IgA2 Antibodi penting pada imunitas mukosa. IgA 3-5 gram/hari disekresi ke lumen intestinal 75% Ig total dihasilkan tubuh 2 Pendahuluan
3. Struktur Ig A Gambar 1: Struktur Ig A (Sumber: Siemel, 2006) 3
26. Pemeriksaan IgA di Instalasi Patologi Klinik RSUD.dr.Soetomo Prinsip pemeriksaan (RID): Ig A serum/plasma manusia membentuk kompleks imun dengan antibodi spesifik pada gel agarose pada cakram Partigen Diameter cincin presipitasi berbanding lurus dengan konsentrasi Ig A dalam sampel Hasil dikonversi ke dalam konsentrasi Ig A menggunakan tabel nilai referensi. 21
27. Reagen: Tiap plate NOR partigen mempunyai 12 sumuran imunodifusi untuk menentukan Ig A serum/plasma. Komposisi: Cakram NOR partigen mengandung lapisan agarose gel yang mengandung antiserum monospesifik terhadap Ig A dalam plasma. Antisera diperoleh dari kelinci, domba, kuda, kambing yang diimunisasi dari manusia. Cakram NOR Partigen juga mengandung sodium azide (<1 g/l) dan sodium timerfonate (<0.1 g/l) sebagai pengawet. 22
29. Penyimpanan dan stabilitas NOR Patigen sebaiknya disimpan pada suhu 2-8°C pada pembungkus yang belum dibuka, stabil sesuai masa berlaku Kontrol Sebaiknya digunakan kontrol sampel yang tersedia komersial 24
30. Spesimen serum maupun plasma segar (24 jam sesudah pengambilan) bila sampel tidak langsung dikerjakan sebaiknya disimpan pada suhu 2-8°C maksimum 8 hari Penyimpanan pada suhu -20°C dapat bertahan hingga 1 tahun. 25
31. Prosedur 5 µl sampel 1 Tes serum 2 8 Antisera dalam agar Tes serum 3 7 UKUR DIAMETER CINCIN PRESIPITASI SETELAH 48 JAM Tes serum 4 6 5 Tes serum Tes serum (Handojo, 2003) 26
36. we defined IgA deficiency as plasma samples with less than 100 ngIgA per mL, decreased level of IgA as plasma samples with 100 ng to600 mg IgA per mL, and normal level of IgA as plasmasamples with more than 0.6 mg per mL as determined by the IgA-ELISA 31
37. IgA measurement by nephelometry Beckman Coulter, Inc. (Fullerton,Calif.). IgA levels were considered deficient when they were less than thelower limit of established age-dependent reference intervals. These limits: 70 mg/dl for teenagers and adults, 23 mg/dl for children 3 to 12 years old, 17mg/dl for children ,3 years old. 32
45. 40 PRINSIP NEPHELOMETRI (6) 2. KINETIK CAIRAN PADA FASE PRESIPITASI Sinartampak (visible) yang mengenaibentukanlatticependarancahaya Rayleigh-Debye. Latticemaksimalzonaekivalen. Nephelometrimengukurkonsentrasi antigen padakeadaankelebihanantibodi, dimanaterdapatjumlahlattice pendarancahaya yang terukursesuaijumlah antigen sampel.
46. Low values Ig A Some people are born with low or absent levels of IgA antibodies. Low levels of IgA occur in some types of leukemia, kidney damage (nephrotic syndrome) a problem with the intestines (enteropathy) a rare inherited disease that affects muscle coordination (ataxia-telangiectasia). 41
47. High values IgA monoclonal gammopathy of unknown significance (MGUS) or multiple myeloma autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (SLE), and in liver diseases, such as cirrhosis and long-term (chronic) hepatitis. 42
48. 3 most commonly used methods for the quantitative determination of serum IgA concentration: nephelometry (or turbidimetry), passive hemagglutination inhibition assay (PHAI), and enzyme-linked immunosorbent assay (ELISA). The basic principle of nephelometric and turbidimetric assays for quantifying IgA concentration is light scatter at various angles by immune complexes formed between IgA (antigen) in the patient's serum and anti-IgA (antibody) antiserum added to the patient's serum. Passive hemagglutination techniques use hemagglutination as the end point of the antigen-antibody reaction, while ELISA methods use a capture antibody bound to the well of a microtiter plate to "capture" antigen (eg, IgA) and a signal antibody, containing an enzyme "signal", that recognizes a different epitope on the antigen than the capture antibody. The addition of substrate specific for the enzyme attached to the signal antibody results in the production of a color whose intensity is directly proportional to the amount of antigen present. 43
49. Among these methods for the quantitation of IgA concentration, ELISA methods provide the best analytical sensitivity [ie, lower limit of detection (LLD) <0.005 mg/dL] ( Table 2 ). Nephelometric or turbidimetric methods are used routinely in most clinical laboratories for the quantitation of serum IgA concentration because these methods are rapid, easy to use, and are amenable to automation. According to the data in the College of American Pathologists (CAP) 2004 Diagnostic Immunology Survey Set-B Summary, 93.6% (712 of 760) of participating laboratories used nephelometric or turbidimetric methods for the quantitation of IgA, with the majority (55.2%) of these laboratories reporting the use of nephelometry over turbidimetry.[2] 44
