Laboratory orientation Ping Chin Lai

HLA – Human Leucocyte Antigen Equivalent to Major Histocompatibility Complex (MHC) in mouse On the short arm of chromosome 6 Highly polymorphic Responsible for recognition of cells as self or foreign. They can present foreign peptides to T-cell receptor in order to initiate immune response.

Antigen Peptide T-Cell Constant Region Variable Region MHC

Peptide Binding Groove Human Leukocyte Antigens (HLA) MHC II MHC I Peptide Binding Groove     2M    B-Cells, Macrophages All Nucleated Cells

HLA Typing in Laboratory Microlymphocytoxicity assays HLA Class I Mixed lymphocyte culture HLA Class II

原理 Microlymphocytotoxicity test (微量淋巴球細胞毒殺試驗) 已知 未知 補體依賴細胞毒殺反應(CDC) – 1964 Terasaki 藉由具特異性抗血清與淋巴球細胞膜上的相對HLA抗原結合,活化補體(新鮮冷凍兔血清),造成細胞膜損傷,致使活性染料進入細胞膜內 已知 Eosin/ Flurorescent Satin Rabbit Complement Read Cell Lysis Serum + Target Lymphocytes Formalin 未知

DNA based Typing Methods RFLP rev. SSOP SSOP SBT PCR SSP

PCR-SSP Method PCR-SSP (Sequence Specific Primer) Characteristic 1. DNA Extraction 2. PCR using SSP’s 3. Agarose Gel Electrophoresis Characteristic Typing Occurs During Amplification Establish linkages between polymorphisms Throughput Depends on the # of thermocyclers Rapid Matched Amplification No Mismatched

PCR-SSP Principle Positive Reaction 5’ G G G G T G C C C T A A A A A T T T T G G G G T C C C C A C G G G A T T T T A G C 5’ 3’

Sequence-Specific Amplification 2 30

X PCR-SSP Principle Negative Reaction G G G G T G C C C T A A A A G T T T T G G G G A C C C C A C G G G A T T T T A G C 5’ 3’ X 3’

PCR-SSP Data Interpretation Negative Positive Positive Blank

Micro SSP Class I Generic Primer Set Tray 96 wells/test Same procedure for both Class I and Class II Allows low resolution typing of Class I alleles A11/23,B49/52, Cw07/12

SSO Sequence Specific Oligonucleotides Probe Hybridisation of amplified PCR products of sample DNA to sequence specific oligonucleotides Assay can be performed manually or automated

Method 4 – Step Process DNA Extraction Any high quality DNA purification system can be used - but need high quality DNA 200ng of purified DNA required in a volume of 15µl PCR Amplification Strip Hybridyzation Results Interpretation

Dynal RELI™ SSO TMB Horseradish Peroxidase Streptavidin Target PCR Nylon Membrane Biotin Streptavidin Horseradish Peroxidase PCR Product TMB Linker = BSA Probe Target H2O2

Method Overview ASSAY CAN BE PERFORMED MANUALLY IN WATERBATH, Baby Bee OR AUTOMATED USING AutoRELI Mk II Add PCR amplicon and hybridisation fluid Hybridize @ 50C for 30min Stringent wash @ 50C Aspirate and add SA-HRP Aspirate & add wash solution Wash 5 min @ room temp x2 Wash @ room temp Aspirate & add wash solution Aspirate & add substrates A & B Shake @ room temp for 10 min Wash @ room temp for 15 min Interpret Results

SBT Method SBT (Sequencing Base Typing) Characteristics 1. DNA Extraction 2. Group-specific PCR 3. Sequencing Reaction 4. Electrophoresis/Fluorescence Detection 5. Sequence Analysis Characteristics Gold Standard Labor Intensive Low Throughput Capital Cost ddA ddC ddG ddT A C G T

CGAT G/T GGATC A/G TTCA CGAT G GGATC A TTCA CGAT G GGATC G TTCA CGAT T GGATC A TTCA CGAT T GGATC G TTCA

Application for HLA Typing Matching suitable donor for recipients awaiting transplant Disease association testing Paternity testing Vaccine efficacy study Disease resistance study

SEROLOGY SPLIT DNA B40 B60 B*4001/07/10/31/34 B61 B*4002~04/06/09/16/27/29 B40 B*4011 B4005 B*4005 B21 B*4026 Unknown Other B*40 alleles

SEROLOGY SPLIT DNA B15 B63 B*1516/17 B70 B*1509/37/51 B71 B*1510/18 B72 B*1503/46 B75 B*1502/08/11/21/31 B76 B*1512/14/19 B77 B*1513 B15 Unknown B*1528~29/33~34/55/58 BLANK B*1501102N B*1526N B35 B*1522 Unknown Other B*15 alleles