Laboratory orientation

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 MHC Variable Region Constant Region

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

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

Microlymphocytotoxicity test(微量淋巴球細胞毒殺試驗) 原理 • 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

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

Amplification Matched No Amplification Mismatched PCR-SSP Method PCR-SSP (Sequence Specific Primer) 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

5’ 3’ G G G G T GC C C TA A A A AT T T T 5’ A G G G G T G C C C C C ACG G G A T T T T 3’ 5’ PCR-SSP Principle Positive Reaction

30 2 Sequence-Specific Amplification

5’ 3’ G G G G T GC C C TA A A A X GT T T T 5’ X A G G G G A G C C C C C ACG G G A T T T T 5’ PCR-SSP Principle Negative Reaction 3’

PCR-SSP Data Interpretation Negative Positive Positive Blank

Micro SSP Class I GenericPrimer 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

SSOSequence 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

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

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

ddT ddG ddC ddA A C G T SBT Method SBT (Sequencing Base Typing) 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

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 B21B*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

Laboratory orientation