Protocol for Analyzing Human Leukocyte Antigen Variants and Sexually Transmitted Infections: From Genotyping to Immunoassays

Abstract

This chapter describes experimental and analytical procedures that can be used to decipher the specific role of human leukocyte antigen (HLA) variants in infectious diseases. The techniques are distilled from more than one decade of active immunogenetics research, primarily on sexually transmitted infections (STIs) caused by viral and bacterial pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and Chlamydia trachomatis. The specific approaches cover (1) sequence-specific oligonucleotide (SSO) probe hybridization for low-resolution genotyping, (2) sequencing-based typing (SBT) for high-resolution, (3) statistical methods for testing associations between HLA variants and phenotypic traits, and (4) enzyme-linked immunospot (ELISpot) assay for enumerating HLA-restricted and epitope-specific T-lymphocyte responses. Proper application of these mature and robust techniques should help establish the importance of individual HLA alleles, haplotypes, and supertypes to host–pathogen interactions.

Appendices

Appendix 1: List of Useful Online Resources Related to HLA (more links can be found at http://www.ashi-hla.org/links/#mhc)

Name of database	Web site
The dbMHC database	http://www.ncbi.nlm.nih.gov/gv/mhc/main.cgi?cmd=init
IMGT/HLA database	www.ebi.ac.uk/imgt/hla
IMGT: HLA allele summary	http://www.ebi.ac.uk/imgt/hla/stats.html
The immunogenomics data analysis working group (IDAWG)	http://www.igdawg.org/
NIH Genetic Association Database	http://geneticassociationdb.nih.gov/
The Immune Epitope Database (IEDB)	http://www.immuneepitope.org/
HIV Molecular Immunology Database	http://hiv-web.lanl.gov/immunology/index.html

Appendix 2: Standard Operation Manual for Semi-automated DNA Hybridization (Part of SSO Assay): 30 Tests per Run

• 1. Turn on Auto LiPA 30 system by pressing the switch button, which is located in the back of machine.

• 2. Verify that there is distilled water in the three beakers for automated rinsing and cleaning.

• 3. Empty all waste bottles at this point.

• 4. Prepare the machine for running a new program: (1) select “Run program;” (2) click on “<” and “>” to until finding “liquid prep” and then press “yes;” (3) choose “auto clean” and press “yes;” (4) after a few seconds, press “yes” sequentially for distilled water and tub cleaning; (5) remove water and press “yes” to start a new assay.

• 5. While the machine is warming up, locate two kits labeled Color dye 40 and Hybridization solution 5×SSPE.

• 6. Place Hybridization solutions and stringent wash solution in a 60 °C water bath until use.

• 7. Prepare the following reagents for one full tray (30 tests): (1) rinse solution (in 1-L glass bottle), consisting of 300 mL ddH₂O and 75 mL concentrated rinse solution (5×); (2) conjugate diluent (in bottle from supplier) containing 70 mL conjugate diluent and 700 μL 100× conjugate; (3) substrate solution with 70 mL substrate buffer and 700 μL substrate (100×).

• 8. When reagents are ready and the auto cleaning function is complete, remove water beakers and replace with reagents from step 7 above.

• 9. Verify that the reagents bottles match seven sets of colored tubes: (1) brown (1 tube) for hybridization solution (kept at 60 °C); (2) red (3 tubes) for stringent wash (was kept at 60 °C); (3) orange (3 tubes) for wash solution (in glass bottle); (4) yellow (1 tube) for diluted conjugate; (6) green (1 tube) for substrate solution; and (7) blue (1 tube) for diluted substrate.

• 10. Set the desired SSO temperatures at 56 °C.

• 11. Press any key to continue and press start button to run program designated as “HLAB56.”

• 12. Answer “Yes” sequentially to all built-in options (e.g., distilled water, sieve check, etc.).

• 13. Wait for ∼20 min until the system reaches the desired SSO conditions.

• 14. Load SSO probe strips (1 and 2) to the 30-well hybridization tray.

• 15. Make sure that all SSO strips have the colored top (probe side) facing up.

• 16. Add 10 μL of denaturing buffer to the top of each trough.

• 17. Load 10 μL of PCR product to the spot of denaturing buffer.

• 18. Insert tray into the Auto LiPA 30 and press any key on display panel to continue.

• 19. Select start position 1 and stop position 30 using the < > keys.

• 20. Select “No” when prompted for “Last aspiration.”

• 21. Select “Yes” when prompted for “Procedure Inc 1.”

• 22. When assay is done, press the “Pause” button then the “Continue” to allow aspiration of washing solution from the troughs.

• 23. Take out the used 30-well tray and load another empty tray.

• 24. Replace all the buffer containers with beakers filled with distilled water.

• 25. Repeat the auto cleaning procedures (see step 4 in this section).

• 26. Press “No” to disallow decanting water from tubes.

• 27. Exit program and switch off the power.