Analytical and Bioanalytical Chemistry

, Volume 403, Issue 3, pp 807–819 | Cite as

Low–medium resolution HLA-DQ2/DQ8 typing for coeliac disease predisposition analysis by colorimetric assay

  • Hamdi Joda
  • Valerio Beni
  • Deirdre Curnane
  • Ioanis Katakis
  • Noora Alakulppi
  • Jukka Partanen
  • Kristina Lind
  • Linda Strömbom
  • Ciara K. O’Sullivan
Original Paper


Coeliac disease is an inflammation of the small intestine, occurring in genetically susceptible individuals triggered by the ingestion of gluten. Human Leukocyte Antigens (HLA) DQ2 and DQ8 gene have been identified as key genetic factors in coeliac disease as they are presented in almost 100 % of the patients. These genes are encoded by the combination of certain alleles in the DQA and DQB region of chromosome 6. Specifically, DQA1*05:01 and DQB1*02:01 alleles for serologically defined leukocyte antigen DQ2 cis, DQA1*05:05 and DQB1*02:02 for DQ2 trans and DQA1*03:01 and DQB1*03:02 alleles for the DQ8. Specific identification of these alleles is a challenge due to the high number of alleles that have been identified so far: 46 in the DQA region and 160 in the DQB region (as of IMGT/HLA Database 10/2011 release). In the reported work, the development of a multiplex colorimetric assay for the low to medium HLA typing of the DQ2 and DQ8 genes is presented. The optimisation of probe design and assay conditions, performed by both surface plasmon resonance and enzyme-linked oligonucleotide assay, are reported. Finally, the performances of the developed typing platform were validated by the analysis of real patient samples and HLA typing, compared with those obtained using hospital based typing technology and an excellent correlation obtained.


Example of the results obtained during the colorimetric HLA typing of a real sample


Coeliac disease HLA typing Sequence specific oligonucleotide probes SPR ELONA 



This work was carried out with the financial support from the Commission of the European Communities, specific RDT program “Coeliac Disease Management Monitoring and Diagnosis using Biosensors and an Integrated Chip System, CD-MEDICs, FP7-2007-ICT-1-216031”. V. Beni acknowledges the European Union’s Seventh Framework Program (FP7/2007-2013) under grant agreement no. PIGF-GA-2008-220928 for financial support. H. Joda thanks Universitst Rovira i Virgili for the doctoral scholarship. We thank Dr. Markku Heikkinen, MD, PhD (Kuopio University Hospital, Kuopio, Finland) for blood samples of Finnish celiac disease patients and Dr. Taina Jaatinen, PhD, Dr. Katri Haimila, PhD and Juha Peräsaari, MS (Finnish Red Cross Blood Service, Helsinki, Finland) for HLA tissue typing expertise.

Supplementary material

216_2012_5898_MOESM1_ESM.pdf (1.2 mb)
ESM 1 (PDF 1.19 Mb)


  1. 1.
    Sollid LM (2002) Coeliac disease: dissecting a complex inflammatory disorder. Nat Rev Immunol 2:647–655CrossRefGoogle Scholar
  2. 2.
    Kaukinen K, Partanen J, Maki M, Collin P (2002) HLA-DQ typing in the diagnosis of celiac disease. Am J Gastroenterol 97:695–699CrossRefGoogle Scholar
  3. 3.
    Catassi C, Fornaroli F, Fasano A (2002) Celiac disease: from basic immunology to bedside practice. Clin Appl Immunol Rev 3:61–71CrossRefGoogle Scholar
  4. 4.
    West J, Logan RFA, Hill PG, Lloyd A, Lewis S, Hubbard R, Reader R, Holmes GKT, Khaw KT (2003) Seroprevalence, correlates, and characteristics of undetected coeliac disease in England. Gut 52:960–965CrossRefGoogle Scholar
  5. 5.
    Megiorni F, Mora B, Bonamico M, Barbato M, Nenna R, Maiella G, Lulli P, Mazzilli MC (2009) HLA-DQ and risk gradient for celiac disease. Hum Immunol 70:55–59CrossRefGoogle Scholar
  6. 6.
    Farrell RJ, CnP K (2002) Celiac Sprue. N Engl J Med 346:180–188CrossRefGoogle Scholar
  7. 7.
    Hill ID, Dirks MH, Liptak GS, Colletti RB, Fasano A, Guandalini S, Hoffenberg EJ, Horvath K, Murray JA, Pivor M, Seidman EG (2005) Guideline for the diagnosis and treatment of celiac disease in children: recommendations of the North American Society for pediatric gastroenterology, hepatology and nutrition. J Pediatr Gastroenterol Nutr 40:1–19CrossRefGoogle Scholar
  8. 8.
    Husby S, Koletzko S, Korponay-Szabó IR, Mearin ML, Phillips A, Shamir R, Troncone R, Giersiepen K, Branski D, Catassi C, Lelgeman M, Mäki M, Ribes-Koninckx C, Ventura A, Zimmer KP, The EWGoCDD (2011) ESPGHAN Guidelines for the diagnosis of coeliac disease in children and adolescents: an evidence-based approach. J Pediatr Gastroenterol Nutr. doi: 10.1097/MPG.1090b1013e31821a31823d31820
  9. 9.
    Guandalini S, Gupta P (2002) Celiac disease A diagnostic challenge with many facets. Clin Appl Immunol Rev 2:293–305CrossRefGoogle Scholar
  10. 10.
    Karell K, Louka AS, Moodie SJ, Ascher H, Clot F, Greco L, Ciclitira PJ, Sollid LM, Partanen J (2003) Hla types in celiac disease patients not carrying the DQA1*05-DQB1*02 (DQ2) heterodimer: results from the european genetics cluster on celiac disease. Hum Immunol 64:469–477CrossRefGoogle Scholar
  11. 11.
    Engelfriet CP, Britten A (1965) The cytotoxic test for leucocyte antibodies. Vox Sang 10:660–674CrossRefGoogle Scholar
  12. 12.
    Jordan BR, Bregegere F, Kourilsky P (1981) Human HLA gene segment isolated by hybridization with mouse H-2 cDNA probes. Nature 290:521–523CrossRefGoogle Scholar
  13. 13.
    Olerup O, Zetterquist H (1992) HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens 39:225–235CrossRefGoogle Scholar
  14. 14.
    Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA (1986) Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature 324:163–166CrossRefGoogle Scholar
  15. 15.
    Saiki RK, Walsh PS, Levenson CH, Erlich HA (1989) Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci USA 86:6230–6234CrossRefGoogle Scholar
  16. 16.
    Bannai M, Tokunaga K, Lin L, Ogawa A, Fujisawa K, Juji T (1996) HLA-B40, B18, B27, and B37 allele discrimination using group-specific amplification and SSCP method. Hum Immunol 46:107–113CrossRefGoogle Scholar
  17. 17.
    Moribe T, Kaneshige T, Inoko H (1997) Complete HLA-A DNA typing using the PCR-RFLP method combined with allele group and sequence-specific amplification. Tissue Antigens 50:535–545CrossRefGoogle Scholar
  18. 18.
    Sayer D, Whidborne R, Brestovac B, Trimboli F, Witt C, Christiansen F (2001) HLA-DRB1 DNA sequencing based typing: an approach suitable for high throughput typing including unrelated bone marrow registry donors. Tissue Antigens 57:46–54CrossRefGoogle Scholar
  19. 19.
    Balazs I, Beekman J, Neuweiler J, Liu H, Watson E, Ray B (2001) Molecular typing of HLA-A, -B, and DRB using a high throughput micro array format. Hum Immunol 62:850–857CrossRefGoogle Scholar
  20. 20.
    Moribe T, Hirai H, Kimura M, Inagawa A, Nakatani S, Kaneshige T, Inoko H (2002) Rapid and simultaneous HLA class I (−A, −B and −C loci) DNA typing using the microtitre plate–reverse hybridization assay (MRHA). Eur J Immunogenet 29:191–204CrossRefGoogle Scholar
  21. 21.
    HLA-Ready Gene Coelic Disease. INNO-TRAIN Diagnostik GmbH.
  22. 22.
  23. 23.
    Engvall E, Perlmann P (1971) Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 8:871–874CrossRefGoogle Scholar
  24. 24.
    Chomel JJ, Thouvenot D, Onno M, Kaiser C, Gourreau JM, Aymard M (1989) Rapid diagnosis of influenza infection of NP antigen using an immunocapture ELISA test. J Virol Methods 25:81–91CrossRefGoogle Scholar
  25. 25.
    Döpel S-H, Porstmann T, Grunow R, Jungbauer A, Von Baehr R (1989) Application of a human monoclonal antibody in a rapid competitive anti-HIV ELISA. J Immunol Methods 116:229–233CrossRefGoogle Scholar
  26. 26.
    Kuno G, Gómez I, Gubler DJ (1991) An ELISA procedure for the diagnosis of dengue infections. J Virol Methods 33:101–113CrossRefGoogle Scholar
  27. 27.
    van der Waart M, Snelting A, Cichy J, Wolters G, Schuurs A (1978) Enzyme-immunoassay in the diagnosis of hepatitis with emphasis on the detection of “e” antigen (HBeAg). J Med Virol 3:43–49CrossRefGoogle Scholar
  28. 28.
    Drolet DW, Moon-McDermott L, Romig TS (1996) An enzyme-linked oligonucleotide assay. Nat Biotechnol 14:1021–1025CrossRefGoogle Scholar
  29. 29.
    Godfroid E, Heinderyckx M, Mansy F, Fayt I, Noël J-C, Thiry L, Bollen A (1998) Detection and identification of human papilloma viral DNA, types 16, 18, and 33, by a combination of polymerase chain reaction and a colorimetric solid phase capture hybridisation assay. J Virol Methods 75:69–81CrossRefGoogle Scholar
  30. 30.
    Acero Sanchez J, Henry O, Mairal T, Laddach N, Nygren A, Hauch S, Fetisch J, O’Sullivan C (2010) Colorimetric quantification of mRNA expression in rare tumour cells amplified by multiple ligation-dependent probe amplification. Anal Bioanal Chem 397:2325–2334CrossRefGoogle Scholar
  31. 31.
    Deng HY, Zhang XE, Mang Y, Zhang ZP, Zhou YF, Liu Q, Lu HB, Fu ZJ (2004) Oligonucleotide ligation assay-based DNA chip for multiplex detection of single nucleotide polymorphism. Biosens Bioelectron 19:1277–1283CrossRefGoogle Scholar
  32. 32.
    Allen M, Eriksson I, Liu L, Gyllensten U (1998) High resolution genetic typing of the class II HLA-DRB1 locus using group-specific amplification and SSO-hybridisation in microplates. Hereditas 129:161–167CrossRefGoogle Scholar
  33. 33.
    Kawai S, Maekawajiri S, Tokunaga K, Kashiwase K, Miyamoto M, Akaza T, Juji T, Yamane A (1996) Routine low and high resolution typing of the HLA-DRB gene using the PCR-MPH (microtitre plate hybridization) method. Int J Immunogenet 23:471–486Google Scholar
  34. 34.
    Legler TJ, Kohler M, Mayr WR, Panzer S, Ohto H, Fischer GF (1996) Genotyping of the human platelet antigen systems 1 through 5 by multiplex polymerase chain reaction and ligation-based typing. Transfusion 36:426–431CrossRefGoogle Scholar
  35. 35.
    Gobi KV, Iwasaka H, Miura N (2007) Self-assembled PEG monolayer based SPR immunosensor for label-free detection of insulin. Biosens Bioelectron 22:1382–1389CrossRefGoogle Scholar
  36. 36.
    Tombelli S, Minunni M, Luzi E, Mascini M (2005) Aptamer-based biosensors for the detection of HIV-1 Tat protein. Bioelectrochemistry 67:135–141CrossRefGoogle Scholar
  37. 37.
    Murphy MB, Fuller ST, Richardson PM, Doyle SA (2003) An improved method for the in vitro evolution of aptamers and applications in protein detection and purification. Nucleic Acids Res 31:e110CrossRefGoogle Scholar
  38. 38.
    Kai E, Sawata S, Ikebukuro K, Iida T, Honda T, Karube I (1999) Detection of PCR products in solution using surface plasmon resonance. Anal Chem 71:796–800CrossRefGoogle Scholar
  39. 39.
    Jiang T, Minunni M, Wilson P, Zhang J, Turner APF, Mascini M (2005) Detection of TP53 mutation using a portable surface plasmon resonance DNA-based biosensor. Biosens Bioelectron 20:1939–1945CrossRefGoogle Scholar
  40. 40.
    Feriotto G, Breveglieri G, Gardenghi S, Carandina G, Gambari R (2004) Surface plasmon resonance and biosensor technology for real-time molecular diagnosis of [beta][degrees]39 Thalassemia Mutation. Mol Diagn 8:33–41CrossRefGoogle Scholar
  41. 41.
    Feriotto G, Ferlini A, Ravani A, Calzolari E, Mischiati C, Bianchi N, Gambari R (2001) Biosensor technology for real-time detection of the cystic fibrosis W1282X mutation in CFTR. Hum Mutat 18:70–81CrossRefGoogle Scholar
  42. 42.
    D’Agata R, Breveglieri G, Zanoli LM, Borgatti M, Spoto G, Gambari R (2011) Direct detection of point mutations in nonamplified human genomic DNA. Anal Chem 83:8711–8717CrossRefGoogle Scholar
  43. 43.
    Avci-Adali M, Paul A, Wilhelm N, Ziemer G, Wendel HP (2009) Upgrading SELEX technology by using lambda exonuclease digestion for single-stranded DNA generation. Molecules 15:1–11CrossRefGoogle Scholar
  44. 44.
    Citartan M, Tang TH, Tan SC, Gopinath SCB (2011) Conditions optimized for the preparation of single-stranded DNA (ssDNA) employing lambda exonuclease digestion in generating DNA aptamer. World J Microbiol Biotechnol 27:1167–1173CrossRefGoogle Scholar
  45. 45.
    Robinson J, Waller MJ, Parham P, Nd G, Bontrop R, Kennedy LJ, Stoehr P, Marsh SGE (2003) IMGT/HLA and IMGT/MHC: sequence databases for the study of the major histocompatibility complex. Nucleic Acids Res 31:311–314CrossRefGoogle Scholar
  46. 46.
    Hurley CK, Fernandez-Vina M, Middleton D, Noreen H, Schmeckpeper B, Smith A, Tag T (2002) HLA 2004: Immunobiology of the Human MHC. The Proceedings of the 13th IHWC. In: Hansen JA, Dupont B (eds), 2002. IHWG Press, Seattle.Google Scholar
  47. 47.
    Nicholas KB, Nicholas HB, Deerfield DW (1997) GeneDoc: analysis and visualization of genetic variation. EMBNEW NEWS 4:1–4Google Scholar
  48. 48.
    Gonzalez-Galarza FF, Christmas S, Middleton D, Jones AR (2010) Allele frequency net: a database and online repository for immune gene frequencies in worldwide populations. Nucleic Acids Res 39:D913–D919CrossRefGoogle Scholar
  49. 49.
    Cucca F, Frau F, Lampis R, Floris M, Argiolas L, Macis D, Cao A, De Virgiliis S, Congia M (1994) HLA-DQB1*0305 and -DQB1*0304 alleles among Sardinians evolutionary and practical implications for oligotyping. Hum Immunol 40:143–149CrossRefGoogle Scholar
  50. 50.
    Nasef H, Beni V, Ozalp VC, O’Sullivan CK (2010) Cystic fibrosis: a label-free detection approach based on thermally modulated electrochemical impedance spectroscopy. Anal Bioanal Chem 396:2565–2574CrossRefGoogle Scholar
  51. 51.
    Chien F-C, Liu J-S, Su H-J, Kao L-A, Chiou C-F, Chen W-Y, Chen S-J (2004) An investigation into the influence of secondary structures on DNA hybridization using surface plasmon resonance biosensing. Chem Phys Lett 397:429–434CrossRefGoogle Scholar
  52. 52.
    Gao Y, Wolf LK, Georgiadis RM (2006) Secondary structure effects on DNA hybridization kinetics: a solution versus surface comparison. Nucleic Acids Res 34:3370–3377CrossRefGoogle Scholar
  53. 53.
    Kushon SA, Jordan JP, Seifert JL, Nielsen H, Nielsen PE, Armitage BA (2001) Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA Hairpins. J Am Chem Soc 123:10805–10813CrossRefGoogle Scholar
  54. 54.
    Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415CrossRefGoogle Scholar
  55. 55.
    Kelley SO, Boon EM, Barton JK, Jackson NM, Hill MG (1999) Single-base mismatch detection based on charge transduction through DNA. Nucleic Acids Res 27:4830–4837CrossRefGoogle Scholar
  56. 56.
    Allawi HT, SantaLucia J (1998) Thermodynamics of internal C·T mismatches in DNA. Nucleic Acids Res 26:2694–2701CrossRefGoogle Scholar
  57. 57.
    Allawi HT, SantaLucia J (1998) Nearest neighbor thermodynamic parameters for internal G·A mismatches in DNA. Biochemistry 37:2170–2179CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Hamdi Joda
    • 1
  • Valerio Beni
    • 1
    • 2
  • Deirdre Curnane
    • 1
  • Ioanis Katakis
    • 1
  • Noora Alakulppi
    • 3
  • Jukka Partanen
    • 3
  • Kristina Lind
    • 4
  • Linda Strömbom
    • 4
  • Ciara K. O’Sullivan
    • 1
    • 5
  1. 1.Departament d’Enginyeria QuimicaUniversitat Rovira i VirgiliTarragonaSpain
  2. 2.Department of Physics, Chemistry and Biology (IFM)Linköping UniversityLinköpingSweden
  3. 3.Finnish Red Cross Blood ServiceHelsinkiFinland
  4. 4.TATAA Biocenter ABGöteborgSweden
  5. 5.Institucio Catalana de Recerca i Estudis AvançatsBarcelonaSpain

Personalised recommendations