Skip to main content

Advertisement

SpringerLink
Log in

Recombinant antibodies and their use in biosensors

  • Review
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically. As such, antibody quality and suitability for use in immunoassays cannot be readily determined or controlled by human intervention. However, the process through which high-quality antibodies can be obtained has been shortened and streamlined by use of genetic engineering and recombinant antibody techniques. Antibodies that traditionally take several months or more to produce when animals are used can now be developed in a few weeks as recombinant antibodies produced in bacteria, yeast, or other cell types. Typically most immunoassays use two or more antibodies or antibody fragments to detect antigens that are indicators of disease. However, a label-free biosensor, for example, a quartz-crystal microbalance (QCM) needs one antibody only. As such, the cost and time needed to design and develop an immunoassay can be substantially reduced if recombinant antibodies and biosensors are used rather than traditional antibody and assay (e.g. enzyme-linked immunosorbant assay, ELISA) methods. Unlike traditional antibodies, recombinant antibodies can be genetically engineered to self-assemble on biosensor surfaces, at high density, and correctly oriented to enhance antigen-binding activity and to increase assay sensitivity, specificity, and stability. Additionally, biosensor surface chemistry and physical and electronic properties can be modified to further increase immunoassay performance above and beyond that obtained by use of traditional methods. This review describes some of the techniques investigators have used to develop highly specific and sensitive, recombinant antibody-based biosensors for detection of antigens in simple or complex biological samples.

Recombinant antibody and its common surface coupling strategies

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23:1126–1136

    Article  CAS  Google Scholar 

  2. Borrebaeck CA, Wingren C (2011) Recombinant antibodies for the generation of antibody arrays. Methods Mol Biol 785:247–262

    Article  Google Scholar 

  3. Roitt IB, Brostoff J, Male D (1998) Immunology, 5th edn. Mosby International Limited, London

    Google Scholar 

  4. Padlan EA (1994) Anatomy of the antibody molecule. Mol Immunol 31:169–217

    Article  CAS  Google Scholar 

  5. Conroy PJ, Hearty S, Leonard P, O'Kennedy RJ (2009) Antibody production, design and use for biosensor-based applications. Semin Cell Dev Biol 20:10–26

    Article  CAS  Google Scholar 

  6. Kreitman RJ, Wilson WH, Bergeron K, Raggio M, Stetler-Stevenson M, FitzGerald DJ, Pastan I (2001) Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia. N Eng J Med 345:241–247

    Article  CAS  Google Scholar 

  7. Weisser NE, Hall JC (2009) Applications of single-chain variable fragment antibodies in therapeutics and diagnostics. Biotechnol Adv 27:502–520

    Article  CAS  Google Scholar 

  8. O'Brien PM, Aitken R (2002) Methods in Molecular Biology. Humana Press, Totowa, New Jersey

    Google Scholar 

  9. Leong SSJ, Chen WN (2008) Preparing recombinant single chain antibodies. Chemical Engineering Science 63:1401–1414

    Article  CAS  Google Scholar 

  10. Breitling F, Dubel S (1999) Recombinant Antibodies. John Wiley & Sons, Inc., New York and Spektrum Akademischer Verlag

    Google Scholar 

  11. Kontermann R, DUbel S (2001) Antibody engineering, Springer-Verlag Berlin Heidelberg. Spring-Verlag Berlin Heidelberg, New York

    Google Scholar 

  12. Pini A, Viti F, Santucci A, Carnemolla B, Zardi L, Neri P, Neri D (1998) Design and Use of a Phage Display Library. HUMAN ANTIBODIES WITH SUBNANOMOLAR AFFINITY AGAINST A MARKER OF ANGIOGENESIS ELUTED FROM A TWO-DIMENSIONAL GEL. J Biol Chem 273:21769–21776

    Article  CAS  Google Scholar 

  13. Jiang B, Liu WL, Qu H, Meng L, Song S, Ouyang T, Shou C (2005) A Novel Peptide Isolated from a Phage Display Peptide Library with Trastuzumab Can Mimic Antigen Epitope of HER-2. J Biol Chem 280:4656–4662

    Article  CAS  Google Scholar 

  14. Carmen S, Jermutus L (2002) Concepts in antibody phage display. Brief Funct Genomic Proteomic 1:189–203

    Article  CAS  Google Scholar 

  15. Saerens D, Frederix F, Reekmans G, Conrath K, Jans K, Brys L, Huang L, Bosmans E, Maes G, Borghs G, Muyldermans S (2005) Engineering camel single-domain antibodies and immobilization chemistry for human prostate-specific antigen sensing. Anal Chem 77:7547–7555

    Article  CAS  Google Scholar 

  16. De Genst E, Handelberg F, Van Meirhaeghe A, Vynck S, Loris R, Wyns L, Muyldermans S (2004) Chemical basis for the affinity maturation of a camel single domain antibody. J Biol Chem 279:53593–53601

    Article  Google Scholar 

  17. Streltsov VA, Varghese JN, Carmichael JA, Irving RA, Hudson PJ, Nuttall SD (2004) Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor. Proc Natl Acad Sci USA 101:12444–12449

    Article  CAS  Google Scholar 

  18. Liu JL, Anderson GP, Delehanty JB, Baumann R, Hayhurst A, Goldman ER (2007) Selection of cholera toxin specific IgNAR single-domain antibodies from a naive shark library. Mol Immunol 44:1775–1783

    Article  CAS  Google Scholar 

  19. Shao CY, Secombes CJ, Porter AJ (2007) Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library. Mol Immunol 44:656–665

    Article  CAS  Google Scholar 

  20. Van Emon JM (2007) Immunoassay and other bioanalytical Techniques. CRC press, Taylor & Francis Group, Roca Raton

    Google Scholar 

  21. Ohiro Y, Ueda H, Shibata N, Nagamune T (2007) Enhanced fluorescence resonance energy transfer immunoassay with improved sensitivity based on the Fab'-based immunoconjugates. Anal Biochem 360:266–272

    Article  CAS  Google Scholar 

  22. Townsend S, Finlay WJJ, Hearty S, O'Kennedy R (2006) Optimizing recombinant antibody function in SPR immunosensing - The influence of antibody structural format and chip surface chemistry on assay sensitivity. Biosens Bioelectron 22:268–274

    Article  CAS  Google Scholar 

  23. Shen Z, Yan H, Parl FF, Mernaugh RL, Zeng X (2007) Recombinant Antibody Piezoimmunosensors for the Detection of Cytochrome P450 1B1. Anal Chem 79:1283–1289

    Article  CAS  Google Scholar 

  24. Shen Z, Yan H, Zhang Y, Mernaugh RL, Zeng X (2008) Engineering Peptide Linkers for scFv Immunosensors. Anal Chem 80:1910–1917

    Article  CAS  Google Scholar 

  25. Capobianco JA, Shih WY, Yuan QA, Adams GP, Shih WH (2008) Label-free, all-electrical, in situ human epidermal growth receptor 2 detection, Review of Scientific Instruments 79

  26. Stich N, Gandhum A, Matyushin V, Raats J, Mayer C, Alguel Y, Schalkhammer T (2002) Phage display antibody-based proteomic device using resonance-enhanced detection. J Nanosci Nanotechnol 2:375–381

    Article  CAS  Google Scholar 

  27. Emanuel PA, Dang J, Gebhardt JS, Aldrich J, Garber EAE, Kulaga H, Stopa P, Valdes JJ, Dion-Schultz A (2000) Recombinant antibodies: a new reagent for biological agent detection. Biosens Bioelectron 14:751–759

    Article  CAS  Google Scholar 

  28. Ferreira GNM, Encarnação JM, Rosa L, Rodrigues R, Breyner R, Barrento S, Pedro L, Aires da Silva F, Gonçalves J (2007) Recombinant single-chain variable fragment and single domain antibody piezoimmunosensors for detection of HIV1 virion infectivity factor. Biosens Bioelectron 23:384–392

    Article  CAS  Google Scholar 

  29. Liu Y, Liu Y, Mernaugh RL, Zeng XQ (2009) Single chain fragment variable recombinant antibody functionalized gold nanoparticles for a highly sensitive colorimetric immunoassay. Biosens Bioelectron 24:2853–2857

    Article  CAS  Google Scholar 

  30. Anderson GP, Bernstein RD, Swain MD, Zabetakis D, Goldman ER (2010) Binding Kinetics of Antiricin Single Domain Antibodies and Improved Detection Using a B Chain Specific Binder. Anal Chem 82:7202–7207

    Article  CAS  Google Scholar 

  31. Mechaly A, Zahavy E, Fisher A (2008) Development and implementation of a single-chain Fv antibody for specific detection of Bacillus anthracis spores. Appl Environ Microbiol 74:818–822

    Article  CAS  Google Scholar 

  32. Zdobnova TA, Dorofeev SG, Tananaev PN, Vasiliev RB, Balandin TG, Edelweiss EF, Stremovskiy OA, Balalaeva IV, Turchin IV, Lebedenko EN, Zlomanov VP, Deyev SM (2009) Fluorescent immunolabeling of cancer cells by quantum dots and antibody scFv fragment. J Biomed Opt 14:021004

    Article  Google Scholar 

  33. Lu RM, Chang YL, Chen MS, Wu HC (2010) Single chain anti-c-Met antibody conjugated nanoparticles for in vivo tumor-targeted imaging and drug delivery. Biomaterials 32:3265–3274

    Article  Google Scholar 

  34. Mackenzie CR, Clark ID, Evans SV, Hill IE, Macmanus JP, Dubuc G, Bundle DR, Narang SA, Young NM, Szabo AG (1995) Bifunctional Fusion Proteins Consisting of a Single-Chain Antibody and an Engineered Lanthanide-Binding. Immunotechnology 1:139–150

    Article  CAS  Google Scholar 

  35. Singh R, Samant U, Hyland S, Chaudhari PR, Wels WS, Bandyopadhyay D (2007) Target-specific cytotoxic activity of recombinant immunotoxin scFv(MUC1)-ETA on breast carcinoma cells and primary breast tumors. Mol Cancer Ther 6:562–569

    Article  CAS  Google Scholar 

  36. Zhang WY, Yip TC, Kwok CS (2004) Rapid purification of a new humanized single-chain Fv antibody/human interleukin-2 fusion protein reactive against HER2 receptor. Acta Biochim Biophys Sinica 36:707–712

    Article  CAS  Google Scholar 

  37. Wang JQ, Ensor CM, Dubuc GJ, Narang SA, Daunert S (2001) Genetically fused single-chain anti-Salmonella antibody with aequorin: a bioluminescence immunoassay for a Salmonella antigen. Anal Chim Acta 435:255–263

    Article  CAS  Google Scholar 

  38. Mousli M, Goyffon M, Billiald P (1998) Production and characterization of a bivalent single chain Fv alkaline phosphatase conjugate specific for the hemocyanin of the scorpion Androctonus australis. Biochim Biophys Acta-Gen Subj 1425:348–360

    Article  CAS  Google Scholar 

  39. Hu WG, Thompson HG, Alvi AZ, Nagata LP, Suresh MR, Fulton RE (2004) Development of immunofiltration assay by light addressable potentiometric sensor with genetically biotinylated recombinant antibody for rapid identification of Venezuelan equine encephalitis virus. J Immunol Methods 289:27–35

    Article  CAS  Google Scholar 

  40. Le HQA, Sauriat-Dorizon H, Korri-Youssoufi H (2010) Investigation of SPR and electrochemical detection of antigen with polypyrrole functionalized by biotinylated single-chain antibody: A review. Anal Chim Acta 674:1–8

    Article  CAS  Google Scholar 

  41. Horacek J, Garrett SD, Skladal P, Morgan MRA (1998) Characterization of the interactions between immobilized parathion and the corresponding recombinant scFv antibody using a piezoelectric biosensor. Food Agric Immunol 10:363–374

    Article  CAS  Google Scholar 

  42. Ferreira GNM, Encarnacao JM, Rosa L, Rodrigues R, Breyner R, Barrento S, Pedro L, da Silva FA, Goncalves J (2007) Recombinant single-chain variable fragment and single domain antibody piezoimmunosensors for detection of HIV1 virion infectivity factor. Biosens Bioelectron 23:384–392

    Article  CAS  Google Scholar 

  43. Nanduri V, Bhunia AK, Tu SI, Paoli GC, Brewster JD (2007) SPR biosensor for the detection of L-monocytogenes using phage-displayed antibody. Biosens Bioelectron 23:248–252

    Article  CAS  Google Scholar 

  44. Dunne L, Daly S (2004) Development of ELISA and biacore-based immunoassays for the detection of aflatoxin B1 using a genetically engineered dimeric SCFV. Abstr Pap Am Chem Soc 228:U60–U60

    Google Scholar 

  45. Dillon PP, Manning BM, Daly SJ, Killard AJ, O'Kennedy R (2003) Production of a recombinant anti-morphine-3-glucuronide single-chain variable fragment (scFv) antibody for the development of a "real-time" biosensor-based immunoassay. J Immunol Methods 276:151–161

    Article  CAS  Google Scholar 

  46. Cortez-Retamozo V, Lauwereys M, Gh GH, Gobert M, Conrath K, Muyldermans S, De Baetselier P, Revets H (2002) Efficient tumor targeting by single-domain antibody fragments of camels. Int J Cancer 98:456–462

    Article  CAS  Google Scholar 

  47. Desmyter A, Decanniere K, Muyldermans S, Wyns L (2001) Antigen specificity and high affinity binding provided by one single loop of a camel single-domain antibody. J Biol Chem 276:26285–26290

    Article  CAS  Google Scholar 

  48. Ghahroudi MA, Desmyter A, Wyns L, Hamers R, Muyldermans S (1997) Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett 414:521–526

    Article  Google Scholar 

  49. Ladenson RC, Crimmins DL, Landt Y, Ladenson JH (2006) Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragment. Anal Chem 78:4501–4508

    Article  CAS  Google Scholar 

  50. Liu XJ, Lin H, Tang Q, Li C, Yang ST, Wang ZC, Wang CJ, He Q, Cao B, Feng ZQ, Guan XH, Zhu J (2011) Characterization of a Human Antibody Fragment Fab and Its Calcium Phosphate Nanoparticles that Inhibit Rabies Virus Infection with Vaccine, Plos One 6

  51. Lowe J, Maia M, Wakshull E, Siguenza P, Liu P, Lakhani S, Rusit J, Elliott R, Quarmby V (2010) Development of a novel homogenous electrochemiluminescence assay for quantitation of ranibizumab in human serum. J Pharm Biomed Anal 52:680–686

    Article  CAS  Google Scholar 

  52. Ito K, Nishimura W, Maeda M, Gomi K, Inouye S, Arakawa H (2007) Highly sensitive and rapid tandem bioluminescent immunoassay using aequorin labeled Fab fragment and biotinylated firefly luciferase. Anal Chim Acta 588:245–251

    Article  CAS  Google Scholar 

  53. Romanazzo D, Ricci F, Volpe G, Elliott CT, Vesco S, Kroeger K, Moscone D, Stroka J, Van Egmond H, Vehniainen M, Palleschi G (2010) Development of a recombinant Fab-fragment based electrochemical immunosensor for deoxynivalenol detection in food samples. Biosens Bioelectron 25:2615–2621

    Article  CAS  Google Scholar 

  54. Ionescu RE, Gondran C, Bouffier L, Jaffrezic-Renault N, Martelet C, Cosnier S (2010) Label-free impedimetric immunosensor for sensitive detection of atrazine. Electrochim Acta 55:6228–6232

    Article  CAS  Google Scholar 

  55. Lu HH, Kreuzer MP, Takkinen K, Guilbault GG (2007) A recombinant Fab fragment-based electrochemical immunosensor for the determination of testosterone in bovine urine. Biosens Bioelectron 22:1756–1763

    Article  CAS  Google Scholar 

  56. Helali S, Martelet C, Abdelghani A, Maaref MA, Jaffrezic-Renault N (2006) A disposable immunomagnetic electrochemical sensor based on functionalised magnetic beads on gold surface for the detection of atrazine. Electrochim Acta 51:5182–5186

    Article  CAS  Google Scholar 

  57. Halamek J, Makower A, Skladal P, Scheller FW (2002) Highly sensitive detection of cocaine using a piezoelectric immunosensor. Biosens Bioelectron 17:1045–1050

    Article  CAS  Google Scholar 

  58. Harris RD, Luff BJ, Wilkinson JS, Piehler J, Brecht A, Gauglitz G, Abuknesha RA (1999) Integrated optical surface plasmon resonance immunoprobe for simazine detection. Biosens Bioelectron 14:377–386

    Article  CAS  Google Scholar 

  59. Stocklein WFM, Warsinke A, Micheel B, Kempter G, Hohne W, Scheller FW (1998) Diphenylurea hapten sensing with a monoclonal antibody and its Fab fragment: Kinetic and thermodynamic investigations. Anal Chim Acta 362:101–111

    Article  CAS  Google Scholar 

  60. Shen Z, Stryker GA, Mernaugh RL, Yu L, Yan HP, Zeng X (2005) Single-chain fragment variable antibody piezoimmunosensors. Anal Chem 77:797–805

    Article  CAS  Google Scholar 

  61. Shen Z, Mernaugh RL, Yan NP, Yu L, Zhang Y, Zeng X (2005) Engineered recombinant single-chain fragment variable antibody for immunosensors. Anal Chem 77:6834–6842

    Article  CAS  Google Scholar 

  62. Shang Y, Singh R, Mernaugh AR, Chisti MM, Zeng X (2011) Immobilization of a Human Epidermal Growth Factor Receptor 2 Mimotope-Derived Synthetic Peptide on Au and Its Potential Application for Detection of Herceptin in Human Serum by Quartz Crystal Microbalance, Anal. Chem. 83, 8928–8936.

    Article  CAS  Google Scholar 

  63. Vanemon JM, Lopezavila V (1992) Immunochemical Methods - for Environmental-Analysis. Anal Chem 64:A79–A88

    Article  Google Scholar 

  64. Walters RR (1985) Affinity-Chromatography. Anal Chem 57:1102A–1114A

    Article  Google Scholar 

  65. Matarraz SMS, Gonzalez-Gonzalez M, Jara M, Orfao A, Fuentes M (2011) New technologies in cancer. Protein microarrays for biomarker discovery. Clin Transl Oncol 13:156–161

    Article  CAS  Google Scholar 

  66. Borrebaeck CAK, Ohlin M (2002) Antibody evolution beyond Nature. Nat Biotechnol 20:1189–1190

    Article  CAS  Google Scholar 

  67. Zhu XX, Kriegel AM, Boustany CA, Blake DA (2010) Single-Chain Variable Fragment (scFv) Antibodies Optimized for Environmental Analysis of Uranium. Anal Chem 83:3717–3724

    Article  Google Scholar 

  68. Deshpande SS (1996) Enzyme Immunoassays From Concept to Product Development. Chapman & Hall, New York

    Google Scholar 

  69. Legendre D, Soumillion P, Fastrez J (1999) Engineering a regulatable enzyme for homogeneous immunoassays. Nat Biotechnol 17:67–72

    Article  CAS  Google Scholar 

  70. Benito A, Feliu JX, Villaverde A (1996) beta-galactosidase enzymatic activity as a molecular probe to detect specific antibodies. J Biol Chem 271:21251–21256

    Article  CAS  Google Scholar 

  71. Brennan CA, Christianson K, Lafleur MA, Mandecki W (1995) A Molecular Sensor System Based on Genetically-Engineered Alkaline-Phosphatase. Proc Natl Acad Sci USA 92:5783–5787

    Article  CAS  Google Scholar 

  72. Doi N, Yanagawa H (1999) Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution. FEBS Lett 453:305–307

    Article  CAS  Google Scholar 

  73. Ramirez E, Mas JM, Carbonell X, Aviles FX, Villaverde A (1999) Detection of molecular interactions by using a new peptide-displaying bacteriophage biosensor. Biochem Biophys Res Commun 262:801–805

    Article  CAS  Google Scholar 

  74. Schultz J, Lin Y, Sanderson J, Zuo Y, Stone D, Mallett R, Wilbert S, Axworthy D (2000) A Tetravalent Single-chain Antibody-Streptavidin Fusion Protein for Pretargeted Lymphoma Therapy. Cancer Res 60:6663–6669

    CAS  Google Scholar 

  75. Cheung N-KV, Modak S, Lin Y, Guo H, Zanzonico P, Chung J, Zuo Y, Sanderson J, Wilbert S, Theodore LJ, Axworthy DB, Larson SM (2004) Single-Chain Fv-Streptavidin Substantially Improved Therapeutic Index in Multistep Targeting Directed at Disialoganglioside GD2. J Nucl Med 45:867–877

    CAS  Google Scholar 

  76. Shen Z, Cheung N-KV, Zeng X, Single-Chian Fv multimers for label free detection of Disialoganglioside GD2 on cancer cells, (In preparation)

  77. Yan HP, Shen ZH, Mernaugh R, Zeng XQ (2011) Single Chain Fragment Variable Recombinant Antibody as a Template for Fc Sensors. Anal Chem 83:625–630

    Article  CAS  Google Scholar 

  78. Riechmann L, Muyldermans S (1999) Single domain antibodies: comparison of camel VH and camelised human VH domains. J Immunol Methods 231:25–38

    Article  CAS  Google Scholar 

  79. Nuttall SD, Irving RA, Hudson PJ (2000) Immunoglobulin VH domains and beyond: design and selection of single-domain binding and targeting reagents. Curr Pharm Biotechnol 1:253–263

    Article  CAS  Google Scholar 

Download references

Acknowledgement

Xiangqun Zeng is grateful for the support of NIH grant (R21 EB006495) and OU-Beaumont Multidisciplinary Research Awards. Ray Mernaugh was supported in part by the Vanderbilt University Institute for Chemical Biology.

Author information

Authors and Affiliations

  1. Department of Chemistry, Oakland University, 2200 Squirrel Road, Rochester, MI, 48309, USA

    Xiangqun Zeng & Zhihong Shen

  2. Department of Biochemistry, Vanderbilt University, Nashville, TN, 37232, USA

    Ray Mernaugh

Authors
  1. Xiangqun Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhihong Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ray Mernaugh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xiangqun Zeng.

Additional information

Published in the topical collection Biomimetic Recognition Elements for Sensing Applications with guest editor María Cruz Moreno-Bondi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zeng, X., Shen, Z. & Mernaugh, R. Recombinant antibodies and their use in biosensors. Anal Bioanal Chem 402, 3027–3038 (2012). https://doi.org/10.1007/s00216-011-5569-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-011-5569-z

Keywords

Search

Navigation