{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,5]],"date-time":"2026-11-05T21:51:25Z","timestamp":1793915485149,"version":"4.8.0"},"reference-count":37,"publisher":"American Chemical Society (ACS)","issue":"23","license":[{"start":{"date-parts":[[2020,11,13]],"date-time":"2020-11-13T00:00:00Z","timestamp":1605225600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_ccbyncnd_termsofuse.html"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2020,12,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Sandwich lateral flow immunoassays (LFIAs) are limited at high antigen concentrations by the hook effect, leading to a contradictory decrease in the test line (T) intensity and false-negative results. The hook effect is mainly associated with the loss of T, and research focuses on minimizing this effect. Nevertheless, the control line (C) intensity is also affected at higher analyte concentrations, undesirably influencing the T\/C ratio in LFIA readers. The main aim of this work is to identify and understand these high antigen concentration effects in order to develop ubiquitous strategies to interpret and mitigate such effects. Four complementary experiments were performed: performance assessment of three different allergen LFIAs (two for hazelnut, one for peanut) over 0.075\u20133500 ppm, LFIAs with C only, surface plasmon resonance (SPR) binding experiments on the immobilized control antibody, and smartphone video recording of LFIAs during their development. As antigen concentrations increase, the C signal decreases before the T signal does, suggesting that distinct mechanisms underlie these intensity reductions. Reduced binding at the C occurred even in the absence of T, so the upfront T does not explain the loss of C. SPR confirmed that the C antibody favors binding with free labeled antibody compared with a labeled antibody\u2013analyte complex, indicating that in antigen excess, binding is reduced at C before T. Finally, a smartphone-based video method was developed for dynamically monitoring the LFIA development in real time to distinguish between different concentration-dependent effects. Digitally analyzing the data allows clear differentiation of highly positive samples and false-negative samples and can indicate whether the LFIA is in the dynamic working range or at critically high concentrations. The aim of this work is to identify and understand such high antigen concentration effects in order to develop ubiquitous strategies to interpret and mitigate such effects.<\/jats:p>","DOI":"10.1021\/acs.analchem.0c03740","type":"journal-article","created":{"date-parts":[[2020,11,13]],"date-time":"2020-11-13T08:11:07Z","timestamp":1605255067000},"page":"15587-15595","source":"Crossref","is-referenced-by-count":154,"title":["Unraveling the Hook Effect: A Comprehensive Study of High Antigen Concentration Effects\nin Sandwich Lateral Flow Immunoassays"],"prefix":"10.1021","volume":"92","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2145-2386","authenticated-orcid":true,"given":"Georgina M. S.","family":"Ross","sequence":"first","affiliation":[{"name":"Wageningen University & Research , , P.O. Box 230, ,","place":["Wageningen, The Netherlands, 6700 AE"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Filippini","sequence":"additional","affiliation":[{"name":"IFM\u2500Linko\u0308ping University , , ,","place":["Linko\u0308ping, Sweden, S58183"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4634-0249","authenticated-orcid":true,"given":"Michel W. F.","family":"Nielen","sequence":"additional","affiliation":[{"name":"Wageningen University & Research , , P.O. Box 230, ,","place":["Wageningen, The Netherlands, 6700 AE"]},{"name":"Wageningen University , , Stippeneng 4 , ,","place":["Wageningen, The Netherlands, 6708 WE"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2870-9084","authenticated-orcid":true,"given":"Gert IJ.","family":"Salentijn","sequence":"additional","affiliation":[{"name":"Wageningen University & Research , , P.O. Box 230, ,","place":["Wageningen, The Netherlands, 6700 AE"]},{"name":"Wageningen University , , Stippeneng 4 , ,","place":["Wageningen, The Netherlands, 6708 WE"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2020,11,13]]},"reference":[{"key":"2026090315382555600_cit1","doi-asserted-by":"publisher","first-page":"5353","DOI":"10.1007\/s00216-018-0989-7","article-title":"Consumer-friendly\nfood allergen detection: moving towards smartphone-based immunoassays","volume":"410","author":"Ross","year":"2018","journal-title":"Anal. Bioanal. Chem."},{"key":"2026090315382555600_cit2","doi-asserted-by":"publisher","first-page":"2311","DOI":"10.1021\/acs.analchem.5b04222","article-title":"Investigation of Reagent Delivery Formats in a Multivalent Malaria\nSandwich Immunoassay and Implications for Assay Performance","volume":"88","author":"Liang","year":"2016","journal-title":"Anal. Chem."},{"key":"2026090315382555600_cit3","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1016\/j.jim.2009.06.003","article-title":"The fundamental\nflaws of immunoassays and potential\nsolutions using tandem mass spectrometry","volume":"347","author":"Hoofnagle","year":"2009","journal-title":"J.\nImmunol. Methods"},{"key":"2026090315382555600_cit4","doi-asserted-by":"publisher","first-page":"209","DOI":"10.1016\/0003-2697(74)90347-9","article-title":"Measurement of serum\nferritin by a 2-site immunoradiometric assay","volume":"61","author":"Miles","year":"1974","journal-title":"Anal. Biochem."},{"key":"2026090315382555600_cit5","doi-asserted-by":"publisher","first-page":"184","DOI":"10.1007\/bf03347299","article-title":"Pituitary\nadenomas secreting large amounts of prolactin may give false low values\nin immunoradiometric assays. The hook effect","volume":"21","author":"Petakov","year":"1998","journal-title":"J. Endocrinol. Invest."},{"key":"2026090315382555600_cit6","doi-asserted-by":"publisher","first-page":"484","DOI":"10.1016\/j.snb.2016.12.008","article-title":"Multiple test zones\nfor improved detection performance in lateral flow assays","volume":"243","author":"Hu","year":"2017","journal-title":"Sens. Actuators, B"},{"key":"2026090315382555600_cit7","doi-asserted-by":"publisher","first-page":"844","DOI":"10.1039\/c9lc00967a","article-title":"Glycation ratio determination through\nsimultaneous detection of human serum albumin and glycated albumin\non an advanced lateral flow immunoassay sensor","volume":"20","author":"Ki","year":"2020","journal-title":"Lab Chip"},{"key":"2026090315382555600_cit8","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/978-1-59745-240-3_1","article-title":"Evolution\nin Lateral Flow\u2013Based Immunoassay Systems","volume-title":"Lateral Flow Immunoassay","author":"O\u2019Farrell","year":"2009"},{"key":"2026090315382555600_cit9","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0169012","article-title":"Field-Usable Lateral Flow Immunoassay for the Rapid\nDetection of White Spot Syndrome Virus (WSSV)","volume":"12","author":"Kulabhusan","year":"2017","journal-title":"PLoS One"},{"key":"2026090315382555600_cit10","doi-asserted-by":"publisher","first-page":"115934","DOI":"10.1016\/j.trac.2020.115934","article-title":"Smartphone-based\noptical assays in the food safety field","volume":"129","author":"Nelis","year":"2020","journal-title":"TrAC,\nTrends Anal. Chem."},{"key":"2026090315382555600_cit11","doi-asserted-by":"publisher","first-page":"4812","DOI":"10.3390\/s19214812","article-title":"Analyte\nQuantity Detection from Lateral Flow Assay Using a Smartphone","volume":"19","author":"Foysal","year":"2019","journal-title":"Sensors"},{"key":"2026090315382555600_cit12","doi-asserted-by":"publisher","first-page":"593","DOI":"10.1007\/s00216-011-5340-5","article-title":"Amorphous carbon nanoparticles: a\nversatile label for rapid diagnostic (immuno)assays","volume":"402","author":"Posthuma-Trumpie","year":"2012","journal-title":"Anal. Bioanal. Chem."},{"key":"2026090315382555600_cit13","doi-asserted-by":"publisher","first-page":"2673","DOI":"10.3390\/s17112673","article-title":"Optimized\nLateral Flow Immunoassay Reader for the Detection\nof Infectious Diseases in Developing Countries","volume":"17","author":"Pilavaki","year":"2017","journal-title":"Sensors"},{"key":"2026090315382555600_cit14","doi-asserted-by":"publisher","first-page":"5095","DOI":"10.1021\/acs.analchem.7b00638","article-title":"Mitigating the Hook\nEffect in Lateral Flow Sandwich Immunoassays Using Real-Time Reaction\nKinetics","volume":"89","author":"Rey","year":"2017","journal-title":"Anal. Chem."},{"key":"2026090315382555600_cit15","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkw136","article-title":"Selection of DNA\nnanoparticles with preferential binding to aggregated protein target","volume":"44","author":"Ruff","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"2026090315382555600_cit16","doi-asserted-by":"publisher","first-page":"2","DOI":"10.3390\/bios9010002","article-title":"Multiplex Lateral Flow Immunoassay:\nAn Overview of\nStrategies towards High-throughput Point-of-Need Testing","volume":"9","author":"Anfossi","year":"2018","journal-title":"Biosensors"},{"key":"2026090315382555600_cit17","doi-asserted-by":"publisher","first-page":"3189","DOI":"10.7150\/thno.24034","article-title":"A hook\neffect-free immunochromatographic assay (HEF-ICA) for measuring the\nC-reactive protein concentration in one drop of human serum","volume":"8","author":"Oh","year":"2018","journal-title":"Theranostics"},{"key":"2026090315382555600_cit18","doi-asserted-by":"publisher","first-page":"269","DOI":"10.3390\/mi11030269","article-title":"Recent\nAdvances of Fluid Manipulation\nTechnologies in Microfluidic Paper-Based Analytical Devices (\u03bcPADs)\ntoward Multi-Step Assays","volume":"11","author":"Kim","year":"2020","journal-title":"Micromachines"},{"key":"2026090315382555600_cit19","doi-asserted-by":"publisher","first-page":"17319","DOI":"10.1038\/s41598-018-35694-9","article-title":"Development a stacking pad design for enhancing the\nsensitivity of lateral flow immunoassay","volume":"8","author":"Tsai","year":"2018","journal-title":"Sci.\nRep."},{"key":"2026090315382555600_cit20","doi-asserted-by":"publisher","first-page":"940","DOI":"10.1021\/acs.analchem.9b03792","article-title":"Transposing\nLateral Flow Immunoassays to Capillary-Driven Microfluidics Using\nSelf-Coalescence Modules and Capillary-Assembled Receptor Carriers","volume":"92","author":"Hemmig","year":"2020","journal-title":"Anal. Chem."},{"key":"2026090315382555600_cit21","doi-asserted-by":"publisher","first-page":"127408","DOI":"10.1016\/j.snb.2019.127408","article-title":"Hook effect detection\nand detection-range-controllable one-step immunosensor for inflammation\nmonitoring","volume":"304","author":"Shin","year":"2020","journal-title":"Sens. Actuators, B"},{"key":"2026090315382555600_cit22","doi-asserted-by":"publisher","first-page":"285","DOI":"10.1016\/j.bios.2014.04.032","article-title":"A three-line\nlateral flow assay strip for the measurement of C-reactive protein\ncovering a broad physiological concentration range in human sera","volume":"61","author":"Oh","year":"2014","journal-title":"Biosens. Bioelectron."},{"key":"2026090315382555600_cit23","doi-asserted-by":"publisher","first-page":"128756","DOI":"10.1016\/j.snb.2020.128756","article-title":"Development of an\nexperimental method to overcome the\nhook effect in sandwich-type lateral flow immunoassays guided by computational\nmodelling","volume":"324","author":"Sathishkumar","year":"2020","journal-title":"Sens. Actuators, B"},{"key":"2026090315382555600_cit24","doi-asserted-by":"publisher","first-page":"119","DOI":"10.1007\/s00216-009-2720-1","article-title":"Biosensor immunoassay\nfor traces\nof hazelnut protein in olive oil","volume":"395","author":"Bremer","year":"2009","journal-title":"Anal. Bioanal.\nChem."},{"key":"2026090315382555600_cit25","doi-asserted-by":"publisher","first-page":"130","DOI":"10.3390\/bios8040130","article-title":"Rapid Antibody Selection Using Surface\nPlasmon Resonance\nfor High-Speed and Sensitive Hazelnut Lateral Flow Prototypes","volume":"8","author":"Ross","year":"2018","journal-title":"Biosensors"},{"key":"2026090315382555600_cit26","doi-asserted-by":"publisher","first-page":"143","DOI":"10.3390\/bios9040143","article-title":"A Critical Comparison\nbetween Flow-through and Lateral Flow Immunoassay Formats for Visual\nand Smartphone-Based Multiplex Allergen Detection","volume":"9","author":"Ross","year":"2019","journal-title":"Biosensors"},{"key":"2026090315382555600_cit27","doi-asserted-by":"publisher","first-page":"126019","DOI":"10.1016\/j.foodchem.2019.126019","article-title":"Thermal processing\nof peanut impacts detection by current analytical techniques","volume":"313","author":"Marsh","year":"2020","journal-title":"Food Chem."},{"key":"2026090315382555600_cit28","doi-asserted-by":"publisher","first-page":"8079561","DOI":"10.34133\/2019\/8079561","article-title":"Geometric Flow Control\nLateral Flow Immunoassay Devices\n(GFC-LFIDs): A New Dimension to Enhance Analytical Performance","volume":"2019","author":"Eriksson","year":"2019","journal-title":"Research"},{"key":"2026090315382555600_cit29","doi-asserted-by":"publisher","first-page":"671","DOI":"10.1038\/nmeth.2089","article-title":"NIH Image\nto ImageJ: 25 years of\nimage analysis","volume":"9","author":"Schneider","year":"2012","journal-title":"Nat. Methods"},{"key":"2026090315382555600_cit30","doi-asserted-by":"publisher","first-page":"83","DOI":"10.1007\/s00216-009-2944-0","article-title":"Allergen\nimmunoassays-considerations for use of naturally incurred standards","volume":"395","author":"Taylor","year":"2009","journal-title":"Anal. Bioanal. Chem."},{"key":"2026090315382555600_cit31","doi-asserted-by":"publisher","first-page":"89","DOI":"10.1016\/j.ab.2003.07.011","article-title":"A mathematical model\nof lateral flow bioreactions applied\nto sandwich assays","volume":"322","author":"Qian","year":"2003","journal-title":"Anal. Biochem."},{"key":"2026090315382555600_cit32","doi-asserted-by":"publisher","first-page":"42","DOI":"10.1177\/0300985813505879","article-title":"When Tissue Antigens and Antibodies\nGet Along:Revisiting\nthe Technical Aspects of Immunohistochemistry\u2500The Red, Brown,\nand Blue Technique","volume":"51","author":"Ramos-Vara","year":"2014","journal-title":"Vet. Pathol."},{"key":"2026090315382555600_cit33","doi-asserted-by":"publisher","first-page":"1390","DOI":"10.1002\/jps.20079","article-title":"Challenges in the development\nof\nhigh protein concentration formulations","volume":"93","author":"Shire","year":"2004","journal-title":"J.\nPharm. Sci."},{"key":"2026090315382555600_cit34","doi-asserted-by":"publisher","first-page":"3475","DOI":"10.1039\/c1em10658a","article-title":"Rapid one-step\nassays for on-site\nmonitoring of mouse and rat urinary allergens","volume":"13","author":"Koets","year":"2011","journal-title":"Journal of Environmental Monitoring"},{"key":"2026090315382555600_cit35","doi-asserted-by":"publisher","first-page":"559","DOI":"10.4155\/bio-2017-0271","article-title":"Surface plasmon resonance as a tool\nfor ligand-binding assay reagent characterization in bioanalysis of\nbiotherapeutics","volume":"10","author":"Duo","year":"2018","journal-title":"Bioanalysis"},{"key":"2026090315382555600_cit36","doi-asserted-by":"publisher","first-page":"193","DOI":"10.4155\/bio-2016-0204","article-title":"Characterization\nof labeled reagents in ligand-binding assays by a surface plasmon\nresonance biosensor","volume":"9","author":"Duo","year":"2017","journal-title":"Bioanalysis"},{"key":"2026090315382555600_cit37","doi-asserted-by":"publisher","first-page":"13493","DOI":"10.1073\/pnas.94.25.13493","article-title":"Computer-based analysis of the binding steps in protein complex formation","volume":"94","author":"Bray","year":"1997","journal-title":"Proc. Natl. Acad. Sci. U.S.A."}],"container-title":["Analytical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.analchem.0c03740","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/92\/23\/15587\/67369430\/acs.analchem.0c03740.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/92\/23\/15587\/67369430\/acs.analchem.0c03740.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T19:38:36Z","timestamp":1788464316000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancham\/article\/92\/23\/15587\/828078\/Unraveling-the-Hook-Effect-A-Comprehensive-Study"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,11,13]]},"references-count":37,"journal-issue":{"issue":"23","published-online":{"date-parts":[[2020,11,13]]},"published-print":{"date-parts":[[2020,12,1]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.analchem.0c03740","relation":{},"ISSN":["0003-2700","1520-6882"],"issn-type":[{"value":"0003-2700","type":"print"},{"value":"1520-6882","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,11,13]]}}}