Chapter Four · failure evidence
What Enzyme-Linked Immunosorbent Assay (ELISA) got wrong, from 53 dissertations
The records describe diverse technical challenges and operational constraints encountered when developing and deploying enzyme linked immunosorbent assays. Reported issues include high background noise from non-specific binding, insufficient analytical sensitivity, high run-to-run variability, and interference from complex sample matrices. These records come from PhD theses at 25 institutions, 2021 to 2026. Each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Non-specific binding and cross-reactivity cause elevated background and false positives
Assays frequently suffer from non-specific plastic adhesion by antibodies and nanobodies as well as cross-reactivity between detection antibodies and same-species capture reagents. Additional background noise stems from whole-cell screening, porous particle entrapment, and automated plate washer artifacts.
Tried and failed
baculovirus particle ELISA using recommended coating dilution applied to antibody specificity screening. Reason: Recommended dilution caused false positive non-specific binding from direct antibody binding to the microplate
Tried and failed
indirect sandwich immunoassay with same-species primary antibodies applied to antigen detection in sandwich ELISA. Reason: secondary detection antibody cross-reacted directly with the capture antibody from the same host species
Tried and failed
ELISA screening of bacterial expression clones applied to single-domain antibody selection. Reason: Screening selected non-specific plastic-binding clones rather than target-specific binders, generating false positives.
Considered and rejected
Considered and rejected: Decided against reliance on commercial ELISA test kits for quantitative neonicotinoid monitoring in environmental mixtures due to high cross-reactivity and matrix false positives.
Considered and rejected
Considered and rejected: Rejected indirect sandwich ELISA using enzyme-labelled secondary antibody because both capture and detection mAbs were murine, causing cross-reactive non-specific binding; biotin-streptavidin was used instead
Considered and rejected
Considered and rejected: Rejected crossed immunoelectrophoresis and ELISA for routine species identification because CIE is too labor-intensive and ELISA showed minor cross-reactivity requiring dilutions.
A study of treponemal antigens · Virginia Tech
Considered and rejected
Considered and rejected: Rejected ELISA-based screening of bacterial supernatants for phage display candidates due to high rates of false positives from plastic-binding nanobodies, switching to flow cytometry.
Considered and rejected
Considered and rejected: Rejected direct ELISA quantification on intact mesoporous particles due to non-specific pore entrapment of assay antibodies.
Entwicklung von Nanopartikeln für das Implant-directed Magnetic Drug Targeting · Leibniz Universität Hannover Repository
Tried and failed
whole-cell ELISA for monoclonal antibody screening applied to bacterial antigen-specific human monoclonal antibodies. Outcome: no signal. Reason: produced high non-specific background binding to negative controls and assay surfaces
Identification of pneumococcal vaccine antigens using Reverse Vaccinology 2.0 · Imperial
Considered and rejected
Considered and rejected: Rejected using automated 96-well plate washers for low-dilution ELISA due to significantly higher non-specific background OD noise compared to manual multichannel washing.
Target concentrations falling below detection limits lead to insufficient analytical sensitivity
Standard ELISA platforms often lack the limit of detection required to quantify low-abundance proteins or attomolar cytokine concentrations in biofluids. Samples frequently fall below reporting thresholds, leading to assay exclusion or failure when diluted for testing.
Considered and rejected
Considered and rejected: Commercial ELISA immunoassays for IFN quantification were rejected due to insufficient sensitivity for attomolar levels and false positives caused by heterophilic rheumatoid factor
ANTIGEN-PRESENTING CELLS AND INTERFERONS IN RHEUMATOID AND JUVENILE IDIOPATHIC ARTHRITIS · DalSpace
Considered and rejected
Considered and rejected: Retired sandwich ELISA for IFN-alpha in favor of multiplex ELISA due to poor sensitivity in detecting low cytokine concentrations
Toll-like Receptor 7 modulation of the immune response in seasonal allergic rhinitis · Imperial
Tried and failed
ELISA detection of shed circulating biomarker applied to subcutaneous tumor-bearing mouse serum. Outcome: no signal. Reason: Shed antigen levels in serum were below detection limit in the subcutaneous xenograft model
Antibody-Based Theranostics in Ovarian Cancer: Targeting MUC16 For Diagnosis and Radioimmunotherapy · Cornell
Considered and rejected
Considered and rejected: Discarded sample dilution in final plasma ELISA due to very low baseline target protein concentrations.
Analyse von potenziellen Protein-Biomarkern in einer Glioblastom-Patientenkohorte Analysis of potential protein biomarkers in a glioblastoma patient cohort · open_UMR Marburg DSpace 10.0
Tried and failed
multiplex ELISA profiling applied to unconcentrated conditioned cell culture media. Outcome: no signal. Reason: analyte concentrations fell below the assay detection threshold
Targeting the aging of tumor-associated neutrophils in lung cancer · EPFL
Lost to a baseline
27 serum samples out of 189 total MPN patient and control samples were excluded from ELISA analysis due to falling below the assay detection threshold (2 pg/ml).
Lost to a baseline
HRP2 ELISA sensitivity (3.3 ng/mL) was roughly an order of magnitude worse than the commercial NxTek Eliminate Malaria P.f. assay (80 pg/mL)
Considered and rejected
Considered and rejected: Commercial ELISA bovine prolactin kit rejected for future work in favor of radioimmunoassay (RIA) due to lack of sensitivity.
Considered and rejected
Considered and rejected: Rejected 1:100 serum dilution for Zonulin ELISA due to over-dilution preventing reliable detection; optimized to 1:30
Mitigating Factors in Inflammatory Bowel Disease Impacting Symptomatology And Quality Of Life · Research Repository UCD
Considered and rejected
Considered and rejected: Rejected standard unmodified Bio-Techne DuoSet ELISA for screening due to high limit of detection (321 pg/mL)
High assay variability and poor signal reproducibility compromise measurement reliability
Assays repeatedly exhibit high coefficients of variation, fluctuating optical density readings, and poor reproducibility between experimental runs. Performance also degrades across distinct antibody clones, sequence variants, or corrupted standard curves.
Tried and failed
competitive ELISA self-ladder assay applied to monoclonal antibody quantification. Outcome: did not generalise. Reason: assay was not robust across different monoclonal antibody clones, failing for majority of tested antibodies
Single Platelet Biomechanics For Translational and Clinical Investigation · Georgia Tech
Tried and failed
direct ELISA for patient plasma antibody detection applied to anti-integrin antibodies in patient plasma. Outcome: unstable. Reason: yielded high variability and inconsistent optical density values between repeated experiments
Single Platelet Biomechanics For Translational and Clinical Investigation · Georgia Tech
Tried and failed
split-GFP and anti-epitope tag ELISA quantification applied to diverse engineered enzyme variants. Outcome: did not generalise. Reason: signal proved inconsistent across diverse sequence variants compared to total protein assay
Tried and failed
using commercial antibody as negative assay control applied to baculovirus particle polyspecificity ELISA. Outcome: unstable. Reason: the reference molecule did not produce consistent low non-specificity baseline readings across assay runs
Considered and rejected
Considered and rejected: Rejected using an ADA2 ELISA for patient plasma analysis due to lack of assay reproducibility.
Considered and rejected
Considered and rejected: Rejected continuing the sandwich ELISA development for canine copeptin due to persistent lack of reproducible signal, choosing instead to suggest a competitive ELISA format for future attempts.
Assessment of Copeptin and Adrenomedullin in Dogs · Research Repository UCD
Considered and rejected
Considered and rejected: Exclusion of Human IL-6 ELISA results from analysis due to standard curve failure/contamination.
Considered and rejected
Considered and rejected: Rejected the use of the Covance/LabCorp sandwich ELISA assay for total CSF alpha-synuclein in future studies due to high assay variability and lack of specificity for aggregated pathogenic isoforms.
Tried and failed
initial rate kinetic ELISA quantification applied to spectrophotometric optical density measurement. Outcome: unstable. Reason: high coefficients of variation and inconsistent measurements during early timepoints (0-2 minutes)
Ultraviolet disinfection of schistosome cercariae in water · Imperial
Considered and rejected
Considered and rejected: Abandoned ELISA-based measurement of phosphorylated ATF2 in favor of the ADP-Glo luminescent assay due to persistent lack of kinase dependence and high variance.
Inhibition of Mitogen-Activated Protein Kinase p38α by Rooperol and Analogues · TXST Digital Repository
Sample matrix components and calibration discrepancies create quantification errors
Unpurified matrices such as cell culture media and fish tissue extract cause severe matrix interference and under-recovery. Discrepancies between standard curve calibrators, universal reagents, or empty non-functional viral particles distort accurate concentration measurements.
Tried and failed
indirect ELISA with universal secondary reagents applied to cross-species antibody affinity comparison. Reason: secondary reagents exhibited differential binding affinities across host species, biasing endpoint titer comparisons
USING SEROLOGY TO UNDERSTAND BAT-VIRUS INTERACTIONS ACROSS SCALES · Cornell
Tried and failed
commercial sandwich ELISA quantification applied to target protein detection in mouse plasma. Outcome: no signal. Reason: antibodies lacked specificity and could not distinguish wild-type from knockout mouse plasma
Lentiviral gene therapy for the production of systemic proteins · Imperial
Considered and rejected
Considered and rejected: Rejected combining non-IBL ELISA datasets (e.g., Cusabio, Cloud-Clone, YH Bioresearch, Eastbiopharm) into the core statistical meta-analysis due to severe cross-assay epitope and calibration discrepancies (up to 1000-fold).
Considered and rejected
Considered and rejected: Discontinued relying on commercial ELISA kits (Eurofins Abraxis) for quantifying DA in fish tissue extracts due to massive matrix interference/under-recovery compared to LC-MS.
Assessing the impact of domoic acid exposure on the zebrafish (Danio rerio) brain across life stages · Woods Hole
Considered and rejected
Considered and rejected: ELISA for initial high-throughput construct screening in mammalian expression was rejected due to cell culture media interfering with sensitive detection, adopting quantitative Western blots instead.
The Degreaser: spot cleaning sequences to maximize protein secretion · ResearchWorks
Considered and rejected
Considered and rejected: Rejected using pure mAb stock solutions for total mAb ELISA standard curves, which inflated signals and led to underreporting total mAb concentrations in ADC samples
Pharmacokinetic and Pharmacodynamic Investigation of Monomethyl Auristatin E (MMAE)-Based Antibody-Drug Conjugates (ADCs) · DSpace at SUNY Buffalo
Considered and rejected
Considered and rejected: Measuring TNFα in serum samples was considered and rejected because available ELISA protocols were validated only for live cell cultures, risking compromised serum results.
Investigating the effect of dexamethasone on productivity, immune function, and behavior in dystocic periparturient dairy cattle · Virginia Tech
Considered and rejected
Considered and rejected: ELISA (p24) and qPCR titration methods were rejected in favor of functional cell culture titration because they overestimated baboon-pseudotyped viral titers due to non-functional/empty particles.
Gene therapy approaches to promote fetal hemoglobin production for the treatment of β-hemoglobinopathies · Publikationssystem UB Tuebingen
Assays underperform in diagnostic accuracy and efficiency compared to alternative methods
Specific ELISA kits demonstrated inferior clinical sensitivity, area under the curve, or sample discrimination when benchmarked against other serological or molecular assays. In other settings, assays were discontinued due to multiplexing complexity, elevated reagent costs, or false positive signals in early fermentations.
Lost to a baseline
Well diffusion bioassay remained superior to ELISA for detecting active bacteriocin production, as ELISA falsely signaled high presence in early non-inhibitory fermentations.
Polyclonal antibodies against the bacteriocin propionicin PLG-1: production, characterization, and assay development · Iowa State
Lost to a baseline
P30 indirect ELISA completely lost to crude extract indirect ELISA and commercial IDVet kit, failing to separate positive from negative samples (SD 0.101 vs 1.246).
Lost to a baseline
CIVTEST SUIS PRRS A/S PLUS (ELISA 2) achieved only 64.9% positivity rate and 0.916 AUC, significantly underperforming serum baseline ELISA S (100%, 1.000 AUC) and IDEXX OF ELISA 1 (100%, 1.000 AUC).
Advances in oral fluid antibody-based surveillance for porcine reproductive and respiratory syndrome virus (PRRSV) · Iowa State
Lost to a baseline
Euroimmun IgG ELISA achieved only 65% sensitivity compared to 90% for Wantai SARS-CoV-2 Total Antibody ELISA.
COVID-9 Detection Strategies: Recent Advances and Future Prospects · YorkSpace
Lost to a baseline
Planar array ELISA (SP-X) measurements of IFN-γ failed to predict HIV rebound, whereas MOSAIC IFN-γ measurements succeeded.
Single-molecule diagnostics to support curative interventions for tuberculosis and HIV · MIT
Considered and rejected
Considered and rejected: Rejected using ELISA-measured proteins in final diagnostic signature due to multiplexing complexity and higher cost compared to Luminex.
Lost to a baseline
Luminex assay (88.8% sensitivity) and single-antigen ELISAs (90.7% sensitivity) were both outperformed by VirScan on matched patient samples.
Considered and rejected
Considered and rejected: Rejected ELISA for routine immunoglobulin screening in favor of radial immunodiffusion (RID) plates due to high reagent cost and low sample throughput.
The use of gnotobiotic SCID pigs for improving humanization methods · Iowa State
Epitope masking and structural alterations disrupt antibody target binding
Recombinant subunits often lack native conformational epitopes, preventing recognition by target antibodies. In addition, N-terminal affinity tags introduce steric interference that blocks antibody binding and whole-cell formats fail to distinguish surface from subcapsular targets.
Tried and failed
recombinant protein ELISA applied to detecting cross-reactive antibodies. Outcome: no signal. Reason: solubilized recombinant subunits lack conformational epitopes preserved in native membrane-bound trimeric surface proteins
Defining determinants of viral membrane protein immunogenicity · Imperial
Tried and failed
N-terminal affinity tagging of recombinant protein applied to homodimeric protein ELISA quantification. Reason: Affinity tags at the N-terminus interfered with antibody binding and quantification in sandwich ELISA
Characterisation of Prodomains in the GDNF Family · Cambridge
Considered and rejected
Considered and rejected: Rejected whole-cell ELISA in favour of flow cytometry for antibody screening because whole-cell ELISA failed to discriminate between surface-exposed and subcapsular antigens.
Identification of pneumococcal vaccine antigens using Reverse Vaccinology 2.0 · Imperial
Considered and rejected
Considered and rejected: Rejected adding N-terminal tags to GDF15 H202D/WT heterodimers because affinity tags interfered with ELISA quantification
Characterisation of Prodomains in the GDNF Family · Cambridge
Left open by the authors
Problems the authors named and did not get to.
Left open
Investigate why negative serum sample NA25505 produced an unexpected signal spike in the EENZ1-IgM ELISA assay. Blocker: Requires access to the specific physical serum sample NA25505 and a wet lab with ELISA reagents.
Left open
Investigate antibody-protein interactions to improve immunoassay selectivity, specificity, and numerical reproducibility for cryptographic key generation. Blocker: Requires a wet lab and physical reagents to perform wet-bench ELISA experiments.
Information Concealment by Use of Antibody-Antigen Interactions · Texas Tech
Left open
Evaluate the VP27 indirect ELISA across a larger swine serum sample size to determine sensitivity and functionality. Blocker: Requires a wet lab, swine serum samples, and biochemical reagents to perform the ELISA assays.
Left open
Investigate specific anti-cardiac autoantibodies and their localisation using autoantibody-specific ELISA and immunofluorescence co-staining. Blocker: Requires wet lab facilities, tissue samples, and specialized assay reagents (ELISA, immunofluorescence).
The effect of systemic autoimmunity on post myocardial infarction electrophysiological remodelling · Imperial
Left open
Perform immunological assays in animal models to confirm the Th2-biased IgG1 versus IgG2a antibody response to iVAX-derived conjugate vaccines. Blocker: Requires wet-lab facilities, animal vaccination models, and immunological serum testing (e.g., ELISA).
Left open
Evaluate cross-reactivity of standard anti-HEV ELISAs against HEV-C antibodies and develop HEV-C-specific serological diagnostic assays. Blocker: Requires wet lab facilities, biological reagents, and serum samples containing HEV-C antibodies.
Epidemiology of Hepatitis E in Guinea: a One Health Approach · Publikationssystem UB Tuebingen
Left open
Evaluate the breast cancer biomarker panel against other cancer types, inflammatory/autoimmune conditions, and in larger non-ER+ cohorts using droplet digital ELISA. Blocker: Requires wet lab facilities, clinical patient blood samples, microfluidic droplet generation apparatus, and ddELISA reagents.
Single Molecule Detection of Blood Biomarkers for Disease Diagnostics · Harvard
Left open
Develop an ELISA assay using the Woodman et al. format with additional MCM protein markers and lateral flow dipstick compatibility. Blocker: Requires wet lab equipment, biological reagents, antibodies, and patient/clinical samples.
Towards the molecular diagnosis of bladder and colorectal cancer : Analysis of CD44 exon splicing · Cranfield
Left open
Confirm IL-36RA protein secretion via ELISA in healthy and COPD monocytes, GM-MDM, and tissue macrophages. Blocker: Requires wet-lab facilities, patient tissue/blood samples, and specific antibodies for ELISA assays.
Investigating IL-36 receptor signalling in COPD: a novel drug target · Imperial
Left open
Evaluate mean titre of negative cows as an indicator for ELISA variability and determine the optimal S/P ratio range to average. Blocker: Requires private dairy farm milk ELISA test-kit and laboratory diagnostic records.
Development and evaluation of predictive models for real-time decision making on dairy farms · University of Nottingham Repository
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