What laboratory methods are used to detect spike protein and how do they differ in sensitivity and specificity?
Executive summary
Laboratory detection of the SARS‑CoV‑2 spike protein and spike‑specific antibodies uses two broad approaches: direct antigen detection (immunoassays, lateral flow, biosensors) and serological assays that detect antibodies against spike (ELISA, CLIA, neutralization surrogate tests), each trading off speed, limit of detection and cross‑reactivity [1] [2]. Reported sensitivity and specificity vary widely across platforms and depend on timing after infection, antigen design (full S, S1, RBD, S2) and assay format, producing sensitivities from modest (<90%) to very high (~98–100%) in specific contexts [3] [4] [5].
1. ELISA / Chemiluminescent immunoassays: the laboratory workhorses
Enzyme‑linked immunosorbent assays (ELISA) and automated chemiluminescent immunoassays (CLIAs) capture spike (full S, S1, RBD subdomains) to measure binding antibodies and report high specificity but variable sensitivity that rises with time after symptom onset; head‑to‑head comparisons show specificities often >94% while sensitivities range from ~86% to >98% depending on antigen and cutoff selection [3] [6] [1]. Manufacturers and comparative studies demonstrate that ROC‑optimized cutoffs can improve diagnostic accuracy but may shift the balance between false positives and false negatives, especially in low‑prevalence settings where high specificity is critical [1] [6].
2. Neutralization and surrogate neutralization tests: functional but complex
Virus neutralization tests (VNTs) measure functional antibodies that block spike‑ACE2 interactions and are the closest correlate of neutralizing activity, but they require live virus or pseudovirus and biosafety capacity, limiting throughput and speed [4]. Surrogate neutralization assays can approximate neutralizing potency and are useful for clinical research, yet studies warn that sensitivity and specificity vary and that sVNTs may miss neutralization against non‑RBD epitopes or detect non‑functional binding antibodies [4].
3. Lateral flow assays (LFAs) and point‑of‑care antigen tests: speed versus accuracy
Lateral flow formats targeting spike or its subunits are rapid and portable but generally show lower sensitivity and, in some reports, lower specificity than lab assays; meta‑analyses and device evaluations conclude LFAs sacrifice analytical sensitivity for accessibility and speed, making them less reliable for low viral load or late‑seroconversion samples [2] [5]. Device design (sandwich versus competitive), antibody pair selection and antigen folding/glycosylation influence both sensitivity and species‑specificity of spike detection [5].
4. High‑sensitivity biosensors and novel platforms: pushing limits of detection
Field‑effect transistor (FET) graphene sensors, electrochemical immunosensors, SPR chips and quantum‑dot enhanced SPR report very low limits of detection—down to picogram or even femtogram ranges in experimental systems—offering superior analytical sensitivity compared with LFAs and many ELISAs [7] [8] [2]. These platforms often claim excellent specificity through surface chemistry and aptamer/antibody selection, but most reports are proof‑of‑concept with limited clinical validation, so real‑world diagnostic specificity and robustness remain to be proven [7] [8].
5. Single‑molecule and amplification‑coupled protein assays: highest sensitivity, higher complexity
Ultra‑sensitive assays such as Simoa (single molecule array) and proximity ligation approaches convert protein binding into amplified nucleic acid signals to achieve exceptional sensitivity for spike or anti‑spike antibodies, enabling detection earlier or at much lower titers than conventional assays [9] [10]. These methods increase analytical sensitivity but require specialized equipment and careful controls to avoid amplification‑related false positives and matrix interferences [10] [4].
6. How sensitivity and specificity trade off in practice and why timing matters
Across platforms, sensitivity is strongly influenced by specimen timing (antibody maturation >14 days improves serology sensitivity) and antigen choice (RBD and properly folded spike improve species specificity), while specificity depends on antigen uniqueness and assay stringency; several studies report spike‑based serology achieving near‑100% specificity with sensitivity rising from ~86% to >98% with optimal antigens and timing [11] [3] [5]. Hidden agendas include commercial pressure to market rapid POC tests despite lower accuracy and academic incentives to promote novel ultra‑sensitive platforms without broad clinical validation [2] [8].