Cross-Platform Comparison of Highly Sensitive Immunoassays for Inflammatory Markers in a COVID-19 Cohort.

A variety of commercial platforms are available for the simultaneous detection of multiple cytokines and associated proteins, often employing Ab pairs to capture and detect target proteins. In this study, we comprehensively evaluated the performance of three distinct platforms: the fluorescent bead-based Luminex assay, the proximity extension-based Olink assay, and a novel proximity ligation assay platform known as Alamar NULISAseq. These assessments were conducted on human serum samples from the National Institutes of Health IMPACC study, with a focus on three essential performance metrics: detectability, correlation, and differential expression. Our results reveal several key findings. First, the Alamar platform demonstrated the highest overall detectability, followed by Olink and then Luminex. Second, the correlation of protein measurements between the Alamar and Olink platforms tended to be stronger than the correlation of either of these platforms with Luminex. Third, we observed that detectability differences across the platforms often translated to differences in differential expression findings, although high detectability did not guarantee the ability to identify meaningful biological differences. Our study provides valuable insights into the comparative performance of these assays, enhancing our understanding of their strengths and limitations when assessing complex biological samples, as exemplified by the sera from this COVID-19 cohort.

Cancer Immunosurveillance Caught in the Act.

The role of the immune system in the control of cancer formation and progression has been a matter of considerable debate over many years. In this issue of Immunity, Mlecnik et al. (2016) show the importance of immunosurveillance in controlling tumors in a series of microsatellite-instable human colon carcinomas.

Integrative Analyses of Colorectal Cancer Show Immunoscore Is a Stronger Predictor of Patient Survival Than Microsatellite Instability.

Microsatellite instability in colorectal cancer predicts favorable outcomes. However, the mechanistic relationship between microsatellite instability, tumor-infiltrating immune cells, Immunoscore, and their impact on patient survival remains to be elucidated. We found significant differences in mutational patterns, chromosomal instability, and gene expression that correlated with patient microsatellite instability status. A prominent immune gene expression was observed in microsatellite-instable (MSI) tumors, as well as in a subgroup of microsatellite-stable (MSS) tumors. MSI tumors had increased frameshift mutations, showed genetic evidence of immunoediting, had higher densities of Th1, effector-memory T cells, in situ proliferating T cells, and inhibitory PD1-PDL1 cells, had high Immunoscores, and were infiltrated with mutation-specific cytotoxic T cells. Multivariate analysis revealed that Immunoscore was superior to microsatellite instability in predicting patients' disease-specific recurrence and survival. These findings indicate that assessment of the immune status via Immunoscore provides a potent indicator of tumor recurrence beyond microsatellite-instability staging that could be an important guide for immunotherapy strategies.

Multiple circulating forms of neprilysin detected with novel epitope-directed monoclonal antibodies.

Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly characterized antibodies supplied in commercial ELISA kits. Validated antibodies with well-defined binding footprints are critical for understanding the biological and clinical context of NEP immunoassay data. To achieve this, we applied in silico epitope prediction and rational peptide selection to generate monoclonal antibodies (mAbs) against spatially distant sites on NEP. One of the selected epitopes contained published N-linked glycosylation sites at N285 and N294. The best antibody pair, mAb 17E11 and 31E1 (glycosylation-sensitive), were characterized by surface plasmon resonance, isotyping, epitope mapping, and western blotting. A validated two-site sandwich NEP ELISA with a limit of detection of 2.15 pg/ml and working range of 13.1-8000 pg/ml was developed with these mAbs. Western analysis using a validated commercial polyclonal antibody (PE pAb) and our mAbs revealed that non-HF and HF plasma NEP circulates as a heterogenous mix of moieties that possibly reflect proteolytic processing, post-translational modifications and homo-dimerization. Both our mAbs detected a ~ 33 kDa NEP fragment which was not apparent with PE pAb, as well as a common ~ 57-60 kDa moiety. These antibodies exhibit different affinities for the various NEP targets. Immunoassay results are dependent on NEP epitopes variably detected by the antibody pairs used, explaining the current discordant NEP measurements derived from different ELISA kits.

p-d Orbital Hybridization-Engineered PdSn Nanozymes for a Sensitive Immunoassay.

Nanozymes with peroxidase-like activity have been extensively studied for colorimetric biosensing. However, their catalytic activity and specificity still lag far behind those of natural enzymes, which significantly affects the accuracy and sensitivity of colorimetric biosensing. To address this issue, we design PdSn nanozymes with selectively enhanced peroxidase-like activity, which improves the sensitivity and accuracy of a colorimetric immunoassay. The peroxidase-like activity of PdSn nanozymes is significantly higher than that of Pd nanozymes. Theoretical calculations reveal that the p-d orbital hybridization of Pd and Sn not only results in an upward shift of the d-band center to enhance hydrogen peroxide (H2O2) adsorption but also regulates the O-O bonding strength of H2O2 to achieve selective H2O2 activation. Ultimately, the nanozyme-linked immunosorbent assay has been successfully developed to sensitively and accurately detect the prostate-specific antigen (PSA), achieving a low detection limit of 1.696 pg mL-1. This work demonstrates a promising approach for detecting PSA in a clinical diagnosis.

Bimetallic Nanozyme: A Credible Tag for In Situ-Catalyzed Reporter Deposition in the Lateral Flow Immunoassay for Ultrasensitive Cancer Diagnosis.

The lateral flow immunoassay (LFIA) is a sought-after point-of-care testing platform, yet the insufficient sensitivity of the LFIA limits its application in the detection of tumor biomarkers. Here, a colorimetric signal amplification method, bimetallic nanozyme-mediated in situ-catalyzed reporter deposition (BN-ISCRD), was designed for ultrasensitive cancer diagnosis. The bimetallic nanozyme used, palladium@iridium core-shell nanoparticles (Pd@Ir NPs), had ultrahigh enzyme-like activity, which was further explained by the electron transfer of Pd@Ir NPs and the change in the Gibbs free energy during catalysis through density functional theory calculations. With gastric cancer biomarkers pepsinogen I and pepsinogen II as model targets, this assay could achieve a cutoff value of 10 pg/mL, which was 200-fold lower than that without signal enhancement. The assay was applied to correctly identify 8 positive and 28 negative clinical samples. Overall, this BN-ISCRD-based LFIA showed great merits and potential in the application of ultrasensitive disease diagnosis.

Plasmonic Annular Nanotrenches with 1 nm Nanogaps for Detection of SARS-CoV-2 Using SERS-Based Immunoassay.

This study represents the synthesis of a novel class of nanoparticles denoted as annular Au nanotrenches (AANTs). AANTs are engineered to possess embedded, narrow circular nanogaps with dimensions of approximately 1 nm, facilitating near-field focusing for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via a surface-enhanced Raman scattering (SERS)-based immunoassay. Notably, AANTs exhibited an exceedingly low limit of detection (LOD) of 1 fg/mL for SARS-CoV-2 spike glycoproteins, surpassing the commercially available enzyme-linked immunosorbent assay (ELISA) by 6 orders of magnitude (1 ng/mL from ELISA). To assess the real-world applicability, a study was conducted on 50 clinical samples using an SERS-based immunoassay with AANTs. The results revealed a sensitivity of 96% and a selectivity of 100%, demonstrating the significantly enhanced sensing capabilities of the proposed approach in comparison to ELISA and commercial lateral flow assay kits.

"Cave Effect" Induces Self-Assembled Bimetallic Hollow Structure for Three-in-One Lateral Flow Immunoassay.

Bimetallic hollow structures have attracted much attention due to their unique properties, but they still face the problems of nonuniform alloys and excessive etching leading to structural collapse. Here, uniform bimetallic hollow nanospheres are constructed by pore engineering and then highly loaded with hemin (Hemin@MOF). Interestingly, in the presence of polydopamine (PDA), the competitive coordination between anionic polymer (γ-PGA) and dimethylimidazole does not lead to the collapse of the external framework but self-assembly into a hollow structure. By constructing the Hemin@MOF immune platform and using E. coli O157:H7 as the detection object, we find that the visual detection limits can reach 10, 3, and 3 CFU/mL in colorimetric, photothermal, and catalytic modes, which is 4 orders of magnitude lower than the traditional gold standard. This study provides a new idea for the morphological modification of the metal-organic skeleton and multifunctional immunochromatography detection.

Palladium/Platinum/Ruthenium Trimetallic Dendritic Nanozymes Exhibiting Enhanced Peroxidase-like Activity for Signal Amplification of Lateral Flow Immunoassays.

Developing ultrasensitive lateral flow immunoassays (LFIAs) has garnered significant attention in the field of point-of-care testing. In this study, a trimetallic dendritic nanozyme (Pd@Pt-Ru) was synthesized through Ru deposition on a Pd@Pt core and utilized to enhancing the sensitivity of LFIAs. Pd@Pt-Ru exhibited a Km value of 5.23 mM for detecting H2O2, which indicates an H2O2 affinity comparable with that of horseradish peroxidase. The Ru surface layer reduces the activation energy barrier, which increases the maximum reaction rate. As a proof of concept, the proposed Pd@Pt-Ru nanozyme was incorporated into LFIAs (A-Pd@Pt-Ru-LFIAs) for detecting human chorionic gonadotropin (hCG). Compared with conventional gold nanoparticle (AuNP)-LFIAs, A-Pd@Pt-Ru-LFIAs demonstrated 250-fold increased sensitivity, thereby enabling a visible detection limit as low as 0.1 IU/L. True positive and negative rates both reached 100%, which renders the proposed Pd@Pt-Ru nanozyme suitable for detecting hCG in clinical samples.

Comparative Evaluation of Commercial Test Kits Cleared for Use in Modified Two-Tiered Testing Algorithms for Serodiagnosis of Lyme Disease.

BACKGROUND: Modified 2-tiered testing (MTTT) for Lyme disease utilizes automatable, high throughput immunoassays (AHTIs) in both tiers without involving western immunoblots, offering performance and practical advantages over standard 2-tiered testing (STTT; first-tier AHTI followed by immunoglobulin M (IgM) and immunoglobulin G (IgG) western immunoblots). For MTTT, Centers for Disease Control and Prevention recommends using AHTI test kits that have been cleared by Food and Drug Administration (FDA) specifically for this intended use. We evaluated performance of FDA-cleared MTTT commercial test kits from 3 manufacturers by comparing with STTT results. METHODS: We performed MTTT (total antibody AHTI with reflex to separate IgM and IgG AHTIs) using test kits from Diasorin, Gold Standard Diagnostics (GSD), and Zeus Scientific on 382 excess serum samples submitted to the clinical laboratory for routine Lyme disease serologic testing in July 2018, measuring agreement between MTTT and STTT using the κ statistic. RESULTS: Overall agreement with STTT was 0.87 (95% confidence interval [CI], .77-.97) using Diasorin assays (almost perfect agreement), 0.80 (95% CI, .68-.93) using GSD assays (substantial agreement) and 0.79 (95% CI, .68-.90) using Zeus assays (substantial agreement). For detection of IgM reactivity, agreement between MTTT and STTT was 0.70 (.51-.90; substantial), 0.63 (95% CI, .44-.82; substantial) and 0.56 (95% CI, .38-.73; moderate), respectively. For detection of IgG reactivity, MTTT/STTT agreement was 0.73 (95% CI,.58-.88), 0.78 (95% CI, .62-.94), and 0.75 (95% CI, .60-.90), respectively (substantial agreement in all cases). CONCLUSIONS: MTTT results obtained using commercial test kits from 3 different manufacturers had substantial to almost perfect agreement with STTT results overall and moderate to substantial agreement for IgM and IgG detection independently. Commercial MTTT tests can be used broadly for the diagnosis of Lyme disease.

Identification and Quantification of Human Relaxin Proteins by Immunoaffinity-Mass Spectrometry.

The human relaxins belong to the Insulin/IGF/Relaxin superfamily of peptide hormones, and their physiological function is primarily associated with reproduction. In this study, we focused on a prostate tissue-specific relaxin RLN1 (REL1_HUMAN protein) and a broader tissue specificity RLN2 (REL2_HUMAN protein). Due to their structural similarity, REL1 and REL2 proteins were collectively named a 'human relaxin protein' in previous studies and were exclusively measured by immunoassays. We hypothesized that the highly selective and sensitive immunoaffinity-selected reaction monitoring (IA-SRM) assays would reveal the identity and abundance of the endogenous REL1 and REL2 in biological samples and facilitate the evaluation of these proteins for diagnostic applications. High levels of RLN1 and RLN2 transcripts were found in prostate and breast cancer cell lines by RT-PCR. However, no endogenous prorelaxin-1 or mature REL1 were detected by IA-SRM in cell lines, seminal plasma, or blood serum. The IA-SRM assay of REL2 demonstrated its undetectable levels (<9.4 pg/mL) in healthy control female and male sera and relatively high levels of REL2 in maternal sera across different gestational weeks (median 331 pg/mL; N = 120). IA-SRM assays uncovered potential cross-reactivity and nonspecific binding for relaxin immunoassays. The developed IA-SRM assays will facilitate the investigation of the physiological and pathological roles of REL1 and REL2 proteins.

False-Reactive Fourth-Generation Human Immunodeficiency Virus Testing in Cancer Patients.

BACKGROUND: The fourth-generation (4th-gen) human immunodeficiency virus (HIV)-1/2 antibody/antigen (Ab/Ag) combination immunoassay currently used for HIV screening offers greater sensitivity than previous assays, but false-reactive results occur in up to 20% of patients. Large-scale observations in cancer patients are lacking. METHODS: We conducted a retrospective study of cancer patients seen at the University of Texas MD Anderson Cancer Center (March 2016-January 2023) who had reactive 4th-gen ARCHITECT HIV-1/2 Ab/Ag combination immunoassay results. We analyzed characteristics of patients with true-reactive and false-reactive results, defined based on Centers for Disease Control and Prevention criteria. RESULTS: A total of 43 637 patients underwent 4th-gen HIV screening, and 293 had reactive 4th-gen HIV test results. Twenty-one patients were excluded because they did not have cancer. Among the remaining 272 patients, 78 (29%) had false-reactive results. None of these patients experienced delays in their cancer treatment, but 26% experienced mental distress. Multivariate logistic regression analysis identified 5 predictors of having false-reactive results: age >60 years (adjusted odds ratio [aOR], 6.983; P < .0001), female sex (aOR, 6.060; P < .0001), race/ethnicity (Black: aOR, 0.274; Hispanic: aOR, 0.236; P = .002), syphilis coinfection (aOR, 0.046; P = .038), and plant alkaloids therapy (aOR, 2.870; P = .013). CONCLUSIONS: False-reactive 4th-gen HIV test results occur in almost one-third of cancer patients. Physicians should be aware of the high rates of false-reactive HIV screening results in this patient population. These findings may have implications for counseling regarding testing, especially among those at low risk for HIV infection.

Challenges of insulin-like growth factor-1 testing.

Insulin-like growth factor 1 (IGF-1), primarily synthesized in the liver, was initially discovered due to its capacity to replicate the metabolic effects of insulin. Subsequently, it emerged as a key regulator of the actions of growth hormone (GH), managing critical processes like cell proliferation, differentiation, and apoptosis. Notably, IGF-1 displays a longer half-life compared to GH, making it less susceptible to factors that may affect GH concentrations. Consequently, the measurement of IGF-1 proves to be more specific and sensitive when diagnosing conditions such as acromegaly or GH deficiency. The recognition of the existence of IGFBPs and their potential to interfere with IGF-1 immunoassays urged the implementation of various techniques to moderate this issue and provide accurate IGF-1 results. Additionally, in response to the limitations associated with IGF-1 immunoassays and the occurrence of discordant IGF-1 results, modern mass spectrometric methods were developed to facilitate the quantification of IGF-1 levels. Taking advantage of their ability to minimize the interference caused by IGF-1 variants, mass spectrometric methods offer the capacity to deliver robust, reliable, and accurate IGF-1 results, relying on the precision of mass measurements. This also enables the potential detection of pathogenic mutations through protein sequence analysis. However, despite the analytical challenges, the discordance in IGF-1 reference intervals can be attributed to a multitude of factors, potentially leading to distinct interpretations of results. The establishment of reference intervals for each assay is a demanding task, and it requires nationwide multicenter collaboration among laboratorians, clinicians, and assay manufacturers to achieve this common goal in a cost-effective and resource-efficient manner. In this comprehensive review, we examine the challenges associated with the standardization of IGF-1 measurement methods, the minimization of pre-analytical factors, and the harmonization of reference intervals. Particular emphasis will be placed on the development of IGF-1 measurement techniques using "top-down" or "bottom-up" mass spectrometric methods.

Standardization of PTH measurement by LC-MS/MS: a promising solution for interassay variability.

Parathyroid hormone measurement is critical in managing chronic kidney disease-mineral bone disorders. However, there are several commercially available immunoassays with interassay variability. To address this, Cavalier et al. developed standardization of parathyroid hormone measurement by establishing regression equations of each assay against the liquid chromatography coupled to tandem mass spectrometry method. The recalibration successfully reduced interassay variability, allowing for more consistent interpretation. The proposed approach may pave the way for accurate interpretation of parathyroid hormone in clinical practice.

Afterglow Nanoprobe-Enabled Quantitative Lateral Flow Immunoassay by a Palm-Size Device for Household Healthcare.

Lateral flow immunoassay (LFIA), a classical point-of-care testing (POCT) technique, plays an important role in disease screening and healthcare monitoring. However, traditional LFIA is either designed for qualitative analysis or requires expensive equipment for quantification, limiting its use in household diagnosis. In this study, we proposed a new generation of LFIA for household health monitoring by using ultralong organic phosphorescence (UOP) nanomaterials as afterglow nanoprobes with a self-developed palm-size sensing device. The UOP nanoprobes exhibit a phosphorescence signal with a second-level lifetime, which completely avoids the interference from excitation light and biological background fluorescence. Therefore, an ultraminiaturized and low-cost UOP nanosensor was successfully designed by eliminating the complex optical path and filtering systems. We chose an inflammatory factor, C-reactive protein (CRP), for household POCT validation. The whole analysis was completed within 9 min. A limit of detection (LOD) of 0.54 ng/mL of CRP antigen was achieved with high stability and good specificity, which is comparable to laboratory instruments and fully satisfying the clinical diagnosis requirement.

Use of a Microfluidic Platform To Evaluate and Predict Reagent Performance in Microtiter Plate-Based Immunoassays.

Selection and characterization of antibodies are critically important in establishing robust immunoassays to support the development efforts of vaccines. Plate-based ELISA can be time- and resource-intensive to select initial antibody clones or characterize downstream resupply lots while providing limited information regarding the binding characteristics of the antibodies beyond concentration-response curves. This work applied the microfluidic Gyrolab to holistically evaluate immunoassay reagents through analyses of concentration-response curves as well as antibody-antigen interactions visualized in column images and affinity estimates. We exploited the automation capability of the Gyrolab to reduce the resources (time, reagents, and scientists) required for screening and evaluating antibody reagents. Using a flexible semi-universal assay format, we compared antibody clones for selection and resupply lots of sera and monoclonal antibodies in a simple "plug-and-play" manner without antibody modifications. We found that the performance of antibodies in the Gyrolab correlated well with the trends observed in traditional ELISAs, while the Gyrolab provided additional advantages over plate-based assays such as column images of antibody binding and affinity measurements.

PEI-Mediated Assembly of Fe3O4 onto SiO2-Encapsulated CsPbBr3 for Highly Sensitive Fluorescent Lateral Flow Immunoassay.

The conventional lateral flow immunoassay (LFIA) method using colloidal gold nanoparticles (Au NPs) as labeling agents faces two inherent limitations, including restricted sensitivity and poor quantitative capability, which impede early viral infection detection. Herein, we designed and synthesized CsPbBr3 perovskite quantum dot-based composite nanoparticles, CsPbBr3@SiO2@Fe3O4 (CSF), which integrated fluorescence detection and magnetic enrichment properties into LFIA technology and achieved rapid, sensitive, and convenient quantitative detection of the SARS-CoV-2 virus N protein. In this study, CsPbBr3 served as a high-quantum-yield fluorescent signaling probe, while SiO2 significantly enhanced the stability and biomodifiability of CsPbBr3. Importantly, the SiO2 shell shows relatively low absorption or scattering toward fluorescence, maintaining a quantum yield of up to 74.4% in CsPbBr3@SiO2. Assembly of Fe3O4 nanoparticles mediated by PEI further enhanced the method's sensitivity and reduced matrix interference through magnetic enrichment. Consequently, the method achieved a fluorescent detection range of 1 × 102 to 5 × 106 pg·mL-1 after magnetic enrichment, with a limit of detection (LOD) of 58.8 pg·mL-1, representing a 13.3-fold improvement compared to nonenriched samples (7.58 × 102 pg·mL-1) and a 2-orders-of-magnitude improvement over commercial colloidal gold kits. Furthermore, the method exhibited 80% positive and 100% negative detection rates in clinical samples. This approach holds promise for on-site diagnosis, home-based quantitative tests, and disease procession evaluation.

A Magnetic-Optical Triple-Mode Lateral Flow Immunoassay for Sensitive and Rapid Detection of Respiratory Adenovirus.

Human respiratory adenovirus (ADV) is a highly infectious respiratory virus with potential for pandemics. There are currently no specific drugs to treat ADV worldwide, so early rapid detection of ADV infection is essential. In this study, we developed an innovative magnetic-optical triple-mode lateral flow immunoassay (LFIA) using magnetic quantum dots as immunomarkers. This novel approach addresses the need for rapid and accurate ADV detection, allowing for multimodal quantitative/semiquantitative analysis of magnetic, fluorescent, and visible signals within a mere 15 min. The lower limit of detection (LOD) for magnetic, fluorescent, and visual signals was determined to be 5.6 × 103, 1.2 × 103, and 1.95 × 104 copies/mL, respectively. The detection range for ADV using this approach was 1.2 × 103-5 × 107 copies/mL. Additionally, semiquantitative analysis, which is user-friendly and does not necessitate specialized equipment, was successfully implemented. Notably, seven respiratory viruses showed no cross-reactivity with the generated LFIA test strips. The intrabatch repeatability exhibited a coefficient of variation (CV) of less than 5%, while the interbatch repeatability had a CV of less than 15%. Furthermore, recovery values ranged from 95% to 106.8% for samples analyzed concurrently with dual signals at the same spiking concentration. The assay developed in this study boasts a wide detection range and exceptional sensitivity and specificity. This technique is exceptionally well-suited for on-site rapid detection, with the potential for personal self-testing and early ADV infection diagnosis. Its versatility extends to a broad array of application scenarios.

High-Density Au Anchored to Ti3C2-Based Colorimetric-Fluorescence Dual-Mode Lateral Flow Immunoassay for All-Domain-Enhanced Performance and Signal Intercalibration.

In this work, a novel MXene-Au nanoparticle (Ti3C2@Au) was synthesized with a high molar extinction coefficient, strong fluorescence quenching ability, ultrahigh antibody affinity, high stability, and good dispersibility, and it was used to develop a colorimetric-fluorescence dual-mode lateral flow immunoassay (LFIA). The detection limits of this method for the detection of dexamethasone in milk, beef, and pork were 0.0018, 0.12, and 0.084 µg/kg in the "turn-off" mode (colorimetric signal), and 0.0013, 0.080, and 0.070 µg/kg in the "turn-on" mode (fluorescent signal), respectively, which was up to 231-fold more sensitive compared with that of the reported LFIAs. The recovery rates ranged from 81.1-113.7%, and 89.2-115.4%, with the coefficients of variation ranging from 1.4-15.0%, and 1.9-14.8%, respectively. The results of the LC-MS/MS confirmation test on 30 real samples had a good correlation with that of our established method (R2 > 0.97). This work not only developed novel nanocarriers for antibody-based LFIA but also ensured high-performance detection.