Enzyme-linked Immunosorbent Assay Protocol Research Articles

Maternal IgG in hemolytic disease of the fetus and newborn-ABO incompatibility

BACKGROUND Hemolytic disease of the fetus and newborn (HDFN) is a type of anemia in the fetus or newborn, characterized by anemia, jaundice, hyperbilirubinemia, and brain damage. IgG is the only antibody that can cross the placenta. The IgG subtypes have a different ability to destroy red blood cells (RBCs). IgG1 and IgG3 can bind to Fc-phagocyte cell receptors and cause hemolysis, while IgG3 has more ability than IgG1. This study aimed to identify the antibody IgG subtype contributing to clinical manifestation differences in HDFN. METHODS This study used blood and umbilical cord blood samples from 30 pairs of mother-baby. The samples were grouped into control (not jaundice/normal bilirubin levels) and jaundice/hyperbilirubinemia groups. A self-developed IgG subtype enzyme-linked immunosorbent assay protocol was performed on maternal samples, resulting in optical density. Statistical analysis was performed using SPSS software version 23. RESULTS Blood type was associated with total bilirubin expression (p = 0.005). IgG1 anti-A, IgG3 anti-A, IgG4 anti-A, IgG1 anti-B, IgG3 anti-B, and IgG4 anti-B significantly affected hyperbilirubinemia in newborns (p = 0.041, 0.013, 0.017, 0.028, 0.001, and 0.007, respectively). CONCLUSIONS IgG1 and IgG3 were more significant in causing clinical problems. IgG4 suppressed IgG activation, resulting in no destruction of the infant’s RBCs.

Surface Acoustic Wave-Driven Enhancement of Enzyme-Linked Immunosorbent Assays: ELISAW.

Enzyme-linked immunosorbent assays (ELISAs) are widely used in biology and clinical diagnosis. Relying on antigen-antibody interaction through diffusion, the standard ELISA protocol can be time-consuming, preventing its use in rapid diagnostics. We present a time-saving and more sensitive ELISA without changing the standard setup and protocol, using surface acoustic waves (SAWs) to enhance performance. Each step of the assay, from the initial antibody binding onto the walls of the well plate to the target analyte molecules' binding for detection─except, notably, for the blocking step─is improved principally via acoustic streaming-driven advection. Using SAWs, the time required for one step of an example ELISA is reduced from 60 to 15 min to achieve the same binding amount. By extending the duration of SAW exposure to 20 min, the sensitivity can be significantly improved over the 60 min, 35 °C ELISA without SAWs. It is also possible to confer beneficial improvements to bead-based ELISA by combining it with SAWs to further reduce the time required for binding to 2 min. By significantly increasing the speed of ELISA, its utility may be improved for a wide range of point-of-care diagnostics applications.

Optimized Enzyme-Linked Immunosorbent Assay for Anti-PEG Antibody Detection in Healthy Donors and Patients Treated with PEGylated Liposomal Doxorubicin.

There is considerable interest in quantifying anti-PEG antibodies, given their potential involvement in accelerated clearance, complement activation, neutralization, and acute reactions associated with drug delivery systems. Published and commercially available anti-PEG enzyme-linked immunosorbent assays (ELISAs) differ significantly in terms of reagents and conditions, which could be confusing to users who want to perform in-house measurements. Here, we optimize the ELISA protocol for specific detection of anti-PEG IgG and IgM in sera from healthy donors and in plasma from cancer patients administered with PEGylated liposomal doxorubicin. The criterion of specificity is the ability of free PEG or PEGylated liposomes to inhibit the ELISA signals. We found that coating high-binding plates with monoamine methoxy-PEG5000, as opposed to bovine serum albumin-PEG20000, and blocking with 1% milk, as opposed to albumin or lysozyme, significantly improve the specificity, with over 95% of the signal being blocked by competition. Despite inherent between-assay variability, setting the cutoff value of the optical density at the 80th percentile consistently identified the same subjects. Using the optimized assay, we longitudinally measured levels of anti-PEG IgG/IgM in cancer patients before and after the PEGylated liposomal doxorubicin chemotherapy cycle (1 month apart, three cycles total). Antibody titers did not show any increase but rather a decrease between treatment cycles, and up to 90% of antibodies was bound to the infused drug. This report is a step toward harmonizing anti-PEG assays in human subjects, emphasizing the cost-effectiveness and optimized specificity.

Difference of Cerebrospinal Fluid Biomarkers and Neuropsychiatric Symptoms Profiles among Normal Cognition, Mild Cognitive Impairment, and Dementia Patient.

The delineation of biomarkers and neuropsychiatric symptoms across normal cognition, mild cognitive impairment (MCI), and dementia stages holds significant promise for early diagnosis and intervention strategies. This research investigates the association of neuropsychiatric symptoms, evaluated via the Neuropsychiatric Inventory (NPI), with cerebrospinal fluid (CSF) biomarkers (Amyloid-β42, P-tau, T-tau) across a spectrum of cognitive states to enhance diagnostic accuracy and treatment approaches. Drawing from the National Alzheimer's Coordinating Center's Uniform Data Set Version 3, comprising 977 individuals with normal cognition, 270 with MCI, and 649 with dementia. To assess neuropsychiatric symptoms, we employed the NPI to understand the behavioral and psychological symptoms associated with each cognitive category. For the analysis of CSF biomarkers, we measured levels of Amyloid-β42, P-tau, and T-tau using the enzyme-linked immunosorbent assay (ELISA) and Luminex multiplex xMAP assay protocols. These biomarkers are critical in understanding the pathophysiological underpinnings of Alzheimer's disease and its progression, with specific patterns indicative of disease stage and severity. This study cohort consists of 1896 participants, which is composed of 977 individuals with normal cognition, 270 with MCI, and 649 with dementia. Dementia is characterized by significantly higher NPI scores, which are largely reflective of mood-related symptoms (p < 0.001). In terms of biomarkers, normal cognition shows median Amyloid-β at 656.0 pg/mL, MCI at 300.6 pg/mL, and dementia at 298.8 pg/mL (p < 0.001). Median P-tau levels are 36.00 pg/mL in normal cognition, 49.12 pg/mL in MCI, and 58.29 pg/mL in dementia (p < 0.001). Median T-tau levels are 241.0 pg/mL in normal cognition, 140.6 pg/mL in MCI, and 298.3 pg/mL in dementia (p < 0.001). Furthermore, the T-tau/Aβ-42 ratio increases progressively from 0.058 in the normal cognition group to 0.144 in the MCI group, and to 0.209 in the dementia group (p < 0.001). Similarly, the P-tau/Aβ-42 ratio also escalates from 0.305 in individuals with normal cognition to 0.560 in MCI, and to 0.941 in dementia (p < 0.001). The notable disparities in NPI and CSF biomarkers among normal, MCI and Alzheimer's patients underscore their diagnostic potential. Their combined assessment could greatly improve early detection and precise diagnosis of MCI and dementia, facilitating more effective and timely treatment strategies.

Development of an Indirect ELISA for the Detection of Lactoferrin in Type 2 Diabetes Plasma: A Novel Approach

Background: In biological systems, lactoferrin (LF) is a crucial protein for protecting the body against diseases and pathogens that can affect both humans and animals. LF is a multifunction protein that binds to different surface receptors to stimulate the innate immune system. In diabetes, lactoferrin has a direct association with inflammation. The effects of inflammation interaction are unknown but reasonably could include changes in LF, a body protein whose changed concentration correlates with type 2 diabetes (T2D). The LF content in plasma has been used as a disease biomarker, and there is a need for convenient and reliable assays. Method: An innovative indirect enzyme-linked immunosorbent assay (ELISA) was developed and applied to measure circulating lactoferrin levels as an inflammation marker in human samples, including healthy and type 2 diabetes. Results: Under optimized conditions, the proposed indirect ELISA was evaluated and linearly responded to LF standards in a 0.05–0.5 µgmL−1 range. The limit of detection (LOD) was 0.05 µgmL−1, and a reliable limit of quantification (LOQ) was 0.240 µgmL−1 . Conclusion: The developed assay showed both specificity and reproducibility, indicating the utility of this indirect ELISA in LF monitoring. This study provides a definitive indirect ELISA protocol to detect various lactoferrin antigens with accurate, reliable, and reproducible data, and it could be applied for diagnosing lactoferrin-related diseases, such as type 2 diabetes. Our innovative approach provides a relatively cost-effective, sensitive, and precise way to assess LF in various human plasmas.

An efficient ELISA protocol for measurement of SARS-CoV-2 spike-specific IgG in human plasma and serum samples

Here, we describe a protocol for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-specific immunoglobulin G (IgG) by enzyme-linked immunosorbent assay (ELISA). The protocol was developed with a keen focus on optimizing several key parameters, including antigen coating concentration, antibody and sample dilutions, and assay development time. The final protocol features the following characteristics:•The capability to detect SARS-CoV-2 spike-specific IgG in both plasma and serum samples.•A streamlined procedure that requires only 1 hour and 20 minutes of hands-on time.•Reliable assay performance, with a remarkable sensitivity of 98.1 % and specificity of 99.5 %.

An Analytical Protocol for Detecting Antibody Titer Levels in Serum/Saliva by Indirect Enzyme-Linked Immunosorbent Assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA) detects qualitatively and quantitatively the presence of antibodies or antigens in a sample. Due to its simplicity, high sensitivity, and user-friendliness, the test is widely used in laboratory research, clinical diagnoses, and food testing. This chapter describes the indirect semiquantitative ELISA protocol used to monitor antibody levels in animals and analyze the titer levels of specific antibodies against a target antigen in serum and saliva.

Dimeric immunoglobulin A as a novel diagnostic marker of measles infection.

The world is facing a measles resurgence, and improved diagnostic tests for measles infection are an urgent World Health Organization research priority. Detection of measles-specific immunoglobulin M (IgM) as a standard diagnostic test has low positive predictive value in elimination settings, and there is a need for new biomarkers of measles infection to enable enhanced surveillance and response to outbreaks. We demonstrate the detection of measles-specific dimeric immunoglobulin A (dIgA) in patients with confirmed measles infections using a new indirect enzyme-linked immunosorbent assay protocol that selects for the dIgA fraction from total IgA in the blood. The magnitude of measles-specific dIgA responses showed a low correlation with IgM responses, and our results highlight the potential of dIgA for further development as an alternative and/or complementary biomarker to IgM for serological diagnosis of measles infection.

Validation Procedures for Quantification of Food Allergens by Enzyme-Linked Immunosorbent Assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA) is a widely used analytical technique for food allergen detection and quantification. Validating ELISA protocols is important for both assay developers and end users as it ensures method reliability. This chapter describes the protocols for validating the sensitivity, specificity, precision, accuracy, robustness, and ruggedness of an ELISA. Example procedures are also provided for sample preparation, allergen extraction, and ELISA operation.

Freeze-drying of few microliters of antibody formulations to implement 384-wells homogeneous instant assays

Enzyme-linked immunosorbent assay protocols have traditionally complex workflows with several intensive wash steps. Analytical tools with both shorter time-to-result and hands-on-time using smaller sample and assays reagents volumes are now investigated. In this context, fluorescence resonance energy transfer (FRET)-based assays are emerging as one of the most promising analytical tools in high-throughput screening (HTS). These immunoassays allow fast quantification of antigens at the nano-gram level in a final assay volume of only a few μL. We used a homogeneous time-resolved FRET (called HTRF) assay to develop a freeze-dried screening and ready-to-use format with only one rehydration step called “instant assay”.To assure optimal performance of the developed homogeneous instant assay, we investigated the critical quality attributes by studying the functionality and stability of the critical reagents and fluorophores. The cyclic adenosine 3′-5′-monophosphate (cAMP) was selected as the antigen target. We tested various formulations (with different buffers, sugars, bulking reagents, surfactants and co-solvants) combined with a slow freezing and the use of an aluminium plate holder during the freeze-drying of few microliter of bioreagents.The optimized freeze-drying procedure permits to preserve more than 70% of Ab recognition properties. The developed off-the-shelf homogeneous FRET immunoassay allows direct and fast quantification of cAMP at a nanogram level.

Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a candidate for biomarker status. CGRP is released from neuronal fibers upon activation and induces sterile neurogenic inflammation and arterial vasodilation in the vasculature that receives trigeminal efferent innervation. The presence of CGRP in the peripheral vasculature has spurred investigations to detect and quantify this neuropeptide in human plasma using proteomic assays, such as the enzyme-linked immunosorbent assay (ELISA). However, its half-life of 6.9 min and the variability in technical details of assay protocols, which are often not fully described, have yielded inconsistent CGRP ELISA data in the literature. Here, a modified ELISA protocol for the purification and quantification of CGRP in human plasma is presented. The procedural steps involve sample collection and preparation, extraction using a polar sorbent as a means of purification, additional steps to block non-specific binding, and quantification via ELISA. Further, the protocol has been validated with spike and recovery and linearity of dilution experiments. This validated protocol can theoretically be used to quantify CGRP concentrations in the plasma of individuals not only with migraine, but also with other diseases in which CGRP may play a role.

Development of a monoclonal antibody specific to burbot (Lota lota) IgM and optimization of an ELISA to measure anti-Aeromonas sp. antibody titers following pathogen challenge

Burbot (Lota lota) are an ideal candidate for cool or cold-water aquaculture and are gaining interest because of their high economic value, low temperature requirements, and fast growth rate. Limited information exists on the innate and adaptive immune systems of this species. This is partly due to the lack of species-specific tools to determine antibody responses following disease or vaccination or to characterize the immune response in general. An anti-IgM monoclonal antibody (mAb 27C) was developed and characterized via enzyme-linked immunosorbent assay (ELISA) and Western blot for species specificity, affinity to the heavy chain of burbot IgM, and cross-reactivity to other reagents used in the analysis. The 27C monoclonal antibody was further utilized to develop an ELISA protocol to measure the specific antibody response of burbot following exposure to two pathogenic strains of Aeromonas sp. (A141 and IR004). This ELISA confirmed that vaccinated burbot that survived the challenge with either strain developed statistically higher titers of anti-Aeromonas antibodies specific for the relative strain when compared to fish that were not vaccinated or challenged. Western blot analysis further demonstrated that burbot surviving challenge had serum IgM that recognized distinct antigens specific to the strain they were challenged with, A141 bound to antigens in the 50-250Kda range and IR004 bound to a distinct 150Kda antigen. Western blots further indicated that each strain shared antigenic regions regardless of experimental Aeromonas strain exposure. Finally, immunofluorescent staining confirmed that mAb 27C binds to membrane-bound IgM (presumably B cells) on burbot head kidney cells. Taken together, results from this study demonstrate that mAb 27C specifically recognized burbot IgM and will be an important tool to further characterize the adaptive and cellular immune responses of this fish species.

Epidemiology of strongyloidiasis determined by parasite-specific IgG detections by enzyme-linked immunosorbent assay on urine samples using Strongyloides stercoralis, S. ratti and recombinant protein (NIE) as antigens in Northeast Thailand.

Detection of anti-Strongyloides IgG in urine by enzyme-linked immunosorbent assay (ELISA) for diagnosis of strongyloidiasis reportedly has comparable performance to conventional serum assays. Initial comparisons of urine assays using commercial ELISA kits designated for serology have shown its diagnostic potential but sub-optimal accuracy. In the present study, we optimized urine ELISA protocols based on different antigen types and evaluated their accuracies in determining the epidemiology of strongyloidiasis in Northeast Thailand. Paired urine and fecal samples of 966 individuals from the study community were collected for three consecutive days and tested for strongyloidiasis. We compared three ELISA protocols using different antigens including crude S. stercoralis antigen (Ss-ELISA), crude S. ratti antigen (Sr-ELISA) and recombinant NIE antigen (NIE-ELISA) and fecal examination by agar plate-culture (APCT) technique and formalin-ethyl acetate concentration technique (FECT). The optimized ELISA protocols using three different antigen sources yielded significantly higher prevalence rates of strongyloidiasis (58.9-65.1%) than those by fecal examination methods (19.7%). The prevalence of strongyloidiasis determined by ELISA protocols significantly increased with age (p value < 0.0001) and males had higher prevalence than females (p value < 0.0001). Diagnostic agreements between ELISA protocols were moderate (κ = 0.461-0.586) and the agreement between each ELISA protocol and fecal examinations were slight (κ = 0.139-0.210). The results obtained by urine ELISA protocols using three different antigens showed comparable diagnostic performances, provided further supports for the utility of urine as an alternative clinical specimen for diagnosis of strongyloidiasis.

A Comparative Study of Prostate Specific Antigen (PSA) Point-of-Care-Testing (POCT) Techniques

Background: Prostate Specific Antigen (PSA) testing is widely used to diagnose and monitor clinical progress in patients with prostate cancer. The availability of various new Point-of-Care-Testing (POCT) equipment for PSA demands that the performance characteristics of these equipment be assessed before introducing them into clinical use to ensure accuracy and reliability. Objectives: To compare the i-CHROMA® automated immunofluorescence serum total PSA assay with the Accubind® Enzyme Linked Immuno-Sorbent Assay (ELISA) as POCT among patients with suspected prostate cancer. Methods: The study was conducted at the Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife, Nigeria. Blood samples drawn from 20 consecutively selected patients were analysed for PSA using the i-CHROMA® immunofluorescence kit once and with the Accubind® ELISA protocol twice. Results: The mean PSA using Accubind® ELISA was 12.9ng/ml, while it was 14.5ng/ml with the i-CHROMA® immunofluorescence assay. The bias between the two methods was 1.6ng/ml. The two methods had a good correlation: Passing Bablok regression equation was y = 1.264604x – 0.0300469, and the Spearman correlation coefficient between the two measurements was high (r = 0.956; Confidence Interval 0.889 - 0.983; p&lt;0.0001). Agreement between the two methods was statistically satisfactory as the mean values of the samples fell within the 95% Confidence Interval of the differences on the Bland Altman plot. Conclusion: The i-CHROMA® POCT assay showed good correlation and agreement with the well-known ELISA method. Therefore, the method is recommended for use in monitoring PSA in patients with prostatic cancer.

Fecal Keratin 8 Is a Noninvasive and Specific Marker for Intestinal Injury in Necrotizing Enterocolitis.

Specific biomarkers of intestinal injury associated with necrotizing enterocolitis (NEC) are needed to diagnose and monitor intestinal mucosal injury and recovery. This study aims to develop and test a modified enzyme-linked immunosorbent assay (ELISA) protocol to detect the total keratin 8 (K8) in the stool of newborns with NEC and investigate the clinical value of fecal K8 as a marker of intestinal injury specifically associated with NEC. We collected fecal samples from five newborns with NEC and five gestational age-matched premature neonates without NEC at the Lucile Packard Children's Hospital Stanford and Washington University School of Medicine, respectively. Fecal K8 levels were measured using a modified ELISA protocol and Western blot, and fecal calprotectin was measured using a commercial ELISA kit. Clinical data, including gestational age, birth weight, Bell stage for NEC, feeding strategies, total white blood cell (WBC) count, and other pertinent clinical variables, were collected and analyzed. Fecal K8 levels were significantly higher in the pre-NEC group (1-2 days before diagnosis of NEC) and NEC group than those in the non-NEC group (p = 0.013, p = 0.041). Moreover, fecal K8 was relatively higher at the onset of NEC and declined after the resolution of the disease (p = 0.019). Results with similar trends to fecal K8 were also seen in fecal calprotectin (p = 0.046), but not seen in total WBC count (p = 0.182). In conclusion, a modified ELISA protocol for the total K8 protein was successfully developed for the detection of fecal K8 in the clinical setting of premature newborns with NEC. Fecal K8 is noted to be significantly increased in premature newborns with NEC and may, therefore, serve as a noninvasive and specific marker for intestinal epithelial injury associated with NEC.

An Enzyme-Linked Immunosorbent Assay to Quantify Poly (ADP-Ribose) Level In Vivo.

PolyADP-ribosylation is a posttranslational modification of proteins that results from enzymatic synthesis of poly(ADP-ribose) with NAD+ as the substrate. A unique characteristic of polyADP-ribosylation is that the poly(ADP-ribose) chain can have 200 or more ADP-ribose residues in branched patterns, and the presence and variety of these chains can have substantive effects on protein function. To understand how polyADP-ribosylation affects biological processes, it is important to know the physiological level of poly(ADP-ribose) in cells. Under normal cell physiological conditions and in the absence of any exogenous DNA damaging agents, we found that the concentration of poly(ADP-ribose) in HeLa cells is approximately 0.04pmol (25pg)/106 cells, as measured with a double-antibody sandwich, enzyme-linked immunosorbent assay protocol that avoids artificial activation of PARP1 during cell lysis. Notably, this system demonstrated that the poly(ADP-ribose) level peaks in S phase and that the average cellular turnover of a single poly(ADP-ribose) is less than 40s.

Light-addressable electrochemical immunoassay for multiplexed detection of antigen

Multi-channel evaluation on a single sensor interface is highly valuable but full of challenge. Herein, a universal light-addressable electrochemical immunoassay based on silicon photoelectrodes was proposed for spatially-resolved detection of prostate-specific antigen (PSA) as a model target. To stabilize and functionalize silicon substrate, a simple and robust antifouling coating was prepared by cross-linking bovine serum albumin (BSA) matrix with glutaraldehyde (GA). For the PSA detection, an electrochemical sandwich-type immunosensor was performed on the basis of BSA@GA functionalized silicon, in which horse radish peroxidase (HRP) was used to catalyze the reaction of H2O2 and hydroquinone (HQ), resulting in in situ generation of benzoquinone (BQ). The light-induced local reduction photocurrent of BQ was then recorded as the sensor response by irradiating the target spot of photoelectrode respectively with a moveable light pointer. The results demonstrated the feasibility of localized measurements of enzymatic reactions. Coupling with light-addressable electrochemistry (LAE) and enzyme-linked immunosorbent assay (ELISA) protocol, multiplexed PSA detection could be achieved on a single sensor chip with good sensitivity and specificity. This proof-of-concept study provides a promising platform for high-throughput and accurate immunoanalysis in diagnostic or biodefense applications.

Rapid magneto-enzyme-linked immunosorbent assay for ultrasensitive protein detection

Protein-based diagnostics are the standard of care for screening and diagnosing a broad range of diseases and medical conditions. The current gold standard method for quantifying proteins in clinical specimens is the enzyme-linked immunosorbent assay (ELISA), which offers high analytical sensitivity, can process many samples at once, and is widely available in many diagnostic laboratories worldwide. However, running an ELISA is cumbersome, requiring multiple liquid handling and washing steps, and time-intensive (∼2 − 4 h per test). Here, we demonstrate a unique magneto-ELISA that utilizes dually labeled magnetic nanoparticles (DMPs) coated with horseradish peroxidase (HRP) and an HRP-conjugated detection antibody, enabling rapid immunomagnetic enrichment and signal amplification. For proof of concept, this assay was used to detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a malaria parasite biomarker, which exhibited a lower limit of detection of 2 pg mL−1 (33 fM) in human serum. Measurements of PfHRP2 in clinical blood samples from individuals with and without P. falciparum infection revealed that this magneto-ELISA offers a superior diagnostic accuracy compared to a commercial PfHRP2 ELISA kit. We also demonstrate the versatility of this platform by adapting it for the detection of SARS-CoV-2 nucleocapsid protein, which could be detected at concentrations as low as 8 pg mL−1 (174 fM) in human serum. In addition to its high analytical performance, this assay can be completed in 30 min, requires no specialized equipment, and is compatible with standard microplate readers and ELISA protocols, allowing it to integrate readily into current clinical practice.

An Electromechanical Lab-on-a-Chip Platform for Colorimetric Detection of Serum Creatinine.

Chronic kidney disease (CKD) is a high-cost disease that affects approximately one in ten people globally, progresses rapidly, results in kidney failure or dialysis, and triggers other diseases. Although clinically used serum creatinine tests are used to evaluate kidney functions, these tests are not suitable for frequent and regular control at-home settings that obstruct the regular monitoring of kidney functions, improving CKD management with early intervention. This study introduced a new electromechanical lab-on-a-chip platform for point-of-care detection of serum creatinine levels using colorimetric enzyme-linked immunosorbent assay (ELISA). The platform was composed of a chip containing microreservoirs, a stirring bar coated with creatinine-specific antibodies, and a phone to detect color generated via ELISA protocols to evaluate creatinine levels. An electromechanical system was used to move the stirring bar to different microreservoirs and stir it inside them to capture and detect serum creatinine in the sample. The presented platform allowed automated analysis of creatinine in ∼50 min down to ∼1 and ∼2 mg/dL in phosphate-buffered saline (PBS) and fetal bovine serum (FBS), respectively. Phone camera measurements in hue, saturation, value (HSV) space showed sensitive analysis compared to a benchtop spectrophotometer that could allow low-cost analysis at point-of-care.

Non-Invasive Detection of SARS-CoV-2 Antigen in Saliva versus Nasopharyngeal Swabs Using Nanobodies Conjugated Gold Nanoparticles.

The development of sensitive, non-invasive tests for the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigens is imperative, and it is still challenging to manage the extent of infection throughout the population. Here, we designed and optimized a sandwich enzyme-linked immunosorbent assay (ELISA) protocol for SARS-CoV-2 S1 antigen detection in saliva. Both saliva samples and nasopharyngeal swabs were collected from 220 real-time quantitative polymerase chain reaction (RT-qPCR)-confirmed positive and negative cases. S1 protein receptor-binding domain (RBD) nanobodies were efficiently conjugated with 40 nm gold nanoparticles (AuNPs) and employed as antigen detection probes in the developed system, while recombinant S1 monoclonal antibodies (S1mAbs) were employed as antigen capture probes. After checkerboard assays and system optimization, the clinical samples were tested. In saliva, the developed ELISA system showed the highest sensitivity (93.3) for samples with cycle threshold (Ct) values ≤ 30; interestingly, high sensitivity (87.5 and 86%) was also achieved for samples with Ct values ≤ 35 and ≤40, respectively, compared with 90, 80 and 88% sensitivity rates for nasopharyngeal swabs with the same categorized Ct values. However, the specificity was 100%, and no cross-reactions were detected with Middle East respiratory syndrome coronavirus (MERS-CoV) or SARS-CoV antigens. These results reveal that our protocol could be established as an efficient and sensitive, non-invasive diagnostic tool for the early detection of SARS-CoV-2 infection using easily collectable saliva samples.