Sensitivity Of Antibody Detection Research Articles

Bioinspired rational spatial-arrangement of antigens enables the accurate and rapid detection of anti-p53 autoantibody.

Ahighly sensitive antibody detection strategy is presentedthat leverages the rational spatial arrangement of antigens at the sensing interface. Specifically, we employed rigid benzene ring-based coupling agents, carefully controlling their density and orientation on the biosensing interface to establish a well-defined spatial arrangement of receptor molecules, thereby enhancing antibody binding efficiency. Additionally, we utilized Au-decorated MoS2 nanosheets as an effective electrode modification, which also function as contact points for regulating the coupling agents. By optimizing both the electrode materials and the spatial arrangement of receptor molecules, our strategy enabled the precise and rapid detection of anti-p53 autoantibodies (anti-p53aAbs) in spiked plasma samples, achieving a broad linear range from 0.05 to 10ng/mL and a low detection limit of 16.67pg/mL, surpassing the performance of most existing methods. Notably, we introduce a biomimetic strategy for the spatial arrangement of antigens, inspired by the bionic recognition mechanism. This design effectively reduces steric hindrance between antibody molecules, enhances binding efficiency, and provides a novel approach for the rapid and sensitive detection of macromolecules, such as antibodies.

Surface Plasmon Resonance Immunosensor for Direct Detection of Antibodies against SARS-CoV-2 Nucleocapsid Protein.

The strong immunogenicity of the SARS-CoV-2 nucleocapsid protein is widely recognized, and the detection of specific antibodies is critical for COVID-19 diagnostics in patients. This research proposed direct, label-free, and sensitive detection of antibodies against the SARS-CoV-2 nucleocapsid protein (anti-SCoV2-rN). Recombinant SARS-CoV-2 nucleocapsid protein (SCoV2-rN) was immobilized by carbodiimide chemistry on an SPR sensor chip coated with a self-assembled monolayer of 11-mercaptoundecanoic acid. When immobilized under optimal conditions, a SCoV2-rN surface mass concentration of 3.61 ± 0.52 ng/mm2 was achieved, maximizing the effectiveness of the immunosensor for the anti-SCoV2-rN determination. The calculated KD value of 6.49 × 10-8 ± 5.3 × 10-9 M confirmed the good affinity of the used monoclonal anti-SCoV2-rN antibodies. The linear range of the developed immunosensor was from 0.5 to 50 nM of anti-SCoV2-rN, where the limit of detection and the limit of quantification values were 0.057 and 0.19 nM, respectively. The immunosensor exhibited good reproducibility and specificity. In addition, the developed immunosensor is suitable for multiple anti-SCoV2-rN antibody detections.

Enhancing antibody detection sensitivity in lateral flow immunoassays using endospores of Bacillus subtilis as signal amplifiers

Antibody detection is the critical first step for tracking the spread of many diseases including COVID-19. Lateral flow immunoassay (LFIA) is the most commonly used method for rapid antibody detection because it is easy-to-use and inexpensive. However, LFIA has limited sensitivity when gold nanoparticles (AuNPs) are used as the signals. In this study, the endospores of Bacillus subtilis were used in combination with AuNP in a LFIA to detect antibodies. The endospores serve as a signal amplifier. The detection limit was about 10−8 M for anti-beta galactosidase antibody detection whereas the detection limit of conventional LFIA is about 10−6 M. Furthermore, the proposed methods have no additional user steps compared with the traditional LFIA. This method, therefore, improved the sensitivity 100-fold without compromising any advantages of LFIA. We believe that the proposed method will be useful for detection of antibodies against HIV, Zika virus, SARS-CoV-2, and so on.

Challenges in Chlamydial Serology: Insights from a Belgian and a Dutch Population Cohort.

Serology routinely serves as a diagnostic tool to confirm Chlamydia infections in humans. Particularly in delayed settings, such as post-outbreak scenarios where the acute phase of infection has subsided, serology is invaluable. Multiple studies, nonetheless, indicate deficiencies in specificity and sensitivity of current chlamydial antibody detection assays. Incorporation of multiple antigens per target is known to improve the accuracy of chlamydial serological assays. We, therefore, used the recomLine test (Mikrogen diagnostics) on serological samples of two cohorts, as it is the only commercially available test allowing detection of antibodies against three human pathogenic Chlamydia species (C. trachomatis, C. pneumoniae and C. psittaci) using multiple antigens per target. The first cohort (n = 156; samples collected between 2008 and 2022 during a C. trachomatis screening initiative) comprised women from the Netherlands (NL) with past exposure to C. trachomatis, while the second cohort (n = 44; samples collected in 2018 in a health examination survey) consisted of Belgian citizens (BE) with occupational or recreational exposure to chickens, representing a risk population for C. psittaci. The test indicated a statistically equivalent C. pneumoniae seroprevalence in both cohorts (39.10% in NL and 34.09% in BE; p = 0.337). As expected C. trachomatis seroprevalence was significantly higher (p < 0.001) in the Dutch cohort (48.72%), as compared to the Belgian cohort (4.55%). Lastly, C. psittaci seroprevalence did not significantly differ between the two groups (2.27% in BE and 1.92% in NL; p = 0.633), even though a higher prevalence was expected for the Belgian cohort. This prompts us to question whether the Belgian cohort truly constituted a C. psittaci risk population or whether the recomLine test is susceptible to cross-reaction of species-specific antibodies, thereby increasing C. psittaci prevalence in the Dutch cohort. We advocate for the development of affordable, highly sensitive antibody detection assays that can effectively distinguish between chlamydial species, addressing the increasing demand for enhanced serological testing methodologies.

Improved detection sensitivity of anti-PRV variant antibodies through preparation of anti-gB and anti-gE monoclonal antibodies and development of blocking ELISAs

Since 2011, PRV has resurged in China and is characterized by a mutated strain with significant alterations in antigenicity and virulence. Therefore, we hypothesized that antibody detection kits based on classic PRV strains may have limitations in detecting PRV variants. For more sensitive antibody detection of PRV variants, two MABs targeting the gB and gE proteins were developed. IFA revealed that these MABs exhibited strong reactivity toward both classic and variant PRV strains. MAB-gE recognizes a novel conserved linear B-cell epitope (41PSAEVWD47), while MAB-gB recognizes a conformational B-cell epitope. The binding of both MABs was effectively inhibited in the PRV-positive pig blood samples. Accordingly, we established blocking-ELISAs to detect anti-PRV gB and gE antibodies, which achieved higher sensitivity than commercial kits. Moreover, the clinical serum samples results of our method and that of IFA were in high agreement, and our test results had a higher coincidence rate than that of a commercial kit. Assessing antibody levels by our methods at various times following immunization and challenge accurately reflected the trend of antibody-level changes and revealed the conversion to positive antibody status before the commercial kit. Our method is crucial for monitoring PRV infections, assessing immune responses, and controlling disease.

Production of recombinant lumpy skin disease virus A27L and L1R proteins for application in diagnostics and vaccine development

Vaccination using live attenuated vaccines (LAVs) is considered the most effective method for control of lumpy skin disease (LSD). However, this method is limited by safety concerns, with reports of adverse reactions following vaccination. This study evaluates A27L and L1R which are essential proteins for virus attachment and membrane fusion as recombinant sub-unit vaccines against LSD. These proteins were recombinantly expressed in Escherichia coli and purified using affinity chromatography. Purified proteins were formulated individually (A27L or L1R) and in combination (A27L and L1R) with 10% (w/w) Montanide™ Gel 01 PR adjuvant at a final antigen dose of 20 µg per protein. The safety and immunogenicity of these formulations were evaluated in rabbits in a 42-day clinical trial. Animals were vaccinated on day 0 and boost injection administered 21 days later. No reduced morbidity, increased temperature and any other clinical signs were recorded in vaccinated animals for all three vaccine formulations. The highest neutralizing antibody response was detected on day 42 post-primary vaccination for all formulations when using serum neutralising assay. The neutralisation data correlates with antibody titres quantified using a whole cell ELISA. Evaluating the combination of A27L and L1R as potential diagnostic reagents showed highest sensitivity for detection of antibodies against LSD when compared to individual proteins. This study reports the immunogenicity of recombinant A27L and L1R combination for successful application in LSD vaccine development. Furthermore, these proteins demonstrated the potential use in LSD diagnostics.

A Miniature Biomedical Sensor for Rapid Detection of Schistosoma japonicum Antibodies.

Schistosomiasis, typically characterized by chronic infection in endemic regions, has the potential to affect liver tissue and pose a serious threat to human health. Detecting and screening for this disease early on is crucial for its prevention and control. However, existing methods encounter challenges such as low sensitivity, time-consuming processes, and complex sample handling. To address these challenges, we report a soluble egg antigen (SEA)-based functionalized gridless and meander-type AlGaN/GaN high electron mobility transistors (HEMT) sensor for the highly sensitive detection of antibodies to Schistosoma japonicum. Immobilization of the self-assembled membrane on the gate surface was verified using a semiconductor parameter analyzer, scanning electron microscope (SEM), and atomic force microscopy (AFM). The developed biosensor demonstrates remarkable performance in detecting anti-SEA, exhibiting a linear concentration range of 10 ng/mL to 100 μg/mL and a sensitivity of 0.058 mA/log (ng/mL). It also exhibits similar excellent performance in serum systems. With advantages such as rapid detection, high sensitivity, miniaturization, and label-free operation, this biosensor can fulfill the requirements for blood defense.

Ferritin-displayed antigen nanoparticles and nanobody-horseradish peroxidase fusions based-competitive ELISA for the rapid and sensitive detection of antibody against African swine fever virus

African swine fever (ASF) is a highly contagious and lethal hemorrhagic disease, which has brought great distress and economic losses to the world pig industry. For lack of approved vaccines and effective treatment, accurate early detection, both ASF virus (ASFV) antigen and antibody detection in the field, is one of the most important aspects for preventing the outbreak and spread of ASFV. Here we screened the specific nanobodies against the ASFV-P30 protein from the library of immunized Bactrian camel and fused them with horseradish peroxidase (HRP) for expression in HEK293T cells. Moreover, the P30 protein was designed and displayed on the surface of ferritin, the fusion nanocages. Then we developed a novel competitive ELISA (cELISA) based on ferritin-displayed P30 nanoparticles protein (P30-Fn) and nanobody-horseradish peroxidase fusions (P30–Nb71-vHRP) for rapid detection of ASFV antibody in serum sample with high sensitivity and specificity. The cELISA can detect as far as 1:200 diluted clinically positive serums and show no cross-reactivity with positive serums against others porcine viruses, such as PRRSV, PPV, CSFV, TGEV, PEDV, PCV and SIV. The agreement rate is 98.1% with the test results of the commercial ELISA kit for the detection of a total of 267 clinical samples, and the kappa value is 0.96. Furthermore, the cELISA exhibits a good repeatability and the intra- and inter-assay coefficient of variation are less than 10%. In summary, the newly developed cELISA is a simple, rapid, sensitive and low-cost immunoassay, which has a great potential for ASFV antibody detection in clinical samples.

A six-month study of anti-SARS-CoV-2 BNT162b2 mRNA vaccination: A comparative analysis of essential trace elements and anti-RBD IgG sera levels

BackgroundAlthough essential trace elements (ETEs) play pivotal roles in life-supporting biochemical processes, their function in innate and adaptive immunity has not been fully elucidated, particularly during immunization. Furthermore, the association between anti-SARS-CoV-2 specific IgG antibodies and ETE levels with vaccine responsiveness has not been investigated. MethodsThe present study explored the status of ETEs (Mn, Cu, Zn, and Se) in sera of healthy women before and after vaccination with the anti-SARS-CoV-2 BNT162b2 mRNA vaccine in a follow-up period of six months. The main aim was to explore links between ETE levels and IgG antibodies produced against Spike glycoprotein's Receptor-Binding Domain (RBD). ResultsA recombinant protein of SARS-CoV-2 comprising the receptor binding domain was successfully expressed in HEK-293 T cells. The purified protein was suitable for producing a sensitive antibody detection assay for human serum and monitored seropositivity, indicating a transient response with peak anti-SARS-CoV-2 IgG levels 2 months after vaccination. In parallel to increasing antibody titers, serum concentrations of Cu, Mn, and Se were not affected by vaccination, and concentrations remained relatively constant at the different sampling times during the 6-month observation period. Total serum Zn concentrations were slightly elevated when compared between the first and last sampling dates. Overall, no consistent effects of vaccination on any of the three trace elements analyzed in our study were observed. ConclusionVaccination of adult healthy female volunteers with an mRNA vaccine was not associated with consistent changes in serum trace element concentrations over a six-month observation period.

Enzyme treatment of red blood cells: use of ficin and papain.

Proteolytic enzymes are used to treat red blood cells (RBCs) to aid in complex antibody identification. Although there are many enzymes that can be used, for the purpose of this method review, enzyme-treated RBCs refers only to RBCs treated with ficin or papain. Ficin and papain can increase the sensitivity of antibody detection by modifying the RBC membrane. Enzyme treatment and test methods can be performed using one-stage or two-stage procedures. Enzyme treatment is especially useful for the differentiation of multiple antibodies, enhancement of detection of weak antibodies, and adsorption methods. In all cases, quality control is required to ensure adequate treatment of RBCs before additional testing. Ficin and papain are useful tools for both immunohematology reference laboratories and transfusion services.

LGI1 antibody-associated encephalitis without evidence of inflammation in CSF and brain MRI.

This study aimed to explore the frequency and distinct characteristics of adult patients with LGI1 antibody-associated encephalitis in the absence of inflammatory abnormalities in both routine CSF analysis and brain MRI. We conducted a retrospective study of adult patients with antibodies targeting LGI1 and then screened patients with no evidence of inflammation in brain MRI and normal results in routine CSF analysis, including white blood cell count, protein concentration, IgG, and oligoclonal bands. Among 80 patients with LGI1 antibody-associated encephalitis in our center, 31 (38.8%) fulfilled the screening criteria. For these patients, the onset age was 57.0 ± 14.7years, and 19 (61.3%) were female. Viral prodrome occurred in 5 patients (16.1%). Faciobrachial dystonic seizures (FBDS) were the most predominant symptom (38.7%), followed by seizure onset (22.6%) and memory deficits (19.4%). The sensitivity of antibody detection in serum was higher than CSF (96.8% vs. 48.4%, p < 0.001). Most patients (30/31, 96.8%) benefited from the first-line immunotherapy, and 23 patients (74.2%) achieved complete recovery, yet 3 patients (9.7%) had clinical relapses in 2-year follow-up after discharge. The patients had a higher prevalence of females (61.9% vs. 26.7%, p = 0.003) and were more frequently associated with FBDS during the disease course (38.7% vs. 10.2%, p = 0.004). However, there was no difference in treatment outcomes and recurrence ratio between the two groups (p = 0.144 and p = 0.515). Moreover, we divided all 80 patients into four groups according to antibody titer levels in serum and CSF at the time of diagnosis, respectively. WBC and protein concentrations in CSF showed no difference among the four groups. The absence of evidence of inflammation in routine CSF analysis and brain MRI did not rule out anti-LGI1 associated encephalitis. FBDS and the subacute onset of cognitive dysfunction should push forward with autoantibody testing for patients even without inflammatory abnormalities. The routine inflammatory indicators in CSF seemed to be unrelated to antibody titer levels.

Sensitive Detection of Antibodies in Patients with Acute Coxiella Burnetii Infection

Sensitive Detection of Antibodies in Patients with Acute Coxiella Burnetii Infection

Detection and quantification of antibody to SARS CoV 2 receptor binding domain provides enhanced sensitivity, specificity and utility

Accurate and sensitive detection of antibody to SARS-CoV-2 remains an essential component of the pandemic response. Measuring antibody that predicts neutralising activity and the vaccine response is an absolute requirement for laboratory-based confirmatory and reference activity.The viral receptor binding domain (RBD) constitutes the prime target antigen for neutralising antibody. A double antigen binding assay (DABA), providing the most sensitive format has been exploited in a novel hybrid manner employing a solid-phase S1 preferentially presenting RBD, coupled with a labelled RBD conjugate, used in a two-step sequential assay for detection and measurement of antibody to RBD (anti-RBD).This class and species neutral assay showed a specificity of 100 % on 825 pre COVID-19 samples and a potential sensitivity of 99.6 % on 276 recovery samples, predicting quantitatively the presence of neutralising antibody determined by pseudo-type neutralization and by plaque reduction. Anti-RBD is also measurable in ferrets immunised with ChadOx1 nCoV-19 vaccine and in humans immunised with both AstraZeneca and Pfizer vaccines. This assay detects anti-RBD at presentation with illness, demonstrates its elevation with disease severity, its sequel to asymptomatic infection and its persistence after the loss of antibody to the nucleoprotein (anti-NP). It also provides serological confirmation of prior infection and offers a secure measure for seroprevalence and studies of vaccine immunisation in human and animal populations.The hybrid DABA also displays the attributes necessary for the detection and quantification of anti-RBD to be used in clinical practice. An absence of detectable anti-RBD by this assay predicates the need for passive immune prophylaxis in at-risk patients.

Albumin Nanoparticles Loaded with Hemin as Peroxidase Mimics for Immunoassay**

AbstractContemporary immunoassays commonly used in clinical diagnostics mostly utilize enzymes, such as horseradish peroxidase, for signal generation. Numerous research is dedicated to the development of artificial peroxidase‐mimicking catalysts with lower cost, high activity, better operational stability, and tunable properties. Herein we synthesized hemin‐loaded bovine serum albumin (BSA) nanoparticles and applied them as catalytic labels (nanozymes) in colorimetric immunoassay of anti‐tetanus antibodies. Hemin is a key part of the peroxidase catalytic center, possessing peroxidase like‐activity. Albumin nanoparticles were loaded with multiple hemin molecules and decorated with Streptococcal protein G. Resulting nanozymes possessed good colloidal stability and allowed for antibody detection in blood serum. The sensitivity of antibody detection was sufficient for the assessment of post‐vaccination immunity.

Ferritin-Displayed Antigen Nanoparticles and Nanobody-Horseradish Peroxidase Fusions Based-Competitive ELISA for the Rapid and Sensitive Detection of Antibody Against African Swine Fever Virus

Ferritin-Displayed Antigen Nanoparticles and Nanobody-Horseradish Peroxidase Fusions Based-Competitive ELISA for the Rapid and Sensitive Detection of Antibody Against African Swine Fever Virus

A Genetically Encoded Bioluminescent System for Fast and Highly Sensitive Detection of Antibodies with a Bright Green Fluorescent Protein.

A method for fast and highly sensitive detection of antibodies in serum would greatly facilitate the early diagnosis of disease and infection and dose optimization of therapeutic antibody. Bioluminescence detection with LUMABS (renamed mNeonG-LUMABS, where mNeonG is short for mNeonGreen) sensors based on bioluminescence resonance energy transfer (BRET) between blue-emitting luciferase Nluc and green fluorescent protein (FP) mNeonGreen has been demonstrated to enable fast detection of antibodies directly in serum with reasonable sensitivity. However, some mNeonG-LUMABS sensors exhibit low sensitivity, and thus, sensitivity improvement remains imperative. Here, we report a bright green FP, Clover4, obtained by structure-guided mutagenesis of green FP Clover. Despite similar brightness and fluorescence spectra of Clover and mNeonGreen, Clover4-LUMABS sensors exhibit a largely increased dynamic range (maximum 20-fold) and much lower limit of detection (LOD) (maximum 5.6-fold), most likely because Clover4 is positioned in a more parallel orientation to Nluc in LUMABS. Due to modular design, Clover4-LUMABS offers a general BRET system for fast and highly sensitive antibody detection in serum.

Approaching the sensitized lung patient: risk assessment for donor acceptance.

The presence of HLA antibodies is widely recognized as a barrier to solid organ transplantation, and for lung transplant candidates, it has a significant negative impact on both waiting time and waiting list mortality. Although HLA antibodies have been associated with a broad spectrum of allograft damage, precise characterization of these antibodies in allosensitized candidates may enhance their accessibility to transplant. The introduction of Luminex-based single antigen bead (SAB) assays has significantly improved antibody detection sensitivity and specificity, but SAB alone is not sufficient for risk-stratification. Functional characterization of donor-specific antibodies (DSA) is paramount to increase donor accessibility for allosensitized lung candidates. We describe here our approach to evaluate sensitized lung transplant candidates. By employing state-of-the-art technologies to assess histocompatibility and determine physiological properties of circulating HLA antibodies, we can provide our Clinical Team a better risk assessment for lung transplant candidates and facilitate a “road map” to transplant. The cases presented in this paper illustrate the “individualized steps” taken to determine calculated panel reactive antibodies (cPRA), titer and complement-fixing properties of each HLA antibody present in circulation. When a donor is considered, we can better predict the risk associated with potentially crossing HLA antibodies, thereby allowing the Clinical Team to approach allosensitized lung patients with an individualized medicine approach. To facilitate safe access of sensitized lung transplant candidates to potential donors, a synergy between the histocompatibility laboratory and the Clinical Team is essential. Ultimately, donor acceptance is a decision based on several parameters, leading to a risk-stratification unique for each patient.

Magnetofluidic immuno-PCR for point-of-care COVID-19 serological testing

Serological tests play an important role in the fight against Coronavirus Disease 2019 (COVID-19), including monitoring the dynamic immune response after vaccination, identifying past infection and determining community infection rate. Conventional methods for serological testing, such as enzyme-linked immunosorbent assays and chemiluminescence immunoassays, provide reliable and sensitive antibody detection but require sophisticated laboratory infrastructure and/or lengthy assay time. Conversely, lateral flow immunoassays are suitable for rapid point-of-care tests but have limited sensitivity. Here, we describe the development of a rapid and sensitive magnetofluidic immuno-PCR platform that can address the current gap in point-of-care serological testing for COVID-19. Our magnetofluidic immuno-PCR platform automates a magnetic bead-based, single-binding, and one-wash immuno-PCR assay in a palm-sized magnetofluidic device and delivers results in ∼30 min. In the device, a programmable magnetic arm attracts and transports magnetically-captured antibodies through assay reagents pre-loaded in a companion plastic cartridge, and a miniaturized thermocycler and a fluorescence detector perform immuno-PCR to detect the antibodies. We evaluated our magnetofluidic immuno-PCR with 108 clinical serum/plasma samples and achieved 93.8% (45/48) sensitivity and 98.3% (59/60) specificity, demonstrating its potential as a rapid and sensitive point-of-care serological test for COVID-19.

A novel epitope-blocking ELISA for specific and sensitive detection of antibodies against H5-subtype influenza virus hemagglutinin

BackgroundH5-subtype highly pathogenic (HP) avian influenza viruses (AIVs) cause high mortality in domestic birds and sporadic infections in humans with a frequently fatal outcome, while H5N1 viruses have pandemic potential. Due to veterinary and public health significance, these HPAIVs, as well as low pathogenicity (LP) H5-subtype AIVs having a propensity to mutate into HP viruses, are under epidemiologic surveillance and must be reported to the World Organization for Animal Health (OIE). Our previous work provided a unique panel of 6 different monoclonal antibodies (mAbs) against H5 hemagglutinin (HA), which meets the demand for high-specificity tools for monitoring AIV infection and vaccination in poultry. In this study, we selected one of these mAbs to develop an epitope-blocking (EB) ELISA for detecting H5 subtype-specific antibodies in chicken sera (H5 EB-ELISA).MethodsIn the H5 EB-ELISA, H5 HA protein produced in a baculovirus-expression vector system was employed as a coating antigen, and the G-7-27-18 mAb was employed as a blocking antibody. The performance characteristics of the assay were evaluated by testing 358 sera from nonimmunized chickens and chickens immunized with AIVs of the H1–H16 subtypes or recombinant H5 HA antigen to obtain the reference and experimental antisera, respectively. The samples were classified as anti-H5 HA positive or negative based on the results of the hemagglutination inhibition (HI) assay, the gold standard in subtype-specific serodiagnosis.ResultsThe H5 EB-ELISA correctly discriminated between the anti-H5 HA negative sera, including those against the non-H5 subtype AIVs, and sera positive for antibodies against the various-origin H5 HAs. Preliminary validation showed 100% analytical and 97.6% diagnostic specificities of the assay and 98.0% and 99.1% diagnostic sensitivities when applied to detect the anti-H5 HA antibodies in the reference and experimental antisera, respectively.ConclusionsThe H5 EB-ELISA performed well in terms of diagnostic estimates. Thus, further optimization and validation work using a larger set of chicken sera and receiver operating characteristic (ROC) analysis are warranted. Moreover, the present assay provides a valuable basis for developing multispecies screening tests for birds or diagnostic tests for humans.

Novel ELISA Protocol Links Pre-Existing SARS-CoV-2 Reactive Antibodies With Endemic Coronavirus Immunity and Age and Reveals Improved Serologic Identification of Acute COVID-19 via Multi-Parameter Detection.

The COVID-19 pandemic has drastically impacted work, economy, and way of life. Sensitive measurement of SARS-CoV-2 specific antibodies would provide new insight into pre-existing immunity, virus transmission dynamics, and the nuances of SARS-CoV-2 pathogenesis. To date, existing SARS-CoV-2 serology tests have limited utility due to insufficient reliable detection of antibody levels lower than what is typically present after several days of symptoms. To measure lower quantities of SARS-CoV-2 IgM, IgG, and IgA with higher resolution than existing assays, we developed a new ELISA protocol with a distinct plate washing procedure and timed plate development via use of a standard curve. Very low optical densities from samples added to buffer coated wells at as low as a 1:5 dilution are reported using this ‘BU ELISA’ method. Use of this method revealed circulating SARS-CoV-2 receptor binding domain (RBD) and nucleocapsid protein (N) reactive antibodies (IgG, IgM, and/or IgA) in 44 and 100 percent of pre-pandemic subjects, respectively, and the magnitude of these antibodies tracked with antibody levels of analogous viral proteins from endemic coronavirus (eCoV) strains. The disease status (HIV, SLE) of unexposed subjects was not linked with SARS-CoV-2 reactive antibody levels; however, quantities were significantly lower in subjects over 70 years of age compared with younger counterparts. Also, we measured SARS-CoV-2 RBD- and N- specific IgM, IgG, and IgA antibodies from 29 SARS-CoV-2 infected individuals at varying disease states, including 10 acute COVID-19 hospitalized subjects with negative serology results by the EUA approved Abbott IgG chemiluminescent microparticle immunoassay. Measurements of SARS-CoV-2 RBD- and N- specific IgM, IgG, IgA levels measured by the BU ELISA revealed higher signal from 9 of the 10 Abbott test negative COVID-19 subjects than all pre-pandemic samples for at least one antibody specificity/isotype, implicating improved serologic identification of SARS-CoV-2 infection via multi-parameter, high sensitive antibody detection. We propose that this improved ELISA protocol, which is straightforward to perform, low cost, and uses readily available commercial reagents, is a useful tool to elucidate new information about SARS-CoV-2 infection and immunity and has promising implications for improved detection of all analytes measurable by this platform.