Antibodies In Human Sera Research Articles

Label-free impedimetric immunosensor for point-of-care detection of COVID-19 antibodies

The COVID-19 pandemic has posed enormous challenges for existing diagnostic tools to detect and monitor pathogens. Therefore, there is a need to develop point-of-care (POC) devices to perform fast, accurate, and accessible diagnostic methods to detect infections and monitor immune responses. Devices most amenable to miniaturization and suitable for POC applications are biosensors based on electrochemical detection. We have developed an impedimetric immunosensor based on an interdigitated microelectrode array (IMA) to detect and monitor SARS-CoV-2 antibodies in human serum. Conjugation chemistry was applied to functionalize and covalently immobilize the spike protein (S-protein) of SARS-CoV-2 on the surface of the IMA to serve as the recognition layer and specifically bind anti-spike antibodies. Antibodies bound to the S-proteins in the recognition layer result in an increase in capacitance and a consequent change in the impedance of the system. The impedimetric immunosensor is label-free and uses non-Faradaic impedance with low nonperturbing AC voltage for detection. The sensitivity of a capacitive immunosensor can be enhanced by simply tuning the ionic strength of the sample solution. The device exhibits an LOD of 0.4 BAU/ml, as determined from the standard curve using WHO IS for anti-SARS-CoV-2 immunoglobulins; this LOD is similar to the corresponding LODs reported for all validated and established commercial assays, which range from 0.41 to 4.81 BAU/ml. The proof-of-concept biosensor has been demonstrated to detect anti-spike antibodies in sera from patients infected with COVID-19 within 1 h.Photolithographically microfabricated interdigitated microelectrode array sensor chips & label-free impedimetric detection of COVID-19 antibody.

Expression and Evaluation of Wb-SXP-1 and Wb-123 Recombinant Antigens as Potential Diagnostic Biomarkers for Lymphatic Filariasis

Lymphatic filariasis (LF) remains a public health concern as it can cause permanent morbidity and disability to those infected. While the global elimination of LF in these endemic areas is ongoing through mass drug administration, there is the need to develop diagnostic tools that would be utilized to track the progress of total global eradication as well as perform surveillance for the recurrence of lymphatic filariasis transmission. Currently, approved LF diagnosis tools are faced with lack of specificity, low sensitivity, and periodicity dependence. Recombinant filarial antigen-based assays can address these drawbacks and offer practical instruments for LF diagnosis and surveillance. This present study, evaluated rWb-SXP-1 and rWb-123 antigens as potential diagnostic biomarker tools for Wuchereria banchrofti in human sera using microspheres-based multiplex serological assay. Based on statistical analysis using XLSTAT 2019 (Addinsoft) on data generated from multiplex technology assay, generated ROC curves for both rWb-SXP-1 and rWb-123 demonstrated 87.1% sensitivity to Wuchereria banchrofti human sera with rWb-SXP-1 antigens having the highest specificity of 96%. Indication that rWb-SXP-1 and rWb-123 antigens are capable of detecting immunoglobulin G4 (IgG4) antibodies in human sera synthesized specifically against W. banchrofti infections. Therefore, rWb-SXP-1 and rWb-123 antigens can be utilized to detect W. banchrofti infections by antibody profiling with excellent diagnostic sensitivity and specificity using microsphere-based multiplex serological tests. This method can be particularly practical for screening a large number of sera samples and/or for quick, extensive field-testing due to the high-throughput and quick formats applied.

Synthesis of methylphosphorylated oligomannosides structurally related to lipopolysaccharide O-antigens of Klebsiella pneumoniae serotype O3 and their application for detection of specific antibodies in rabbit and human sera.

Methylphosphorylated mono-, di- and trimannosides structurally related to the lipopolysaccharide (LPS) O-antigens of Klebsiella pneumoniae of serotype O3 were synthesized and conjugated with a biotin tag. The stereo- and regioselective assembly of target carbohydrate chains was conducted using uniform monosaccharide synthetic blocks. After that, a methylphosphate group was introduced by coupling with a methyl-H-phosphonate reagent followed by oxidation and deprotection to give the target oligosaccharides. The 1H and 13C NMR spectra of the obtained compounds showed a good fit with the spectrum of the corresponding natural polysaccharide. The newly prepared biotinylated oligosaccharides along with the previously reported biotinylated glycoconjugates related to galactan I and galactan II of K. pneumoniae LPS were used for the ELISA detection of antibodies in anti-K. pneumoniae rabbit sera. Anti-O3 serum antibodies specifically recognized the synthesized oligosaccharide ligands with terminal methylphosphomannosyl residues, whereas anti-O1 serum antibodies recognized the oligosaccharide related to K. pneumoniae galactan II. The analysis of human sera from patients with confirmed Klebsiella infection also revealed the presence of antibodies against the synthesized oligosaccharides in clinical cases. Thus, the described compounds together with other Klebsiella related antigenic oligosaccharides could be potentially used as molecular probes for K. pneumoniae serological diagnostics development and strain serotyping.

An Optimised Indirect ELISA Protocol for Detection and Quantification of Anti-viral Antibodies in Human Plasma or Serum: A Case Study Using SARS-CoV-2.

Advanced immunoassays are crucial in assessing antibody responses, serving immune surveillance goals, characterising immunological responses to evolving viral variants, and guiding subsequent vaccination initiatives. This protocol outlines an indirect ELISA protocol to detect and quantify virus-specific antibodies in plasma or serum after exposure to viral antigens. The assay enables the measurement of IgG, IgA, and IgM antibodies specific to the virus of interest, providing qualitative and quantitative optical densities and concentration data. Although this protocol refers to SARS-CoV-2, its methodology is versatile and can be modified to assess antibody responses for various viral infections and to evaluate vaccine trial outcomes. Key features • This protocol builds upon previously described methodology [1] explicitly tailored for SARS-CoV-2 and broadens its applicability to other viral infections. • The protocol outlines establishing antibody responses to SARS-CoV-2 infections by determining optical densities and concentrations from blood plasma or serum.

Development and Validation of the ELISA Method for Anti-trastuzumab Antibodies Determination in Human Serum

Introduction. One of the widely used specific anti-HER2 (human epidermal growth factor receptor 2) MAb drugs is trastuzumab. Trastuzumab is highly effective for malignant HER2 hyperexpression reduction, which results in HER2 oncogenicity decrease. As any other biotherapeutics trastuzumab can cause immunological adverse reactions, e.g. immunogenicity or anti-drug antibodies (ADAs) production.Aim. The aim of this study was to develop and validate the analytical method for anti-trastuzumab antibodies determination in human blood serum.Materials and methods. The semi-quantitative anti-trastuzumab antibody determination was carried out by the ELISA method combined with ACE technique, using spectrophotometric detection in the visible range of the spectrum.Results and discussion. The developed method was validated for cut point, selectivity, sensitivity, "hook" effect, drug tolerance, precision and stability (short-term and long-term). To decrease the background noise from non-specific binding of sera components, the minimum required dilution value was determined at 10 % serum. The calculated values for screening cut point (normalization factor) and confirmatory cut point were 0.004 and 34.59 %, respectively. The sensitivity of the developed method was estimated at 99.5 ng/mL of anti-trastuzumab antibodies.Conclusion. The obtained results allow us to use the developed ACE ELISA method for the determination of anti-trastuzumab antibodies in human serum during trastuzumab safety clinical trials.

The SARS-CoV-2 specific IgG antibodies biophotonic sensor.

In this paper, we present the design and the principle of operation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific immunoglobulin G (IgG) biophotonic sensor, which is based on the single-mode telecommunication fiber. We fabricated the sensor head at the face of the single mode fiber-28. Due to the process of bio-functionalization, our sensor has the ability to selectively detect the SARS-CoV-2 specific IgG antibodies. The results of preliminary tests allowed us to correctly determine the presence of antibodies in less than 1 min in 5 μl in a volume sample of concentration of 10 μg/ml, which according to studies, corresponds to the concentration of IgG antibodies in human serum. Additionally, the tested sample can be smaller than 5 μl in volume.

Electrochemical Sensing of Cortisol in Human Saliva and Serum Using DNA-Steroid Conjugation with a Versatile DNA Nanostructure Sensor

Having a generalizable point of care (POC) method for clinically relevant biomolecules would greatly enhance healthcare management and disease diagnosis. Recently our group developed a novel DNA nanostructure architecture for versatile detection of analytes which is generalizable for assaying several small molecules and their larger protein binding partners (e.g. antibodies) in human serum. The DNA nanostructure is built through on-electrode enzymatic ligation of three oligos for electrode attachment, anchor binding, and electrochemical signaling. In this study, we developed an economical synthetic approach for making oligonucleotide-steroid conjugates, and we explored the capability of these DNA nanostructure sensors for small molecule/steroid detection.Cortisol is a steroid hormone secreted by the hypothalamic-pituitary-adrenal system in response to the body’s stress level. As the main stress hormone, it controls processes such as immune, adrenal, circulatory, and metabolic. Moreover, anomalies of cortisol levels can result in serious conditions such as Addison’s disease, Cushing’s syndrome, and adrenal insufficiencies. Every year, 120,000 deaths are attributed, in part, to elevated levels of stress. To minimize these issues, a simplified method for cortisol sensing is vital. In this study, we conjugated cortisol to amine-tagged DNA and used the conjugates in our sensors. Anti-cortisol antibodies induced a 67% signal drop, validating conjugation. A calibration curve for cortisol showed a limit of detection of 800 pM and a dynamic range of 1 – 100 nM. The sensor was validated in saliva and human serum samples using the gold standard method, ELISA. Changing cortisol levels in two human patients' saliva samples were successfully detected after several collection times throughout a single day. As a potential point-of-care (POC) detection device, our novel cortisol biosensor could detect the hormone in human serum and saliva samples within 6 minutes.

Target-templated molecular nano-machine for auto-catalytic recycling amplification detection of low-abundant antibody in human serum

The development of convenient and sensitive sensing method capable of monitoring low-abundant antibody is appealing for pathogens diagnosis and vaccine immunotherapy evaluation. With regards to this, we herein constructed a simple molecular nano-machine for ultrasensitive monitoring of Anti-Digoxigenin (Anti-Dig) antibody via a target-initiated auto-cycling proximity recording (APR) amplification. One target bivalent Anti-Dig antibody can simultaneously recognize two Dig-labeled catalytic hairpin DNAs (Dig-HP1) and bring them into close distance to form the proximity catalytic probe pairs. Subsequently, the 3′-overhang of the FAM/Dabycl labeled hairpin signal probes hybridize on the exposure primer binding domain at Dig-HP1 template and initiate the APR reaction to cross open and extend Dig-HP1 to generate many elongated DNA duplexes with the help of Bst DNA polymerase. These DNA duplexes make the fluorophore and quencher away from each other and thereby produce dramatically enhanced fluorescence signal for sensitive detection of target Anti-Dig antibody down to 0.18 nM. Moreover, this method can selectively monitor Anti-Dig antibody against other interference proteins, and also be extended to detect Anti-Dig antibody in serum samples, confirming its potential for ultrasensitive detection of low-abundant antibodies for early disease therapy and vaccine evaluation.

Development of colorimetric cellulose-based test-strip for the rapid detection of antibodies against SARS-CoV2 virus.

Given the pandemic situation, there is an urgent need for an accurate test to monitor antibodies anti-SARS-CoV-2, providing crucial epidemiological and clinical information to monitor the evolution of coronavirus disease in 2019 (COVID-19) and to stratify the immunized and asymptomatic population. Therefore, this paper describes a new cellulose-based test strip for rapid and cost-effective quantitative detection of antibodies to SARS-CoV2 virus by colorimetric transduction. For this purpose, Whatman paper was chemically modified with sodium metaperiodate to introduce aldehyde groups on its surface. Subsequently, the spike protein of the virus is covalently bound by forming an imine group. The chemical control of cellulose paper modification was evaluated by Fourier transform infrared spectroscopy, thermogravimetry and contact angle analysis. Colorimetric detection of the antibodies was performed by a conventional staining method using Ponceau S solution as the dye. Color analysis was performed after image acquisition with a smartphone using Image J software. The color intensity varied linearly with the logarithm of the anti-S concentration (from 10 ng/mL to 1 μg/mL) in 500-fold diluted serum samples when plotted against the green coordinate extracted from digital images. The test strip was selective in the presence of nucleocapsid antibodies, urea, glucose, and bovine serum albumin with less than 15% interference, and detection of antibodies in human serum was successfully performed. Overall, this is a simple and affordable design that can be readily used for mass population screening and does not require sophisticated equipment or qualified personnel.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10570-022-04808-y.

Target-triggered catalytic hairpin assembly activation of CRISPR/Cas12a for amplified detection of therapeutic monoclonal antibody

It is of great significance to monitor therapeutic antibodies in human serum to obtain the pharmacokinetic data for assessing their therapeutic efficacy at the dosage of administration. Here, based on the target-triggered proximity binding approach, as well as the catalytic hairpin assembly (CHA) and CRISPR/Cas12a cleavage dual signal amplification strategy, we describe the establishment of a highly sensitive method for detecting panitumumab, a cancer therapeutic monoclonal antibody, in diluted human serums. The two proximity probes bind the target panitumumab simultaneously to form initiation sequences for subsequent CHA for the cyclic yield of many dsDNAs, which further activate the specific trans-cleavage activity of Cas12a to digest lots of fluorescently quenched ssDNAs to restore drastically enhanced fluorescence for detecting panitumumab with a 0.62 pM detection limit. Besides, the sensing approach can selectively discriminate panitumumab against other monoclonal antibodies and realize low levels of panitumumab detection in diluted serums. Moreover, by simple replacement of the proximity recognition probes, the established method can be easily expanded for the development of versatile sensing platforms for different biomolecules in addition to antibodies.

A lateral flow assay for the immunodiagnosis of human cat-transmitted sporotrichosis.

BackgroundCat‐transmitted sporotrichosis (CTS) caused by Sporothrix brasiliensis has emerged as an important zoonosis in Brazil and neighbouring countries.ObjectivesEvaluate the performance of a lateral flow assay (LFA) for the detection of anti‐Sporothrix antibodies in human sera.MethodsA LFA for the detection of anti‐Sporothrix antibodies (Anti‐Sporo LFA) in human sera, developed by IMMY, was evaluated using 300 human sera collected prospectively at the Hospital de Clínicas, Federal University of Paraná (HC‐UFPR), in Curitiba, Brazil. These specimens included 100 sera from patients with CTS. CTS cases were classified as follows: 59 lymphocutaneous, 27 fixed cutaneous,13 ocular, and one mixed form. One‐hundred specimens from patients with other mycoses, including cryptococcosis (n = 32), candidemia (n = 27), paracoccidioidomycosis (n = 14), aspergillosis (n = 10), histoplasmosis (n = 9), fusariosis (n = 4), lobomycosis (n = 1), chromoblastomycosis (n = 1), mucormycosis (n = 1) and trichosporonosis (n = 1). And 100 specimens from apparently healthy volunteers (AHV).ResultsThe Anti‐Sporo LFA showed a global sensitivity of 83% (95% confidence interval [CI] = 74%–90%), a global specificity of 82% (95% CI = 76%–87%), and accuracy of 82% (95% CI = 77%–86%). By clinical form sensitivity was as follows: Mixed form 100%, ocular 92%, lymphocutaneous 83% and fixed cutaneous 78%. False‐positive results were observed in 11 specimens from people with other mycoses and 26 specimens from AHV.Conclusion and discussionThis study presents the results of the evaluation of the first lateral flow assay for the detection of anti‐Sporothrix antibodies in human sera. The findings here show evidence that IMMY's Anti‐Sporo LFA is a promising tool for the rapid diagnosis of CTS.

Ultrabright nanoparticle-labeled lateral flow immunoassay for detection of anti-SARS-CoV-2 neutralizing antibodies in human serum

The level of anti-SARS-CoV-2 neutralizing antibodies (NAb) is an indispensable reference for evaluating the acquired protective immunity against SARS-CoV-2. Here, we established an ultrabright nanoparticles-based lateral flow immunoassay (LFIA) for one-step rapid semi-quantitative detection of anti-SARS-CoV-2 NAb in vaccinee's serum. Once embedded in polystyrene (PS) nanoparticles, the aggregation-induced emission (AIE) luminogen, AIE490, exhibited ultrabright fluorescence due to the rigidity of PS and severe inhibition of intramolecular motions. The ultrabright AIE490-PS nanoparticle was used as a fluorescent marker of LFIA. Upon optimized conditions including incubation time, concentrations of coated proteins and conjugated nanoparticles, amounts of antigens modified on the surface of nanoparticles, dilution rate of serum samples, and so on, the ultrabright nanoparticles-based LFIA could accurately identify 70 negative samples and 63 positive samples from human serum (p < 0.0001). The intra- and inter-assay precisions of the established method are above 13% and 16%, respectively. The established LFIA has tremendous practical value of generalization as a rapid semi-quantitative detection method of anti-SARS-CoV-2 NAb. Meanwhile, the AIE490-PS nanoparticle is also promising to detect many other analytes by altering the protein on the surface.

Development and validation of an enzyme-linked immunoassay kit for diagnosis and surveillance of COVID-19

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli. Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-CoV-2 antibodies in human sera with high reliability.

DUPLICATE: DEVELOPMENT AND VALIDATION OF AN ENZYME-LINKED IMMUNOASSAY KIT FOR DIAGNOSIS AND SURVEILLANCE OF COVID-19

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli. Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-CoV-2 antibodies in human sera with high reliability.

An impedimetric biosensor for COVID-19 serology test and modification of sensor performance via dielectrophoresis force

Coronavirus disease 2019 (COVID-19) has caused significant global morbidity and mortality. The serology test that detects antibodies against the disease causative agent, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has often neglected value in supporting immunization policies and therapeutic decision-making. The ELISA-based antibody test is time-consuming and bulky. This work described a gold micro-interdigitated electrodes (IDE) biosensor for COVID antibody detection based on Electrochemical Impedance Spectroscopy (EIS) responses. The IDE architecture allows easy surface modification with the viral structure protein, Spike (S) protein, in the gap of the electrode digits to selectively capture anti-S antibodies in buffer solutions or human sera. Two strategies were employed to resolve the low sensitivity issue of non-faradic impedimetric sensors and the sensor fouling phenomenon when using the serum. One uses secondary antibody-gold nanoparticle (AuNP) conjugates to further distinguish anti-S antibodies from the non-specific binding and obtain a more significant impedance change. The second strategy consists of increasing the concentration of target antibodies in the gap of IDEs by inducing an AC electrokinetic effect such as dielectrophoresis (DEP). AuNP and DEP methods reached a limit of detection of 200 ng/mL and 2 μg/mL, respectively using purified antibodies in buffer, while the DEP method achieved a faster testing time of only 30 min. Both strategies could qualitatively distinguish COVID-19 antibody-positive and -negative sera. Our work, especially the impedimetric detection of COVID-19 antibodies under the assistance of the DEP force presents a promising path toward rapid, point-of-care solutions for COVID-19 serology tests.

Mutational analysis of SARS-CoV-2 variants of concern reveals key tradeoffs between receptor affinity and antibody escape.

SARS-CoV-2 variants with enhanced transmissibility represent a serious threat to global health. Here we report machine learning models that can predict the impact of receptor-binding domain (RBD) mutations on receptor (ACE2) affinity, which is linked to infectivity, and escape from human serum antibodies, which is linked to viral neutralization. Importantly, the models predict many of the known impacts of RBD mutations in current and former Variants of Concern on receptor affinity and antibody escape as well as novel sets of mutations that strongly modulate both properties. Moreover, these models reveal key opposing impacts of RBD mutations on transmissibility, as many sets of RBD mutations predicted to increase antibody escape are also predicted to reduce receptor affinity and vice versa. These models, when used in concert, capture the complex impacts of SARS-CoV-2 mutations on properties linked to transmissibility and are expected to improve the development of next-generation vaccines and biotherapeutics.

Detection of Bourbon Virus-Specific Serum Neutralizing Antibodies in Human Serum in Missouri, USA.

ABSTRACTBourbon virus (BRBV) was first discovered in 2014 in a fatal human case. Since then it has been detected in the tick Amblyomma americanum in the states of Missouri and Kansas in the United States. Despite the high prevalence of BRBV in ticks in these states, very few human cases have been reported, and the true infection burden of BRBV in the community is unknown. Here, we developed two virus neutralization assays, a vesicular stomatitis virus (VSV)-BRBV pseudotyped rapid assay and a BRBV focus reduction neutralization assay, to assess the seroprevalence of BRBV neutralizing antibodies in human sera collected in 2020 in St. Louis, MO. Of 440 human serum samples tested, three (0.7%) were able to potently neutralize both VSV-BRBV and wild-type BRBV. These findings suggest that human infections with BRBV are more common than previously recognized.IMPORTANCE Since the discovery of the Bourbon virus (BRBV) in 2014, a total of five human cases have been identified, including two fatal cases. BRBV is thought to be transmitted by the lone star tick, which is prevalent in the eastern, southeastern, and midwestern United States. BRBV has been detected in ticks in Missouri and Kansas, and serological evidence suggests that it is also present in North Carolina. However, the true infection burden of BRBV in humans is not known. In the present study, we developed two virus neutralization assays to assess the seroprevalence of BRBV-specific antibodies in human sera collected in 2020 in St. Louis, MO. We found that a small subset of individuals are seropositive for neutralizing antibodies against BRBV. Our data suggest that BRBV infection in humans is more common than previously thought.

Both Natural and Induced Anti-Sda Antibodies Play Important Roles in GTKO Pig-to-Rhesus Monkey Xenotransplantation.

Sda, produced by the B4GALNT2 enzyme, has been recognized as an important xenoantigen for pig-to-nonhuman primate xenotransplantation. However, little is known about Sda expression in pigs and its immunogenicity in xenotransplantation. In this study, peripheral blood mononuclear cells (PBMCs) were isolated from wildtype, GTKO (with high, moderate, and low Sda expression), GTKO/β4GalNT2KO, GTKO/CMAHKO, or GTKO/CMAHKO/β4GalNT2KO pigs. Anti-pig IgM/IgG binding and complement-dependent cytotoxicity (CDC) to pig PBMCs was measured by flow cytometry using pooled rhesus monkey sera (n=20) or human sera (n=20). As compared to wild-type pigs (n=12), GTKO pigs (n=17) had a significantly higher mean level of Sda expression on PBMCs and showed a greater individual difference in expression. Both the overall binding of monkey serum IgM/IgG antibody to GTKO pig PBMCs and CDC against these PBMCs decreased significantly with a progressive reduction in Sda expression, showing a clear dose-effect relationship. Both the monkey serum antibody binding and CDC decreased significantly after the additional deletion of Sda, whereas the binding of human serum antibody and CDC against the GTKO pig PBMCs were markedly reduced after the deletion of Neu5Gc in the pigs. In addition, anti-Sda antibody accounted for > 50% of the induced anti-non-Gal antibody at the time of rejection in two rhesus monkeys that received GTKO/hCD55 pig kidney xenotransplantation, and the anti-Sda antibody showed significant cytotoxic activity against GTKO pig cells. We conclude that both natural and induced anti-Sda antibodies play important roles in GTKO pig-to-rhesus monkey xenotransplantation, thus providing further evidence for GTKO/β4GalNT2KO pigs as the preferred organ source for rhesus monkeys as a preclinical model of xenotransplantation.

Sensitive and specific serological ELISA for the detection of SARS-CoV-2 infections

BackgroundThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered the worldwide coronavirus disease 2019 (COVID-19) pandemic. Serological assays for the detection of SARS-CoV-2 infections are important to understand the immune response in patients and to obtain epidemiological data about the number of infected people, especially to identify asymptomatic persons not aware of a past infection.MethodsWe recombinantly produced SARS-CoV-2 nucleocapsid (N)-protein in Escherichia coli. We used the purified protein to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV-2 specific antibodies. This ELISA method was optimized and validated with serum samples collected from 113 patients with RT-PCR-confirmed SARS-CoV-2 infections including hospitalized COVID-19 patients and 1500 control sera mostly collected before 2015 with different clinical background.ResultsThe optimized N-protein-ELISA provided a sensitivity of 89.7% (n = 68) for samples collected from patients with confirmed SARS-CoV-2 infections and mild to severe symptoms more than 14 days after symptom onset or a positive PCR test. The antibody levels remained low for serum samples collected in the first six days (n = 23) and increased in the second week (n = 22) post symptom onset or PCR confirmation. At this early phase, the ELISA provided a sensitivity of 39.1% and 86.4%, respectively, reflecting the time of an IgG immune response against pathogens. The assay specificity was 99.3% (n = 1500; 95% CI 0.995–0.999). Serum samples from persons with confirmed antibody titers against human immunodeficiency viruses 1/2, parvovirus B19, hepatitis A/B virus, cytomegalovirus, Epstein Barr virus, and herpes simplex virus were tested negative.ConclusionsWe conclude that the N-protein-based ELISA developed here is well suited for the sensitive and specific serological detection of SARS-CoV-2 specific IgG antibodies in human serum for symptomatic infections. It may also prove useful to identify previous SARS-CoV-2 infections in vaccinated people, as all currently approved vaccines rely on the SARS-CoV-2 spike (S-) protein.

Robust validation and performance comparison of immunogenicity assays assessing IgG and neutralizing antibodies to SARS-CoV-2.

To enable benchmarking of immunogenicity between candidate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, there is a need for standardized, validated immunogenicity assays. In this article, we report the design and criteria used to validate immunogenicity assays and the outcome of the validation of serologic and functional assays for the evaluation of functional immune response and antibody titers in human serum. A quantitative cell-based microneutralization (MNT) assay, utilizing a reference standard, for detecting anti-SARS-CoV-2 spike protein-neutralizing antibodies in human serum and Meso Scale Discovery’s multiplex electrochemiluminescence (MSD ECL) assay for immunoglobulin G (IgG) antibodies to SARS-CoV-2 spike, nucleocapsid, and receptor-binding domain (RBD) proteins were assessed for precision, accuracy, dilutional linearity, selectivity, and specificity using pooled human serum from coronavirus disease 2019 (COVID-19)-confirmed recovered donors. Both assays met prespecified acceptance criteria for precision, relative accuracy, dilutional linearity, selectivity, and specificity. Both assays demonstrated high specificity for the different SARS-CoV-2 antigens or virus tested, and no significant cross-reactivity with seasonal coronaviruses. An evaluation to compare the neutralizing activity in the MNT assay to the IgG measured using the MSD ECL assay showed a strong correlation between the presence of neutralizing activity and amount of antibodies against the spike and RBD proteins in sera from both convalescent and vaccinated individuals. Finally, the MNT assay was calibrated to the WHO reference standard to enable reporting of results in international units, thus facilitating comparison of immunogenicity data generated by different assays and/or laboratories. The MSD ECL assay has previously been calibrated. In conclusion, these validated assays for the evaluation of functional immune response and antibody titers following SARS-CoV-2 vaccination could provide a relatively simple standardized approach for accurately comparing immune responses to different vaccines and/or vaccination regimens.