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1.
Epidemiol Infect ; 150: e177, 2022 Oct 19.
Article in English | MEDLINE | ID: covidwho-2106269

ABSTRACT

Limited prospective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) data in children regarding the impact of Omicron variant in seropositivity have been reported. We investigated SARS-CoV-2 seropositivity in children between 1 September 2021 and 30 April 2022, representing Delta and Omicron predominance periods. Serum samples from children admitted to the major tertiary Greek paediatric hospital for any cause, except for COVID-19, were randomly collected and tested for SARS-CoV-2 natural infection antibodies against nucleocapsid antigen (Elecsys® Anti-SARS-CoV-2 reagent). A total of 506/1312 (38.6%) seropositive children (0-16 years) were detected (males: 261/506(51.6%); median age (IQR): 95.2 months(24-144)). Seropositivity rates (%) increased from Delta to Omicron period from 29.7% to 48.5% (P-value<0.0001). Seropositivity increased for all age groups, except for the age group of 0-1 year (P-value:0.914). The highest seropositivity rate was detected in April 2022 (52.6%) and reached 73.9% specifically for the age group 12-16 years. No significant differences were detected in seropositivity with respect to gender, origin, or hospitalisation status. Median (IQR) antibody titres were higher in the Omicron vs. Delta period in all age groups, especially in 12-16 years [32.2 COI (7-77.1) vs. 11.4 COI(2.8-50.2), P-value:0.009). During Omicron variant period increased SARS-CoV-2 seropositivity was detected in paediatric population, especially in adolescents, implicating either increased transmissibility or reinfection rates.


Subject(s)
COVID-19 , SARS-CoV-2 , Adolescent , Child , Humans , Infant , Infant, Newborn , Male , Antibodies, Viral , COVID-19/epidemiology , Enzyme-Linked Immunosorbent Assay/methods , Prospective Studies , Seroepidemiologic Studies , Female , Child, Preschool
2.
Int J Mol Sci ; 23(20)2022 Oct 17.
Article in English | MEDLINE | ID: covidwho-2071517

ABSTRACT

Transmembrane glycoprotein integrins play crucial roles in biochemical processes, and by their inhibition or activation, different signal pathways can be disrupted, leading to abnormal physiological functions. We have previously demonstrated the inhibitory effect of glyphosate herbicide's active ingredient on cell adhesion and its αvß3 integrin antagonist effect. Therefore, it appeared particularly exciting to investigate inhibition of glyphosate and its metabolites on a wider range of Arg-Gly-Asp (RGD) binding integrins, namely αvß3, α5ß1 and αllbß3. Thus, the purpose of this study was to assess how extended the inhibitory effect observed for glyphosate on the integrin αvß3 is in terms of other RGD integrins and other structurally or metabolically related derivatives of glyphosate. Five different experimental setups using enzyme-linked immunosorbent assays were applied: (i) αvß3 binding to a synthetic polymer containing RGD; (ii) αvß3 binding to its extracellular matrix (ECM) protein, vitronectin; (iii) α5ß1 binding to the above polymer containing RGD; (iv) αllbß3 binding to its ECM protein, fibrinogen and (v) αvß3 binding to the SARS-CoV-2 spike protein receptor binding domain. Total inhibition of αvß3 binding to RGD was detected for glyphosate and its main metabolite, aminomethylphosphonic acid (AMPA), as well as for acetylglycine on α5ß1 binding to RGD.


Subject(s)
COVID-19 , Herbicides , Humans , Integrin alphaVbeta3/metabolism , Vitronectin , Herbicides/pharmacology , SARS-CoV-2 , Oligopeptides/chemistry , Enzyme-Linked Immunosorbent Assay , Fibrinogen , Polymers
3.
Anal Biochem ; 658: 114902, 2022 Dec 01.
Article in English | MEDLINE | ID: covidwho-2031059

ABSTRACT

The development of the Coronavirus disease 2019 (COVID-19) vaccine is one of the most important efforts in controlling the pandemic. Serological tests are used to identify highly reactive human donors for convalescent plasma therapy, measuring vaccine efficacy and durability. This review article presents a review of serology tests and how antibody titers in response to vaccines have been developed. Some of the serological test methods discussed are Plaque Reduction Neutralization Test (PRNT), Enzyme-Linked Immunosorbent Assay (ELISA), Lateral flow immunoassay (LFIA), chemiluminescent immunoassay (CLIA), and Chemiluminescent Micro-particle Immunoassay (CMIA). This review can provide an understanding of the application of the body's immune response to vaccines to get some new strategies for vaccines.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , COVID-19/diagnosis , COVID-19/prevention & control , Clinical Laboratory Techniques/methods , Antibodies, Viral , Serologic Tests/methods , Enzyme-Linked Immunosorbent Assay/methods , Vaccination , Antibodies, Neutralizing
4.
Vopr Virusol ; 67(4): 331-340, 2022 09 12.
Article in Russian | MEDLINE | ID: covidwho-2030645

ABSTRACT

INTRODUCTION: The main approach to the rabies prevention is the vaccination of domestic and wild carnivores. For the routine evaluation the anti-rabies vaccination effectiveness, World Organization for Animal Health (OIE) recommends various enzyme-linked immunosorbent assays (ELISA).The aim of the study was to design and validate a competitive ELISA (cELISA) test system for the detection of antibodies to the rabies virus (RABV). MATERIALS AND METHODS: The development of the cELISA was carried out following the OIE recommendations. RESULTS: The repeatability of the cELISA results within one laboratory was satisfactory (coefficient of variation 7.95-13.61%). The coefficient of determination (CD) between the results of the virus neutralization reaction (FAVN) and cELISA was 0.988, p < 0.001. The lower threshold for antibody detection was less than 0.02 IU/ml. The cELISA did not demonstrate cross-reactivity against antibodies to canine distemper virus, parainfluenza virus, parvovirus, coronavirus, and canine adenovirus (types I and II). During the study of 137 dog blood sera, diagnostic specificity (DSp) and diagnostic sensitivity (DSe) for the cELISA were 83.1% and 94.9%, respectively, and CD between the cELISA and FAVN results was 0.968, p < 0.001. DISCUSSION: Indirect ELISA test systems for determining the level of antibodies to RABV are not sensitive enough compared to reference tests, unlike cELISA. The developed test system is not inferior for its DSp and DSe to the commercial cELISA BioPro ELISA Rabies Ab (DSp 66.7%, DSe 94.4%). CONCLUSION: The developed cELISA test system can be used to detect antibodies to RABV in the blood serum of dogs for evaluating the effectiveness of mass vaccination programs.


Subject(s)
Lyssavirus , Rabies virus , Rhabdoviridae , Animals , Antibodies, Viral , Dogs , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/veterinary
5.
Sci Rep ; 12(1): 11298, 2022 07 04.
Article in English | MEDLINE | ID: covidwho-2028705

ABSTRACT

Reliable serological assays are needed to understand the real impact of COVID-19. In order to compare the efficiency of different COVID-19 vaccines used in the National Vaccination Program in Tunisia, we have developed a quantitative in-house ELISA. The ELISA is based on the ectodomain of the SARS-CoV-2 Spike Baculovirus recombinant protein. We used a panel of 145 COVID-19 RT-PCR positive serum samples and 116 pre-pandemic serum samples as a negative panel. The validation was carried out by comparison to four commercial techniques (Vidas SARS-CoV-2 IgG anti-RBD Biomérieux, Elecsys Anti-Nucleocapsid of SARS-CoV-2 Roche, cPass GenScript and the quantitative Elecsys Anti-RBD of SARS-CoV-2, Roche). For the evaluation of the National Vaccination campaign, we have included 115 recipients who received one of the approved vaccines. The qualitative performances of the developed ELISA gave 96% sensitivity, 97.5% specificity and 0.968 accuracy. For the evaluation of the different brand of vaccines in recipients not previously infected with SARS-CoV-2, it seems that mRNA vaccine of Pfizer/BioNTech has shown a higher efficacy compared to inactivated virus vaccines. COVID-19 convalescent individuals have generated poor antibody responses. Nevertheless, when they are vaccinated with any brand of the COVID-19 vaccines, many of them mounted an exponential increase of the induced immune responses, qualified as a "hybrid vigor immunity". Our developed in-house ELISA seems to be very efficient in evaluating the effectiveness of anti-COVID-19 vaccination. Platforms based on mRNA vaccine are better performing than those based on inactivated virus.


Subject(s)
COVID-19 , Viral Vaccines , COVID-19/prevention & control , COVID-19 Vaccines , Enzyme-Linked Immunosorbent Assay , Humans , SARS-CoV-2 , Vaccines, Inactivated , Vaccines, Synthetic , mRNA Vaccines
6.
Int J Infect Dis ; 122: 576-584, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-2015433

ABSTRACT

OBJECTIVES: Observing the serological cross-reactivity between SARS-CoV-2 and dengue virus (DV), we aimed to elucidate its effect on dengue serodiagnosis and infectivity in a highly dengue-endemic city in India. METHODS: A total of 52 COVID-19 (reverse transcription-polymerase chain reaction [RT-PCR] positive) serum samples were tested in rapid lateral flow immunoassays and DV immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) to detect DV or SARS-CoV-2 IgG/immunoglobulin M. The COVID-19 antibody (Ab) positive samples were subjected to a virus neutralization test (Huh7 cells) using DV type 1 (DV1) clinical isolate. RESULTS: Most (93%) of the SARS-CoV-2 Ab-positive serum samples cross-reacted with DV in rapid or ELISA tests. All were DV RNA and nonstructural protein 1 (NS1) antigen-negative. COVID-19 serum samples that were DV cross-reactive neutralized DV1. Of these, 57% had no evidence of DV pre-exposure (DV NS1 Ab-negative). The computational study also supported potential interactions between SARS-CoV-2 Ab and DV1. CONCLUSION: DV serodiagnosis will be inconclusive in areas co-endemic for both viruses. The COVID-19 pandemic appears to impart a protective response against DV in DV-endemic populations.


Subject(s)
COVID-19 , Dengue Virus , Dengue , Antibodies, Viral , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G , Immunoglobulin M , Neutralization Tests , Pandemics , SARS-CoV-2 , Sensitivity and Specificity , Serologic Tests
7.
Viruses ; 14(8)2022 08 18.
Article in English | MEDLINE | ID: covidwho-2010308

ABSTRACT

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen which mainly causes diarrhea, dehydration and death in nursing piglets, threatening the global swine industry. Moreover, it can infect multiple animal species and humans. Hence, reliable diagnostic assays are needed to better control this zoonotic pathogen. Here, a blocking ELISA was developed using a recombinant nucleocapsid (N) protein as the coating antigen paired with an N-specific monoclonal antibody (mAb) as the detection antibody. The percent inhibition (PI) of the ELISA was determined using 384 swine serum samples, with an indirect immunofluorescence assay (IFA) as the reference method. Through receiver operating characteristic analysis in conjunction with Youden's index, the optimal PI cut-off value was determined to be 51.65%, which corresponded to a diagnostic sensitivity of 98.79% and a diagnostic specificity of 100%. Of the 330 serum samples tested positive via IFA, 326 and 4 were tested positive and negative via the ELISA, respectively, while the 54 serum samples tested negative via IFA were all negative via the ELISA. The overall coincidence rate between the two assays was 98.96% (380/384). The ELISA exhibited good repeatability and did not cross-react with antisera against other swine pathogens. Overall, this is the first report on developing a blocking ELISA for PDCoV serodiagnosis.


Subject(s)
Coronavirus Infections , Swine Diseases , Animals , Antibodies, Viral , Coronavirus Infections/diagnosis , Coronavirus Infections/veterinary , Deltacoronavirus , Enzyme-Linked Immunosorbent Assay/methods , Humans , Nucleocapsid Proteins , Swine
9.
Tuberculosis (Edinb) ; 136: 102253, 2022 09.
Article in English | MEDLINE | ID: covidwho-2004564

ABSTRACT

Tuberculosis (TB) stays a major cause of death globally after COVID-19 and HIV. An early diagnosis to control TB effectively, needs a fast reliable diagnostic method with high sensitivity. Serodiagnosis involving polyclonal antibodies detection against an antigen of Mycobacterium tuberculosis (Mtb) in serum samples can be instrumental. In our study, Rv3874 and Rv3875 antigens were cloned, expressed, and purified individually and as a chimeric construct in Escherichia coli BL21. Enzyme-Linked Immunosorbent Assay (ELISA) based findings revealed that the Rv3874-Rv3875 chimeric construct was two-fold more sensitive (59.7%) than the individual sensitivities of Rv3874 (28.4%) and Rv3875 (24.9%) for 201 serum TB positive samples. Furthermore, the fusion construct was a little more sensitive (60.4%) for male subjects than that for females (58.8%). Lastly, our preliminary findings, molecular insights of secondary structure, and statistical and in silico analysis of each construct also advocate that CEP can be considered a better immunodiagnostic tool in addition to previously reported EC skin test.


Subject(s)
COVID-19 , Mycobacterium tuberculosis , Tuberculosis , Antigens, Bacterial , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli , Female , Humans , Male , Mycobacterium tuberculosis/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sensitivity and Specificity , Serologic Tests , Tuberculosis/diagnosis
10.
BMC Vet Res ; 18(1): 319, 2022 Aug 18.
Article in English | MEDLINE | ID: covidwho-2002179

ABSTRACT

BACKGROUND: Porcine epidemic diarrhea (PED), a swine epidemic disease caused by porcine epidemic diarrhea virus (PEDV), is characterized by severe watery diarrhea, vomiting, dehydration and high mortality in piglets, and has caused serious economic losses to the global porcine industry. The level of PEDV IgA antibody is a key marker to assess the extent of passive immunity of the resistance against PEDV infection. However, current commercial structure proteins-based kits for detection of PEDV antibody are not affordable, and those kits require complicated antigen preparation procedures, which cannot meet the scope of economic benefits of many large-scale pig companies in China. Therefore, there is an urgent need to develop an accurate, simple, and economical method for IgA detection in clinical samples. In this study, an indirect ELISA (i-ELISA) method was developed based on a purified PEDV epidemic strain (NH-TA2020). RESULTS: The results show that optimal working dilution ratios of PEDV antigen and HRP anti-swine IgA are at 1: 1000 and 1:15000 respectively. The sensitivity of this method is high with the maximum dilution of samples up to 1:160, and coefficients of variation (CV) of both the intra assays and inter assays were no more than 15%. In addition, the relative sensitivities of the i-ELISA were above 90% compared with values from commercial kits in both serum and oral fluid samples. CONCLUSIONS: Our results suggested that the i-ELISA developed in this study was an accurate, simple, and economical method for PEDV-IgA detection in clinical samples.


Subject(s)
Coronavirus Infections , Porcine epidemic diarrhea virus , Swine Diseases , Animals , Antibodies, Viral , Coronavirus Infections/diagnosis , Coronavirus Infections/epidemiology , Coronavirus Infections/veterinary , Diarrhea/veterinary , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/veterinary , Immunoglobulin A , Swine
11.
J Clin Virol ; 155: 105270, 2022 10.
Article in English | MEDLINE | ID: covidwho-1996329

ABSTRACT

Dried Blood Spots (DBS) are broadly used in SARS-CoV-2 surveillance studies, reporting either the presence or absence of SARS-CoV-2 antibodies. However, quantitative follow-up has become increasingly important to monitor humoral vaccine responses. Therefore, we aimed to evaluate the performance of DBS for the detection of anti-spike SARS-CoV-2 antibody concentrations using a commercially available assay, reporting in a standardised unitage (International Units/mL; IU/mL). To assess the sensitivity and specificity of the ImmunoDiagnostics ELISA on serum and DBS for SARS-CoV-2 antibody detection, we analysed 72 paired DBS and serum samples. The SARS-CoV-2 S1 IgG ELISA kit (EUROIMMUN) on serum was used as the reference method. We performed a statistical assessment to optimise the cut-off value for DBS and serum and assessed the correlation between DBS and serum antibody concentrations. We found that anti-spike SARS-CoV-2 antibody concentrations detected in DBS are highly correlated to those detected in paired serum (Pearson correlation 0.98; p-value < 0.0001), allowing to assess serum antibody concentration using DBS. The optimal cut-off for antibody detection on DBS was found to be 26 IU/mL, with 98.1% sensitivity and 100% specificity. For serum, the optimal cut-off was 14 IU/mL, with 100% sensitivity and 100% specificity. Therefore, we conclude that the ImmunoDiagnostics ELISA kit has optimal performance in the detection of SARS-CoV-2 antibodies on both DBS and serum. This makes DBS ideal for large-scale follow-up of humoral SARS-CoV-2 immune responses, as it is an easy but valuable sampling method for quantification of SARS-CoV-2 antibodies, compared to serum.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , COVID-19/diagnosis , COVID-19 Testing , Enzyme-Linked Immunosorbent Assay/methods , Humans , Immunoglobulin G , Sensitivity and Specificity
12.
ScientificWorldJournal ; 2022: 7754329, 2022.
Article in English | MEDLINE | ID: covidwho-1993134

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that causes coronavirus disease 2019 (COVID-19) is a disease with a high rate of transmission. Serological tests are important to perform surveys and to determine the immunological status of the population. Based on this, we evaluated three enzyme-linked immunoassays (ELISAs) using different antigens from SARS-CoV-2 in a cohort of 161 patients. The performance of the ELISA developed for immunoglobulin G (IgG) measurement against SARS-CoV-2 was evaluated based on sensitivity, specificity, and accuracy. We found specificities of 0.98, 0.98, and 0.99 and sensitivities of 0.99, 0.91, and 0.87 for the nucleocapsid (N) protein, spike protein, and receptor binding domain (RBD) fraction, respectively. The accuracy assessment indicated the N protein (accuracy = 0.98) as the antigen most likely to give a correct diagnosis. Overall, the antibody responses were present for all three proteins in subjects with confirmed SARS-CoV-2 infections, showing a similar pattern of antibody production for different antigens. In summary, these highly sensitive and specific ELISAs, with a more competitive price, appear to be a valid approach for the serodiagnosis of COVID-19.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , COVID-19/diagnosis , COVID-19/immunology , COVID-19 Testing , Enzyme-Linked Immunosorbent Assay , Humans , SARS-CoV-2/immunology , Sensitivity and Specificity , Serologic Tests , Spike Glycoprotein, Coronavirus
13.
Appl Microbiol Biotechnol ; 106(18): 6225-6238, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-1990607

ABSTRACT

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has adversely affected humankind and caused millions of deaths globally since January 2020. Robust and quick serological tests such as antibody detection assays for SARS-CoV-2 provide relevant information and aid in the process of vaccine development and diagnostics, as well as in sero-epidemiological monitoring of antibody response to the virus. The receptor-binding domain (RBD) of spike and nucleocapsid protein are specific targets for detecting SARS-CoV-2 antibodies. Here, we present the development of a stable spike (S) and nucleocapsid (N) protein-based ELISA antibody detection test "CoroSuchak," with 99% sensitivity, 98% specificity, cost-effective, and detection in a minimum time for serodiagnosis and mass screening of the population for antibodies against SARS-CoV-2. Blood samples were analyzed from 374 SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) positive, 772 negative and asymptomatic, and 874 random groups of subjects. We found that the antibody titer was significantly higher (p < 0.0001) in infected and vaccinated group compared to the only vaccinated and only infected group. Using enzyme-linked immunosorbent assay (ELISA), we detected SARS-CoV-2 immunoglobulin G (IgG) antibodies in 118/123 (96%) infected individuals, 570/653 (87%) non-infected but vaccinated individuals, 231/237 (97%) individuals who were both infected and vaccinated, and 499/874 (57%) from randomly selected individuals from the first and second waves of the pandemic. Similarly in the third wave, 14/14 (100%) infected and 16/20 (80%) RT-PCR-negative but symptomatic subjects were detected. Thus, the highly sensitive and specific in-house developed ELISA antibody detection kit "CoroSuchak" is extremely useful to determine the seroprevalence of SARS-CoV-2 antibodies in the coronavirus-exposed population. KEY POINTS: •Indigenous kit using a combination of spike and nucleocapsid proteins and peptide sequences. •High sensitivity and specificity to detect variants. •Highly sensitive for mass screening.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G , Mass Screening , Nucleocapsid Proteins , Sensitivity and Specificity , Seroepidemiologic Studies , Spike Glycoprotein, Coronavirus
14.
Anal Chim Acta ; 1225: 340246, 2022 Sep 08.
Article in English | MEDLINE | ID: covidwho-1982438

ABSTRACT

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.


Subject(s)
COVID-19 , Malaria, Falciparum , Nanoparticles , Enzyme-Linked Immunosorbent Assay/methods , Humans , Malaria, Falciparum/diagnosis , Malaria, Falciparum/parasitology , Plasmodium falciparum , SARS-CoV-2
15.
Eur Rev Med Pharmacol Sci ; 26(14): 5307-5310, 2022 07.
Article in English | MEDLINE | ID: covidwho-1975732

ABSTRACT

OBJECTIVE: Antinuclear antibodies (ANA) are detected in approximately a quarter of COVID-19 patients when assessed by indirect immunofluorescence. Since there is no information, our study investigated the presence of ANA detected by Enzyme-Linked Immunosorbent Assay (ELISA) and its clinical and laboratory associations. PATIENTS AND METHODS: A longitudinal study was conducted on 92 patients with severe COVID-19, 20 patients with acute myocardial infarction, and 25 healthy subjects. Blood samples were obtained at hospital admission. Commercial ELISA was used to detect ANA, while flow cytometry was used to measure serum interferons. RESULTS: ANAs were positive in 8.6% of COVID-19 patients, 10% of myocardial infarction patients, and 4% in healthy individuals (p=0.676). COVID-19 patients with ANA+ had less ferritin, troponin, and neutrophils but more albumin and lymphocytes than ANA- patients. Serum levels of type I, II, and III interferons were similar between groups. At follow-up, all ANA+ patients survived, while mortality was significant in ANA- patients (0 vs. 36%; p=0.048). CONCLUSIONS: ANA detection is not increased in severe cases of COVID-19 when assessed by ELISA. However, its presence appears to be associated with a less aggressive disease phenotype, regardless of circulating levels of interferons.


Subject(s)
Antibodies, Antinuclear , COVID-19 , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay , Humans , Interferons , Longitudinal Studies
16.
PLoS One ; 17(2): e0262591, 2022.
Article in English | MEDLINE | ID: covidwho-1968842

ABSTRACT

SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/chemistry , COVID-19/blood , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , Immunoglobulin G/blood , Inclusion Bodies/chemistry , Protein Refolding , SARS-CoV-2/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Hydrogen-Ion Concentration , Hydrostatic Pressure , Immunoglobulin G/immunology , Phosphoproteins/chemistry , Phosphoproteins/immunology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Solubility
17.
PLoS One ; 17(7): e0272298, 2022.
Article in English | MEDLINE | ID: covidwho-1963049

ABSTRACT

Virus neutralization assays provide a means to quantitate functional antibody responses that block virus infection. These assays are instrumental in defining vaccine and therapeutic antibody potency, immune evasion by viral variants, and post-infection immunity. Here we describe the development, optimization and evaluation of a live virus microneutralization assay specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this assay, SARS-CoV-2 clinical isolates are pre-incubated with serial diluted antibody and added to Vero E6 cells. Replicating virus is quantitated by enzyme-linked immunosorbent assay (ELISA) targeting the SARS-CoV-2 nucleocapsid protein and the standardized 50% virus inhibition titer calculated. We evaluated critical test parameters that include virus titration, assay linearity, number of cells, viral dose, incubation period post-inoculation, and normalization methods. Virus titration at 96 hours was determined optimal to account for different growth kinetics of clinical isolates. Nucleocapsid protein levels directly correlated with virus inoculum, with the strongest correlation at 24 hours post-inoculation. Variance was minimized by infecting a cell monolayer, rather than a cell suspension. Neutralization titers modestly decreased with increasing numbers of Vero E6 cells and virus amount. Application of two different normalization models effectively reduced the intermediate precision coefficient of variance to <16.5%. The SARS-CoV-2 microneutralization assay described and evaluated here is based on the influenza virus microneutralization assay described by WHO, and are proposed as a standard assay for comparing neutralization investigations.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , Enzyme-Linked Immunosorbent Assay , Humans , Neutralization Tests/methods , Nucleocapsid Proteins , Spike Glycoprotein, Coronavirus
18.
Viruses ; 14(7)2022 Jul 18.
Article in English | MEDLINE | ID: covidwho-1939023

ABSTRACT

We aimed to review the existing literature on the different types of neutralization assays and international standards for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We comprehensively summarized the serological assays for detecting neutralizing antibodies against SARS-CoV-2 and demonstrated the importance of an international standard for calibrating the measurement of neutralizing antibodies. Following the coronavirus disease outbreak in December 2019, there was an urgent demand to detect neutralizing antibodies in patients or vaccinated people to monitor disease outcomes and determine vaccine efficacy. Therefore, many approaches were developed to detect neutralizing antibodies against SARS-CoV-2, such as microneutralization assay, SARS-CoV-2 pseudotype virus assay, enzyme-linked immunosorbent assay (ELISA), and rapid lateral flow assay. Given the many types of serological assays for quantifying the neutralizing antibody titer, the comparison of different assay results is a challenge. In 2020, the World Health Organization proposed the first international standard as a common unit to define neutralizing antibody titer and antibody responses against SARS-CoV-2. These standards are useful for comparing the results of different assays and laboratories.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay , Humans , Neutralization Tests/methods , Spike Glycoprotein, Coronavirus
20.
Int J Mol Sci ; 23(14)2022 Jul 08.
Article in English | MEDLINE | ID: covidwho-1928574

ABSTRACT

Avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is the causative agent of infectious bronchitis (IB) that has brought great threat and economic losses to the global poultry industry. Rapid and accurate diagnostic methods are very necessary for effective disease monitoring. At the present study, we screened a novel nanobody against IBV-N protein for development of a rapid, simple, sensitive, and specific competitive ELISA for IBV antibody detection in order to enable the assessment of inoculation effect and early warning of disease infection. Using the phage display technology and bio-panning, we obtained 7 specific nanobodies fused with horseradish peroxidase (HRP) which were expressed in culture supernatant of HEK293T cells. Out of which, the nanobody of IBV-N-Nb66-vHRP has highly binding with IBV-N protein and was easily blocked by the IBV positive serums, which was finally employed as an immunoprobe for development of the competitive ELISA (cELISA). In the newly developed cELISA, we reduce the use of enzyme-conjugated secondary antibody, and the time of whole operation process is approximately 1 h. Moreover, the IBV positive serums diluted at 1:1000 can still be detected by the developed cELISA, and it has no cross reactivity with others chicken disease serums including Newcastle disease virus, Fowl adenovirus, Avian Influenza Virus, Infectious bursal disease virus and Hepatitis E virus. The cut-off value of the established cELISA was 36%, and the coefficient of variation of intra- and inter-assay were 0.55-1.65% and 2.58-6.03%, respectively. Compared with the commercial ELISA (IDEXX kit), the agreement rate of two methods was defined as 98% and the kappa value was 0.96, indicating the developed cELISA has high consistency with the commercial ELISA. Taken together, the novel cELISA for IBV antibody detection is a simple, rapid, sensitive, and specific immunoassay, which has the potential to rapidly test IBV antibody contributing to the surveillance and control of the disease.


Subject(s)
Coronavirus Infections , Infectious bronchitis virus , Poultry Diseases , Animals , Antibodies, Viral , Chickens , Coronavirus Infections/diagnosis , Coronavirus Infections/veterinary , Enzyme-Linked Immunosorbent Assay/methods , HEK293 Cells , Horseradish Peroxidase , Humans
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