Performance evaluation of novel fluorescent-based lateral immune flow assay (LIFA) for rapid detection and quantitation of total anti-SARS-CoV-2 S-RBD binding antibodies in infected individuals

1.AbstractBackgroundThe vast majority of the commercially available LFIA is used to detect SARS-CoV-2 antibodies qualitatively. Recently, a novel fluorescence-based LIFA test was developed for quantitative measurement of the total binding antibody units (BAU/mL) against the receptor-binding domain of the SARS-CoV-2 spike protein (S-RBD).AimTo evaluate the performance of the fluorescence LIFA Finecare™ 2019-nCoV S-RBD test along with its reader (Model No.: FS-113).MethodsPlasma from 150 RT-PCR confirmed-positive individuals and 100 pre-pandemic samples were tested by FinCare™ to access sensitivity and specificity. For qualitative and quantitative validation of the FinCar™ measurements, the BAU/mL results of FinCare™ were compared with results of two reference assays: the surrogate virus-neutralizing test (sVNT, GenScript, USA), and the VIDAS®3 automated assay (BioMérieux, France).ResultsFinecare™ showed 92% sensitivity and 100% specificity compared to PCR. Cohen’s Kappa statistic denoted moderate and excellent agreement with sVNT and VIDAS®3, ranging from 0.557 (95% CI: 0.32-0.78) to 0.731 (95% CI: 0.51-0.95), respectively. A strong correlation was observed between Finecare™/sVNT (r=0.7, p<0.0001) and Finecare™/VIDAS®3 (r=0.8, p<0.0001).ConclusionFinecare™ is a reliable assay and can be used as a surrogate to assess binding and neutralizing antibody response post-infection or vaccination, particularly in none or small laboratory settings.

International collaborative study to evaluate and calibrate two recombinant L chain Ferritin preparations for use as a WHO International Standard

Objectives To evaluate and calibrate two candidate preparations for the 4th International Standard for Ferritin (Human, Recombinant) (codes: 19/118 and 19/162) against the 3rd International Standard for Ferritin (Human, Recombinant) (code: 94/572), and three serum commutability samples in an international collaborative study involving 12 laboratories in nine countries. Methods Eleven of the 12 participating laboratories performed Ferritin quantitation using automated assay platforms and one laboratory used a manual ELISA kit. Results There was better overall agreement between all laboratories and between assay methods for the potency of preparation 19/118 than for preparation 19/162. The overall geometric mean potency (from all methods) of the candidate 4th International Standard, 19/118, was 10.5 µg/ampoule, with inter-laboratory variability, expressed as % geometric coefficient of variation (GCV), of 4.7%. Accelerated stability studies have predicted both 19/118 and 19/162 to be very stable for long term storage at −20 °C. Conclusions The candidate 4th International Standard for Ferritin (Human, Recombinant) (19/118) has been shown to be immunologically similar to the 3rd International Standard for Ferritin (Human, Recombinant) (94/572). It was recommended to and accepted by the WHO Expert Committee on Biological Standardization that 19/118 be established as the 4th International Standard for Ferritin (Human, Recombinant) with an assigned potency of 10.5 µg/ampoule and expanded uncertainty limits 10.2–10.8 µg/ampoule (95% confidence; k=2.23).

Multicenter Technical Validation of 30 Rapid Antigen Tests for the Detection of SARS-CoV-2 (VALIDATE)

During COVID19 pandemic, SARS-CoV-2 rapid antigen tests (RATs) were marketed with minimal or no performance data. We aimed at closing this gap by determining technical sensitivities and specificities of 30 RATs prior to market release. We developed a standardized technical validation protocol and assessed 30 RATs across four diagnostic laboratories. RATs were tested in parallel using the Standard Q® (SD Biosensor/Roche) assay as internal reference. We used left-over universal transport/optimum media from nasopharyngeal swabs of 200 SARS-CoV-2 PCR-negative and 100 PCR-positive tested patients. Transport media was mixed with assay buffer and applied to RATs according to manufacturer instructions. Sensitivities were determined according to viral loads. Specificity of at least 99% and sensitivity of 95%, 90%, and 80% had to be reached for 107, 106, 105 virus copies/mL, respectively. Sensitivities ranged from 43.5% to 98.6%, 62.3% to 100%, and 66.7% to 100% at 105, 106, 107 copies/mL, respectively. Automated assay readers such as ExDia or LumiraDx showed higher performances. Specificities ranged from 88.8% to 100%. Only 15 of 30 (50%) RATs passed our technical validation. Due to the high failure rate of 50%, mainly caused by lack of sensitivity, we recommend a thorough validation of RATs prior to market release.

Capture the Tag: Mitigation of Biotin Interference in ELISA and Automated Immunoassays by Pre-Conjugating Biotinylated Antibodies to the Streptavidin Surface

Introduction Streptavidin to biotin binding is one of the strongest non-covalent interactions in nature, and is therefore, successfully incorporated into many immunoassays to facilitate antibody capture. Biotin-streptavidin coupling assays are susceptible to interference from free biotin in patient specimens, which may falsely decrease or increase the result depending on assay format. Currently, biotin-stripping methods capture free biotin in patient specimens by pre-incubation with streptavidin-coated microparticles. However, this approach increases turnaround time, test cost, and testing steps, which increases the chance of error. Our objective was to determine if pre-conjugating biotinylated antibodies to the assay’s streptavidin solid surface before adding patient specimen could mitigate biotin interference in ELISA and automated sandwich immunoassays. Methods We performed this study on 3 different ELISAs (CYFRA-21, NSE, S100B) and one automated assay (thyroglobulin); all are a sandwich immunoassay format. Serum pools were spiked with biotin (25 - 1000 µg/L) or PBS control. Manufacturer protocols were followed in both the ELISAs and automated assay to evaluate baseline concentration-dependent biotin interference. Mitigation of biotin interference by pre-incubation was then evaluated in the ELISAs by adding biotinylated antibody to the streptavidin-coated wells 0, 10, 15, or 60 min before adding biotin- or PBS-spiked serum specimens. For the automated assay, streptavidin-coated beads and biotinylated antibody were removed from the reagent cartridge, mixed, and incubated 4.5 hours. The mixture containing biotin-tagged antibody bound to streptavidin beads was added back to the cartridge and placed on the analyzer to evaluate spiked specimens. Lastly, we compared the pre-incubation method to a standard biotin-stripping protocol in the ELISAs to compare the effectiveness of mitigating biotin interference. Results We observed biotin interference across the three ELISAs, where 400 µg/L biotin spiked in serum pools reduced analyte detection to between 10 – 15% of the total activity using the standard assay format. Our time-course studies showed 84 – 95% recovery of the total activity when the biotinylated antibody was pre-incubated in the streptavidin-coated wells for 1 hour prior to addition of serum specimens, compared to 69 – 99% by a standard biotin stripping protocol. We extended this concept to the automated immunoassay where at 1000 µg/L biotin in the specimen, only 9 ±0.01% of the total analyte was measured by the conventional method. However, pre-mixing biotinylated antibody and streptavidin beads resulted in 97 ±0.01% of the total analyte recovery in the presence of 1000 µg/L biotin. Conclusion We have demonstrated that pre-conjugating the biotin antibody to streptavidin is as effective as biotin-stripping methods to avoid biotin interference in sandwich immunoassays that utilize the biotin-streptavidin system, with the additional benefit of optimizing turnaround times, cost, and labor. A simple change in manufacturer assay design could make immunoassays more robust against biotin interference in patient samples.

The influence of direct oral anticoagulants on thrombin generation on Ceveron TGA

Introduction: Monitoring of direct oral anticoagulants (DOACs) with calibrated anti-Xa assay is limited by the high intra- and interindividual variation of the test results. Thrombin generation (TG) is a global hemostatic assay that reflects the patient´s individual coagulation status. The aim of this study was to investigate the influence of DOACs on TG measured with a fully automated assay system. Methods: All consecutive patients under apixaban and rivaroxaban coming to the outpatient coagulation center MVZ Limbach, Magdeburg, Germany between October 2017 and April 2020 were included. DOAC plasma levels were correlated with TG assessed using the fully automated Ceveron TG analyser. Results: A total of 703 rivaroxaban and 252 apixaban containing plasma samples were included. There was a significant correlation between DOAC plasma levels and all TG parameters except for lag time regarding apixaban. Time to peak and peak thrombin followed an exponential regression curve, while this was linear for the endogenous thrombin potential (ETP). Apixaban showed a lower correlation coefficient for all TG parameters compared to rivaroxaban and thrombin generation was less influenced by apixaban than rivaroxaban at plasma levels > 100 ng/mL. The sensitivity and negative predictive value of normal TG parameters for the prediction of DOAC plasma levels < 30 ng/mL was > 85%. Conclusion: The present data show a moderate predominantly non-linear correlation between TG parameters and plasma levels of apixaban and rivaroxaban. Rivaroxaban has a stronger effect on TG than apixaban.

EXPRESS: Clinical performance of the Elecsys® Anti-SARS-CoV-2 combined in an algorithm with two specific anti-IgG immunoassays for the evaluation of the serological response of patients with COVID-19 in a population with a high prevalence of infection

Background: Anti-SARS-CoV-2 antibodies have been used in the study of the immune response in infected patients. However, differences in sensitivity and specificity have been reported, depending on the method of analysis. The aim of the present study was to evaluate the diagnostic accuracy of an algorithm in which a high-throughput automated assay for total antibodies was used for screening and two semi-automated IgG-specific methods were used to confirm the results, and also to correlate the analytical results with the clinical data and the time elapsed since infection. Methods: We studied 306 patients, some hospitalized and some outpatients, belonging to a population with a high prevalence of COVID-19. One-hundred and ten patients were classified as SARS-CoV-2 negative and 196 as positive by polymerase chain reaction. Results: The algorithm and automated assay alone had a specificity and a positive predictive value of 100%, although the sensitivity and negative predictive value of the algorithm was higher. Both methods showed a good sensitivity from day 11 of the onset of symptoms in asymptomatic and symptomatic patients. The absorbance of the total antibodies was significantly higher in severely symptomatic than in asymptomatic or mildly symptomatic patients, which suggests the antibody level was higher. We found 15 patients that did not present seroconversion at 12 days from the onset of symptoms or the first polymerase chain reaction test. Conclusion: This study highlights the proper functioning of algorithms in the diagnosis of the immune response to COVID-19, which can help to define testing strategies against this disease.

Analytical validation of an automated assay for the measurement of adenosine deaminase (ADA) and its isoenzymes in saliva and a pilot evaluation of their changes in patients with SARS-CoV-2 infection

Objectives The aim of the present study was to validate a commercially available automated assay for the measurement of total adenosine deaminase (tADA) and its isoenzymes (ADA1 and ADA2) in saliva in a fast and accurate way, and evaluate the possible changes of these analytes in individuals with SARS-CoV-2 infection. Methods The validation, in addition to the evaluation of precision and accuracy, included the analysis of the effects of the main procedures that are currently being used for SARS-CoV-2 inactivation in saliva and a pilot study to evaluate the possible changes in salivary tADA and isoenzymes in individuals infected with SARS-CoV-2. Results The automated assay proved to be accurate and precise, with intra- and inter-assay coefficients of variation below 8.2%, linearity under dilution linear regression with R2 close to 1, and recovery percentage between 80 and 120% in all cases. This assay was affected when the sample is treated with heat or SDS for virus inactivation but tolerated Triton X-100 and NP-40. Individuals with SARS-CoV-2 infection (n=71) and who recovered from infection (n=11) had higher mean values of activity of tADA and its isoenzymes than healthy individuals (n=35). Conclusions tADA and its isoenzymes ADA1 and ADA2 can be measured accurately and precisely in saliva samples in a rapid, economical, and reproducible way and can be analyzed after chemical inactivation with Triton X-100 and NP-40. Besides, the changes observed in tADA and isoenzymes in individuals with COVID-19 open the possibility of their potential use as non-invasive biomarkers in this disease.