An Evaluation of Serological Tests to Determine Postvaccinal Immunity to SARS-CoV-2 by mRNA Vaccines.

BACKGROUND: The duration of the protective efficacy of vaccines against SARS-CoV-2 is unknown. Thus, an evaluation of the clinical performance of available tests is required. OBJECTIVES: To evaluate the clinical performance of LFIA immunoassay compared to ELIA and CLIA immunoassays available in Europe for the detection of IgG antibodies generated by mRNA vaccines against SARS-CoV-2. METHODS: Two automated immunoassays (the EUROIMMUN anti-SARS-CoV-2 IgG S1 ELISA and the LIAISON de Diasorin anti-SARS-CoV-2 IgG S1/S2 test) and a lateral flow immunoassay (the Livzon LFIA anti-SARS-CoV-2 IgG S test) were tested. We analyzed 300 samples distributed in three groups: 100 subjects aged over 18 years and under 45 years, 100 subjects aged between 45 and 65 years, and 100 subjects aged over 65 years. The samples were collected before vaccination; at 21 days; and then at 1, 2, 3, and 6 months after vaccination. The sensitivity, specificity, positive predictive value, negative predictive value, positive probability quotient, negative probability quotient, and concordance (kappa index) were calculated for each serological test. RESULTS: The maximum sensitivity values for IgG were 98.7%, 98.1%, and 97.8% for the EUROIMMUN ELISA, Abbott CLIA, and Livzon LFIA tests, respectively, and the maximum specificity values for IgG were 99.4%, 99.9%%, and 98.4% for the ELISA, CLIA, and LFIA tests, respectively, at the third month after vaccination, representing a decrease in the antibody levels after the sixth month. The best agreement was observed between the ELISA and CLIA tests at 100% (k = 1.00). The agreement between the ELIA, CLIA, and LFIA tests was 99% (k = 0.964) at the second and third month after vaccination. Seroconversion was faster and more durable in the younger age groups. CONCLUSION: Our study examined the equivalent and homogeneous clinical performance for IgG of three immunoassays after vaccination and found LFIA to be the most cost-effective, reliable, and accurate for routine use in population seroconversion and seroprevalence studies.

Improving Chinese hamster ovary host cell protein ELISA using Ella®: an automated microfluidic platform.

Chinese hamster ovary (CHO) cells are a mammalian cell line used in the production of therapeutic proteins. Host cell proteins (HCPs) are process-related impurities that are derived from the host cell expression system. During biopharmaceutical drug development, removal of HCPs is required. Enzyme-linked immunosorbent assay (ELISA) is a common technique to quantitate HCPs, but is a labor-intensive process that takes up to 7 h. Ella® is an automated instrument that utilizes microfluidics and glass nanoreactors to quantitate HCPs in 75 min using similar ELISA reagents. The antibodies and antigens are captured on three distinct glass nanoreactors, resulting in sensitive reproducible data. Our results indicate that Ella quantitates CHO HCPs with precision, accuracy, sensitivity and trends comparable with our traditional CHO HCP ELISA.

Assessment of SARS-CoV-2 serological tests for the diagnosis of COVID-19 through the evaluation of three immunoassays: Two automated immunoassays (Euroimmun and Abbott) and one rapid lateral flow immunoassay (NG Biotech).

BACKGROUND: The emergence of new SARS-CoV-2 has promoted the development of new serological tests that could be complementary to RT-PCR. Nevertheless, the assessment of clinical performances of available tests is urgently required as their use has just been initiated for diagnose. OBJECTIVES: The aim of this study was to assess the performance of three immunoassays for the detection of SARS-CoV-2 antibodies. METHODS: Two automated immunoassays (Abbott SARS-CoV-2 CLIA IgG and Euroimmun Anti-SARS-CoV-2 ELISA IgG/IgA assays) and one lateral flow immunoassay (LFIA NG-Test® IgG-IgM COVID-19) were tested. 293 specimens were analyzed from patients with a positive RT-PCR response, from patients with symptoms consistent with COVID-19 but exhibiting a negative response to the RT-PCR detection test, and from control group specimens. Days since symptoms onset were collected from clinical information sheet associated with respiratory tract samples. RESULTS: Overall sensitivity for IgG was equivalent (around 80 %) for CLIA, ELISA and LFIA. Sensitivity for IgG detection, >14 days after onset of symptoms, was 100.0 % for all assays. Overall specificity for IgG was greater for CLIA and LFIA (more than 98 %) compared to ELISA (95.8 %). Specificity was significantly different between IgA ELISA (78.9 %) and IgM LFIA (95.8 %) (p < 0.05). The best agreement was observed between CLIA and LFIA assays (97 %; k = 0.936). CONCLUSION: Excellent sensitivity for IgG detection was obtained >14 days after onset of symptoms for all immunoassays. Specificity was also excellent for IgG CLIA and IgG LFIA. Our study shows that NG-Test® is reliable and accurate for routine use in clinical laboratories.

A Rapid and Simple Liquid-Chromatography-Tandem Mass Spectrometry Method for Measuring 25-Hydroxyvitamin D2 and 25-Hydroxyvitamin D3 in Human Serum: Comparison with Two Automated Immunoassays.

BACKGROUND: Total 25-hydroxyvitamin D (25(OH)D) is well-known to be a reliable biomarker of human vitamin D status, with the recognition of widespread vitamin D insufficiency in general populations. The aims of this study are to validate a fast and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying 25(OH)D2 and 25(OH)D3 in serum and to compare two automated immunoassays with the LC-MS/MS method. METHODS: Samples were prepared by protein precipitation with ethanol including 25(OH)D3-d6, followed by a liquid-liquid extraction with hexane. The analytes were separated within a total run time of 3 min. Accuracy was evaluated with standard reference materials (SRM) 972a. Using 150 samples, the LC-MS/MS method was compared with the LIAISON® assay and ADVIA Centaur® assay. RESULTS: The LC-MS/MS method had a limit of quantitation of 1 ng/mL for the 25(OH)D2 and 25(OH)D3 with linear responses between 1 and 100 ng/mL. Intra- and inter-assay precision were <8.8% and <13.2%, respectively. It also showed a smallest mean difference (+0.9%) for the SRM level 1 to 3, compared to the two immunoassays. Compared to the LC-MS/MS, the mean biases of the RIAISON and ADVIA were +2.4 and +7.9 ng/mL, respectively. Also, the agreement of the LC-MS/MS with the RIAISON was better than that with the ADVIA. CONCLUSION: This study suggests that the LC-MS/MS method traceable to the SRM can be reliably applied in routine quantification of 25(OH)D2 and 25(OH)D3.

Differential extraction of endogenous and exogenous 25-OH-vitamin D from serum makes the accurate quantification in liquid chromatography-tandem mass spectrometry assays challenging.

BACKGROUND: Extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is the method of choice when it comes to the accurate quantification of 25-OH-vitamin D in blood samples. It is generally assumed that the addition of exogenous internal standard allows for the determination of the endogenous analyte concentration. In this study we investigated the extraction properties of endogenous and exogenous 25-OH-vitamin D. METHODS: Eight samples were used for the evaluation of the extraction procedure and 59 patients' samples for a method comparison. The methanol-to-sample ratio (v/v) and the sample-to-hexane ratio (v/v) were varied and the LC-MS/MS signals of endogenous 25-OH-vitamin D3, spiked 25-OH-vitamin D2 and internal standard of the extracts recorded. The optimized 'in-house' LC-MS/MS assay was compared to two automated chemiluminescence immunoassays from DiaSorin and Abbott. RESULTS: Mathematical analysis of the data revealed a differential extraction of endogenous 25-OH-vitamin D3, spiked 25-OH-vitamin D2 and non-equilibrated internal standard. Exogenous 25-OH-vitamin D can be measured accurately if a definite methanol-to-sample ratio is used. Endogenous 25-OH-vitamin D is affected by critical quantification issues due to a differential slope in the extraction profile. The actual 25-OH-vitamin D concentration can be one-third above the measured extractable concentration. Results confirm that the 'in-house' LC-MS/MS assay provides reproducible 25-OH-vitamin D results. CONCLUSIONS: Discordant concentrations of 25-OH-vitamin D from LC-MS/MS assays can be caused by selection of suboptimal extraction conditions. Furthermore, a different sample pretreatment or solvent extraction system may result in a different dissociation and extraction yield of endogenous 25-OH-vitamin D and therefore contribute to variations of LC-MS/MS results.