A magnetic bead immunoassay to detect human IgG reactive to SARS-CoV-2 Spike S1 RBD produced in Escherichia coli

Immunological assays to detect SARS-CoV-2 Spike receptor binding domain antigen seroconversion in humans are important tools to monitor the levels of protecting antibodies in the population in response to infection and/or immunization. Here we describe a simple, low cost and high throughput Ni2+ magnetic bead immunoassay to detect human IgG reactive to Spike S1 RBD Receptor Binding Domain produced in Escherichia coli. A 6xHis tagged Spike S1 RBD was expressed in E. coli and purified by affinity chromatography. The protein was mobilized on the surface of Ni2+ magnetic beads and used to investigate the presence of reactive IgG in the serum obtained from pre-pandemic and COVID-19 confirmed cases. The method was validated with a cohort of 290 samples and an area under the receiver operating characteristics curve of 0.94 was obtained. The method operated with>82% sensitivity at 98% specificity and was also able to track human IgG raised in response to vaccination with Comirnaty with 85% sensitivity. The IgG signal obtained with the described method was well correlated with the signal obtained when pre fusion Spike produced in HEK cell lines were used as antigen. This novel low-cost and high throughput immunoassay may act as an important tool to investigate protecting IgG antibodies against SARS-CoV-2 in the human population.

Ultra-fast, high throughput and inexpensive detection of SARS-CoV-2 seroconversion

ABSTRACTA technique allowing high throughput, fast and low-cost quantitative analysis of human IgG antibodies reacting to SARS-CoV-2 antigens will be required to understand the levels of protecting antibodies in the population raised in response to infections and/or to immunization. We described previously a fast, simple, and inexpensive Ni2+ magnetic bead immunoassay which allowed detection of human antibodies reacting against the SARS-CoV-2 nucleocapsid protein using a minimal amount of serum or blood. A major drawback of the previously described system was that it only processed 12 samples simultaneously. Here we describe a manually operating inexpensive 96 well plate magnetic extraction / homogenization process which allows high throughput analysis delivering results of 96 samples in chromogenic format in 12 minutes or in fluorescent ultrafast format which takes only 7 minutes. We also show that His tag antigen purification can be performed on the fly while loading antigens to the Ni2+ magnetic beads in a process which takes only 12 min reducing the pre analytical time and cost. Finally, we show that the magnetic bead immunoassay is antigen flexible and can be performed using either Nucleocapsid, Spike or Spike RBD. The method performed with low inter and intra assay variability using different antigens and detection modes and was able to deliver >99.5% specificity and >95% sensitivity for a cohort of 203 pre pandemic and 63 COVID-19 positive samples.

Evaluation of Humoral Immunity to SARS-CoV-2: Diagnostic Value of a New Multiplex Addressable Laser Bead Immunoassay

Despite efforts to develop anti–severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody (Ab) immunoassays, reliable serological methods are still needed. We developed a multiplex addressable laser bead immunoassay (ALBIA) to detect and quantify anti-Spike S1 and nucleocapsid N Abs. Recombinant S1 and N proteins were bound to fluorescent beads (ALBIA-IgG-S1/N). Abs were revealed using class-specific anti-human Ig Abs. The performances of the test were analyzed on 575 serum samples including 192 from SARS-CoV-2 polymerase chain reaction–confirmed patients, 13 from seasonal coronaviruses, 70 from different inflammatory/autoimmune diseases, and 300 from healthy donors. Anti-S1 IgM were detected by monoplex ALBIA-IgM-S1. Comparison with chemiluminescent assays or enzyme-linked immunosorbent assays was performed using commercial tests. Multiplex ALBIA-IgG-S1/N was effective in detecting and quantifying anti–SARS-CoV-2 IgG Abs. Two weeks after first symptoms, sensitivity and specificity were 97.7 and 98.0% (anti-S1), and 100 and 98.7% (anti-N), respectively. Agreement with commercial tests was good to excellent, with a higher sensitivity of ALBIA. ALBIA-IgG-S1/N was positive in 53% of patients up to day 7, and in 75% between days 7 and 13. For ALBIA-IgM-S1, sensitivity and specificity were 74.4 and 98.7%, respectively. Patients in intensive care units had higher IgG Ab levels (Mann–Whitney test, p < 0.05). ALBIA provides a robust method for exploring humoral immunity to SARS-CoV-2. Serology should be performed after 2 weeks following first symptoms, when all COVID-19 (coronavirus disease 2019) patients had at least one anti-S1 or anti-N IgG Ab, illustrating the interest of a multiplex test.

Expression of Serum Cytokines Profile in Neonatal Sepsis

Sepsis remained a major cause of neonatal death, but the pathologic mechanisms were poorly understood. The objective of this study was to characterize the serum cytokine/chemokine profile in neonates with sepsis. In this study, we enrolled 40 full-term neonates with sepsis and 19 neonates without infection as controls. Serum 40 cytokines/chemokines were analyzed using Luminex Bead Immunoassay System. Serum IL-17 was measured using enzyme-linked immune-absorbent assay. Our results showed that serum IL-6, IL-8, TNF-α, IL-1β, MIF, CXCL13, CXCL1, CXCL2, CXCL5, CXCL6, CXCL16, CCL27, CCL2, CCL8, CCL3, CCL20, CCL23, CCL27 and CX3CL1 were significantly increased in neonates with sepsis compared to controls (all p<0.05). The levels of serum CXCL6, CXCL1, IL-6, CXCL10, CXCL11, CCL20, and IL-17 were higher in LOS than those in EOS (all p<0.05). Conversely, serum IL-16, CXCL16, CCL22 were lower in LOS than those in EOS (all p<0.05). The levels of CX3CL1, CXCL2, CCL8 and TNF-α were all positively correlated with SOFA scores. We suggest that excessive pro-inflammatory cytokines might involve in the damage of neonatal sepsis. In addition, chemokines significantly increased the recruitment of immune cells after infection to participate in the anti-infection defense of neonates, but they might lead to damage.

Zearalenone Contamination in Corn, Corn Products, and Swine Feed in China in 2016–2018 as Assessed by Magnetic Bead Immunoassay

In total, 405 samples of corn, corn products, and swine feed from China in 2016–2018 were surveyed for zearalenone (ZEN) contamination using a magnetic bead immunoassay-coupled biotin–streptavidin system (BAS-MBI). The developed BAS-MBI had a limit of detection (LOD) of 0.098 ng mL−1, with half-maximal inhibition concentration (IC50) of 0.71 ng mL−1 in working buffer, and an LOD of 0.98 ng g−1; the detection range was from 0.98 to 51.6 ng g−1 in authentic agricultural samples. The BAS-MBI has been demonstrated to be a powerful method for the rapid, sensitive, specific, and accurate determination of ZEN. The ZEN positivity rate reached the highest level of 40.6% in 133 samples in 2016; ZEN levels ranged from 1.8 to 1100.0 ng g−1, with an average level of 217.9 ng g−1. In 2017, the ZEN positivity rate was the lowest at 24.5% in 143 samples; ZEN levels ranged from 1.1 to 722.6 ng g−1, with an average of 166.7 ng g−1. In 2018, the ZEN positivity rate was 31.8% in 129 samples; ZEN levels ranged from 1.3 to 947.8 ng g−1, with an average of 157.0 ng g−1. About 20% of ZEN-positive samples exceeded maximum limit levels. An alternative method of ZEN detection and a valuable reference for ZEN contamination in corn and its related products in China are provided. This survey suggests the need for prevention of serious ZEN contamination, along with management for food safety and human health.

Droplet-based magnetic bead immunoassay using microchannel-connected multiwell plates (μCHAMPs) for the detection of amyloid beta oligomers

Automated operation of droplet-based magnetic bead immunoassay in the μCHAMPs.

Evaluation of a Multiplex Bead Immunoassay for Determination of Immune Status to Varicella-Zoster Virus in Medical Center Students and Employees

ABSTRACTThis study evaluated an enzyme immunoassay, a multiplex bead immunoassay (MBIA), and the anticomplement immunofluorescence (ACIF) test for detecting varicella-zoster virus IgG antibodies in sera from medical center students and employees. The agreement between methods was ≥95%. The MBIA was less sensitive than was the ACIF test, with a negative predictive value of 66.7%.

Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines

Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT). Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA) was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA) and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA) tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL). These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required.

Identification of Chemokines and Growth Factors in Proliferative Diabetic Retinopathy Vitreous

Associations were investigated between levels of chemokines and growth factors in the vitreous and proliferative diabetic retinopathy (PDR). Enrolled were 58 patients (58 eyes) requiring pars plana vitrectomy (PPV), with PDR (n=32, none with traction retinal detachment) or not (non-PDR). In the latter, 16 had macular hole (MH) and 10 had epiretinal membrane (ERM). With a multiplex bead immunoassay, levels of 11 chemokines and growth factors were measured from the undiluted vitreous sample from each patient. In the non-PDR eyes, the levels of the 11 chemokines and growth factors tested were similar between patients with MH and those with ERM. However, the levels of all 11 were significantly higher in the PDR eyes relative to the non-PDR; CCL17, CCL19, and TGFβ3 were markedly upregulated and have not been investigated in PDR previously. The significantly higher levels of CCL4 and CCL11 in PDR contradict the results of previous reports. Based on Spearman’s nonparametric test, moderate-to-strong correlations were found between VEGF and other mediators. Our results indicate that these chemokines and growth factors could be candidates for research into targeted therapies applied either singly or in combination with anti-VEGF drugs for the treatment of PDR.