Detection and Quantification of SARS-CoV-2 Receptor Binding Domain Neutralization by a Sensitive Competitive ELISA Assay

This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include (1) serum heat treatment to prevent non-specific interactions, (2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, (3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and (4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 h for >30 serum samples; this includes the 7.5 h of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the ‘gold standard’ assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious and time consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R2 = 0.975, n = 25), demonstrating high sensitivity.

The Changes of Prostatic Microvessel Density in Sprague-Dawleyrats After Castration Under the Effect of Estrogen/ Androgen at Different Concentrations

Background: Currently, there are relatively few studies on the effects of changes in estrogen and androgen levels on prostatic MVD.This article aimed to study the changes of prostatic MVD in SD rats after castration under the effect of estrogen/androgen at different concentrations.Methods: Male Sprague-Dawley(SD) rats aged 3-4 months were randomly divided into the control group, castration group, and different concentrations of estrogen/ androgen treatment after castration. Dihydrotestosterone(DHT) and estradiol(E) were administered daily by subcutaneous injection for one month. All the rats were sacrificed by cervical dislocation after one month, and the serum DHT and E concentrations of the rats in each group were measured by ELISA assay. Prostate tissues specimens were immunohistochemically stained with monoclonal antibodies against CD-34 and factor VIII for the MVD.Results: Compared with the control group, the MVD decreased significantly in the castration group (P<0.05). When the exogenous E concentration was constant, in general, the MVD of rats in all the groups increased with the increase of exogenous DHT concentration; Among them, compared with the castration group, the MVD increased significantly in the E0.05+DHT0.015 mg/kg group, E0.05+DHT0.05 mg/kg group, E0.05+DHT0.15 mg/kg group, E0.05+DHT0.5 mg/kg group, and E0.05+DHT1.5 mg/kg group (P<0.05). In addition, when the exogenous DHT concentration was constant, the MVD increased with the increase of exogenous E concentration in all the groups; Among them, compared with the control and castration group, the MVD increased significantly in the DHT0.15+E0.015 mg/kg group, DHT0.15+E0.15 mg/kg group, and DHT0.15+E0.5 mg/kg group (P<0.05).Conclusions: Androgens carried an important role in the regulation of prostatic MVD in SD rats, and the decrease of DHT concentration can induce a decrease in prostatic MVD. In contrast, prostatic MVD can be increased with the increase of DHT concentration. In addition, prostatic MVD can be increased gradually with the increase of estrogen concentration.

SARS-CoV-2 Antibodies Are Persisting in Saliva for More Than 15 Months After Infection and Become Strongly Boosted After Vaccination

SARS-CoV-2 antibodies in saliva serve as first line of defense against the virus. They are present in the mucosa, more precisely in saliva, after a recovered infection and also following vaccination. We report here the antibody persistence in plasma and in saliva up to 15 months after mild COVID-19. The IgG antibody response was measured every two months in 72 participants using an established and validated in-house ELISA assay. In addition, the virus inhibitory activity of plasma antibodies was assessed in a surrogate virus neutralization test before and after vaccination. SARS-CoV-2-specific antibody concentrations remained stable in plasma and saliva and the response was strongly boosted after one dose COVID-19 vaccination.

Long non-coding RNA LINC01194 promotes the inflammatory response and apoptosis of LPS-treated MLE 12 cells through the miR-203a-3p /MIP-2 axis

Acute lung injury (ALI) induced by bacteria LPS is characterized by the upregulation of the apoptosis rate of tissue cells and aggravation of inflammatory response. Although many studies have focused on the pathogenesis of this disease, its mechanism remains unknown. This study examined the regulatory role of long non-coding RNA (lncRNA) LINC01194 in the progression of ALI through various bioinformatics analyses and experimental work, including ELISA assay, dual-luciferase reporter assay, biotinylated RNA pull-down assay, and western blot analysis. The result showed that the LINC01194 was overexpressed in the ALI-induced mice model. We observed a significant upregulation of LINC01194 in LPS-treated Mouse lung epithelial type II cells (MLE-12 cells) after 24 hrs of induction. Bioinformatics analysis, Elisa assay, qRT-PCR analysis, Biotinylated RNA pull-down assay, apoptosis test, and western blot analysis demonstrated that the LINC01194 could act as a miR-203a-3p sponge to activate the inflammatory response in LPS-induced ALI model through post-transcriptional upregulation of MIP-2. We showed that LINC01194 regulates the inflammatory response and apoptosis of LPS-induced mice and MLE-12 cells via the miR-203a-3p/MIP-2 axis. LINC01194 could be a potential biomarker for early diagnosis and the treatment of ALI.

Improved diagnosis of viable parenchymal neurocysticercosis by combining antibody banding patterns on enzyme-linked immunoelectrotransfer blot (EITB) with antigen ELISA assay

The diagnosis of NCC depends on neuroimaging and serological confirmation. While antibody detection by enzyme-linked immunoelectrotransfer blot (EITB) fails to predict viable NCC, EITB banding patterns provide information about the host’s infection course. Adding antigen ELISA results on EITB banding patterns may improve their ability to predict or rule out of viable NCC. We assessed whether combining EITB banding patterns with Ag-ELISA improves discrimination of viable infection in imaging-confirmed parenchymal NCC. EITB banding patterns were grouped into classes using latent class analysis. True-positive and false-negative Ag-ELISA results in each class were compared using Fisher’s exact test. Four classes were identified: 1 (EITB-negative or positive to GP50 alone [GP50 antigen family]), 2 (positive to GP42-39 and GP24 [T24/42 family], with or without GP50), 3 and 4 (positive to GP50, GP42-39 and GP24, and reacting to bands in the 8-kDa family). Most cases in classes 3 and 4 had viable NCC (82% and 88%) compared to classes 2 and 1 (53% and 5%). Adding positive Ag-ELISA results to class 2 predicted all viable NCC cases (22/22 [100%]), whereas 11/40 patients (27.5%) Ag-ELISA negative had viable NCC ( P < 0.001). Only 1/4 patients (25%) Ag-ELISA positive in class 1 had viable NCC, whereas 1/36 patients (2.8%) Ag-ELISA negative had viable NCC ( P = 0.192). In classes 3 and 4, adding Ag-ELISA was not contributory. Combining Ag-ELISA with EITB banding patterns improves discrimination of viable from non-viable NCC, particularly for class-2 responses. Together, these complement neuroimaging more appropriately for the diagnosis of viable NCC.

Immunoreactivity of Polish Lyme Disease Patient Sera to Specific Borrelia Antigens—Part 1

The diverse clinical picture and the non-specificity of symptoms in Lyme disease (LD) require the implementation of effective diagnostics, which should take into account the heterogeneity of Borrelia antigens. According to available guidelines, laboratories should use a two-tier serological diagnosis based on the enzyme-linked immunosorbent (ELISA) screening test and confirmation of the immunoblot (IB). The aim of the study was to investigate the immunoreactivity of LD patient sera to Borrelia antigens and to attempt to identify the genospecies responsible for LD using an ELISA–IB assay combination. Eighty patients with suspected LD and 22 healthy people participated in the study. All samples were tested with ELISA and IB assays in both IgM and IgG antibodies. In the case of the ELISA assay, more positive results were obtained in the IgM class than in the IgG class. In the case of the IB assay, positive results dominated in the IgG class. Positive results obtained in the IB assay most often showed IgM antibodies against the OspC and flagellin antigens, whereas the IgG antibodies were against VlsE, BmpA, OspC, p41, and p83 antigens. The IB assay is an important part of LD serodiagnosis and should be mandatory in diagnostic laboratories.

A Novel ELISA-Based Peptide Biosensor Assay for Screening ABL1 Activity in vitro: A Challenge for Precision Therapy in BCR-ABL1 and BCR-ABL1 Like Leukemias

The pathogenic role of the overactivated ABL1 tyrosine kinase (TK) pathway is well recognized in some forms of BCR-ABL1 like acute lymphoblastic leukemia (ALL); TK inhibitors represent a useful therapeutic choice in these patients who respond poorly to conventional chemotherapy. Here we report a novel peptide biosensor (PABL)-ELISA assay to investigate ABL1 activity in four immortalized leukemic cell lines with different genetic background. The PABL sequence comprises an ABL1 tyrosine (Y) phosphorylation site and a targeting sequence that increases the specificity for ABL1; additional peptides (Y-site-mutated (PABL-F) and fully-phosphorylated (PPHOSPHO-ABL) biosensors) were included in the assay. After incubation with whole cell lysates, average PABL phosphorylation was significantly increased (basal vs. PABL phosphorylation: 6.84 ± 1.46% vs. 32.44 ± 3.25%, p-value &lt; 0.0001, two-way ANOVA, Bonferroni post-test, percentages relative to PPHOSPHO-ABL in each cell line). Cell lines expressing ABL1-chimeric proteins (K562, ALL-SIL) presented the higher TK activity on PABL; a lower signal was instead observed for NALM6 and REH (p &lt; 0.001 and p &lt; 0.05 vs. K562, respectively). Phosphorylation was ABL1-mediated, as demonstrated by the specific inhibition of imatinib (p &lt; 0.001 for K562, NALM6, ALL-SIL and p &lt; 0.01 for REH) in contrast to ruxolitinib (JAK2-inhibitor), and occurred on the ABL1 Y-site, as demonstrated by PABL-F whose phosphorylation was comparable to basal levels. In order to validate this novel PABL-ELISA assay on leukemic cells isolated from patient’s bone marrow aspirates, preliminary analysis on blasts derived from an adult affected by chronic myeloid leukaemia (BCR-ABL1 positive) and a child affected by ALL (BCR-ABL1 negative) were performed. Phosphorylation of PABL was specifically inhibited after the incubation of BCR-ABL1 positive cell lysates with imatinib, but not with ruxolitinib. While requiring further optimization and validation in leukemic blasts to be of clinical interest, the PABL-based ELISA assay provides a novel in vitro tool for screening both the aberrant ABL1 activity in BCR-ABL1 like ALL leukemic cells and their potential response to TK inhibitors.

The G505A Nonsynonymous Single-Nucleotide Polymorphism (SNP) in TAFI, the Gene Encoding Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) Is Pleiotropically Associated with TAFI Antigen Levels and Coronary Heart Disease (CHD) in Mexican Americans of South Texas

Studies have reported that the G505A nonsynonymous (ns)-SNP encoding the Ala147Thr amino acid substitution in TAFI, is associated with a reduction in risk of venous thrombosis and myocardial infarction. Interestingly, homozygosity for the A-allele (AA) of G505A is associated with increased TAFI antigen (TAFI:Ag) levels in plasma. In our study of the genetic determinants of cardiovascular disease (CVD) in Mexican Americans of South Texas, we performed an exome-wide scan in relation to plasma TAFI antigen levels in 784 individuals. While accounting for age, sex, and their interactions as confounders in linear mixed model, we found a heritability of 59% for TAFI:Ag levels (p=1.4E-19), and that ns-SNP G505A was the only variant showing exome-wide significance (p=3.5E-14; Figure A). Figure B shows the quantile-quantile distribution of the p-values from all the exome-wide tests. Clearly, the p-value distribution reveals that there is no systematic bias that may act to skew the p-values, and that the lone exception¾in agreement between observed and expected quantiles¾is due to this TAFI SNP, which strongly suggests a truly significant effect. Consistent with previous reports, the regression coefficient for G505A as a predictor of TAFI:Ag levels showed them to be increasing from the homozygous for the major G-allele (GG), to the heterozygous individuals (GA), to the individuals homozygous for the minor A-allele (AA) (Figure B). We also investigated if G505A is associated with our CHD variable. Using a statistical genetic model for the liability to disease conditional on a threshold, which is equivalent to a probit mixed model, we found that the G505A ns-SNP in TAFI, encoding TAFI Ala147Thr, is significantly associated with a reduction in the risk of CHD (p=0.002). As observed in existing literature, potential limitations of this study include the ELISA assay used for the quantification of TAFI levels. Some ELISA assays measure proTAFI, TAFIa, and TAFIi. However, recent evidence suggests that there may be cross reactivity between TAFIa and TAFIi. This can result in measuring ongoing TAFI activation peptides and elevated levels of TAFIa which ultimately goes on to make resistant fibrin. This is likely the marker that results in the increased risk of venous thromboembolism. To mitigate this confounding factor, an ELISA assay specific to measuring TAFI antigen is needed. In conclusion, we found that TAFI G505A is pleiotropically associated with TAFI:Ag levels and risk of CHD. Figure 1 Figure 1. Disclosures DeFronzo: AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; Intarcia: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding.

Diagnostic Utility of High Dose Heparin Confirmation Step in Heparin Induced Thrombocytopenia ELISA Assay

Introduction: Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening condition that could occur following exposure to heparin. Accurate and timely diagnosis is critical for appropriate clinical management. Laboratory testing for suspected cases is based on screening for the presence of serum anti-PF4/heparin antibodies using solid-phase enzyme-linked immunosorbent assay (ELSIA) which is known to be sensitive but less specific. A positive ELISA test is followed by functional testing to demonstrate the platelet activating properties and heparin dependence of the pathogenic antibodies. Serotonin release assay (SRA) is considered the gold standard functional test for the diagnosis of HIT. In most anti-PF4/heparin ELISA assays, a high-dose heparin buffer (100U/mL) confirmation step is recommended to demonstrate heparin dependence of detected antibodies and increase the specificity of the assay. The necessity of this confirmation step is controversial with some reports suggesting it could lead to misinterpreting positive ELISA results as negative or indeterminate, especially in cases of very strong and high titer HIT activating antibodies. We hence aimed to investigate the utility of applying this confirmation step as part of an inhouse validation study of a mass spectrometry-coupled SRA (Mayo-SRA). Materials: Three hundred archived serum samples were tested using anti-PF4/heparin IgG antibody ELISA (Immucor Diagnostics, GA, USA). High-dose heparin (100U/mL) confirmation step was performed on all samples with OD units ≥0.4 as recommended by the manufacturer. Samples with OD ≥ 0.4 and ≥50% OD inhibition in the high dose heparin confirmation step are interpreted positive. Mayo-SRA results were compared to a reference 14C SRA method. The 4T clinical score was retrospectively calculated for all patient (range 0-8 points). Results: Of the 300 tested samples, 57 samples were interpreted positive by the anti-PF4/heparin screening ELISA. 33 of the 57 samples were positive using the reference 14C SRA method, whereas 43 samples were positive by Mayo-SRA assay (≥20% serotonin release). Three additional samples were positive by Mayo-SRA, but negative by both screening ELISA and the reference 14C SRA method. All samples with OD units ≥0.4 displayed &gt;50% inhibition in the high-dose heparin regardless of the intensity of the initial OD value or the HIT 4T score, with the exception of one that was negative by both SRA methods and of 1.35 OD value and 6 4T HIT score (Fig-1A). Importantly, thirteen samples were anti-PF4/heparin positive, but SRA negative (by Mayo-SRA and reference method). These samples also displayed positive %heparin inhibition (≥50% OD inhibition) (Fig-1B). Lastly, there were no differences in the degree of %inhibition in samples positive by both reference and Mayo-SRA or Mayo-SRA only (Fig-1B). Conclusion: In our patient cohort, addition of the high dose heparin inhibition confirmation step to the screening anti-PF4/heparin ELISA assay was of no additional diagnostic utility. We hence propose eliminating the heparin inhibition step which would improve laboratory turnaround time, reduce costs, and importantly speed up urgent clinical management decisions. Figure Legend: Fig-1A. No correlation between initial OD values and %OD inhibition using high dose heparin. Scatter plot of all ELISA positive samples (OD ≥0.4) grouped according to OD values. Samples include all SRA-positive and thirteen SRA-negative ELISA-positive samples. Fig-1B. Scatter plot of %OD inhibition comparing samples positive by Mayo-SRA and reference SRA method, positive by Mayo-SRA only, and ELISA-positive SRA-negative by both methods. Figure 1 Figure 1. Disclosures Pruthi: Bayer Healthcare AG: Honoraria; CSL Behring: Honoraria; Merck: Honoraria; Genentech: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Synthetic Heparan Sulfate Compounds Attenuate Vascular Complications Associated with Sickle Cell Disease

In sickle cell disease (SCD), a mutation of the β-globin gene leads to abnormal polymerization of hemoglobin, resulting in formation of sickled red blood cells, hemolytic anemia, and vaso-occlusive crisis (VOC). These primary events produce clinical complications and trigger additional multiple pathologies driven by chronic oxidative stress, sterile inflammation, and activation of coagulation. Heparins, highly sulfated forms of heparan sulfate (HS), are a group of polysaccharide compounds with great variance in structure. In addition to their anticoagulant effect, heparins have anti-adhesive and anti-inflammatory properties. These are determined by both sulfation pattern and length of the polysaccharide chain, which influence the ability to bind HMGB-1, histones, and P-selectin (Psel). This heterogeneity together with the short half-life and dosing regimen based on anticoagulant activity, limit the use of heparins as anti-adhesive and anti-inflammatory agents. To overcome these limitations, we previously developed a chemoenzymatic approach to synthesize a structurally defined HS oligosaccharides and demonstrated their ability to reduce sterile inflammation in animal models by binding HMGB-1 and histones. In the present study, we investigated the compound's anti-Psel properties in vitro and in a mouse model of SCD. First, using a Psel inhibition ELISA assay, we determined that a heptadecasaccharide (17-mer) is the minimum polysaccharide chain length required for inhibition of Psel binding to Sialyl Lewis X polyacrylamide. Based on these data, we synthesized three 18-mer compounds with a different sulfation position on each monosaccharide ring (NS2S, NS6S and NS2S6S). To obtain 18-mers with no anticoagulant activity, we omitted 3-O-sulfation of glucosamine, which is important for binding antithrombin III (confirmed by anti-FXa activity assay). In the Psel inhibition ELISA assay, all compounds demonstrated dose dependent (0.1 - 1000 µg/mL) anti-Psel activity comparable to that observed for low molecular weight heparin (LMWH). Psel is a key molecule mediating VOC by promoting formation of multicellular aggregates. Therefore, we evaluated the effect of 18-mers on Psel-mediated platelet/leukocyte aggregates (PLA) formation ex vivo. Leukocytes and platelets isolated from healthy donors were stimulated with PMA (100 nM) for 1 hour or thrombin (5 µg/mL) for 30 minutes, respectively, then incubated with vehicle, 18-mer compounds, or LMWH (0.5, 5, 50 and 500 µg/mL) for 15 minutes. After incubation, cells were combined to allow PLA formation for 15 minutes and analyzed by flow cytometry. At the highest tested 18-mer concentration, all compounds attenuated PLA formation. However only NS2S6S, the most highly sulfated compound, showed significant inhibition at all concentrations. NS2S6S decreased PLA formation to 82.8% 90.8%, 76.3% and 68.3%, lowest to highest concentrations respectively (p&lt;0.01 for all concentrations versus vehicle). LMWH demonstrated significant decreases only at the two highest concentrations (82.1% and 63.8%, p&lt;0.001). The number of circulating PLA was increased in sickle Townes HbSS mice by 6.1-fold (p&lt;0.01) compared to non-sickle Townes HbAA controls. In ex vivo experiments, addition of NS2S6S (1 mg/ml) to the HbSS blood decreased PLA formation to 66.4% (p=0.03) compared to untreated HbSS blood. Finally, we determined the effect of NS2S6S on heme-induced microvascular stasis in Townes HbSS mice. Sickle mice were implanted with a dorsal skinfold chamber to visualize dermal microvessels. PBS or NS2S6S (3 mg/kg, s.c.) were injected 15 min before infusion of heme (1.2 µmol/kg, iv). 0ne, 2, 3 and 4 hours after heme infusion, microvascular stasis was observed in 31.5, 20.5, 18.9 and 14.2% of preselected vessels in PBS treated sickle mice, and NS2S6S treatment reduced that numbers to 10.9, 6.2, 3.1 and 3.2%, respectively (p&lt;0.01 for all time points). In summary, we showed that NS2S6S prevents Psel dependent formation of PLA ex vivo and reduces heme-induced stasis in sickle mice. Together with previously described anti-HMGB1 and anti-histone effects, this compound is a good candidate for multi-modal therapy to mitigate the pathophysiology of SCD. However, like LMWH, NS2S6S has a short half-life which makes prophylactic treatment of SCD patients impractical. Studies to extend the half-life of HS are currently ongoing in our group. Disclosures Xu: Glycan Therapeutics: Current Employment. Belcher: Mitobridge/Astellas: Consultancy, Research Funding; CSL Behring: Research Funding. Vercellotti: CSL Behring: Research Funding; Mitobridge, an Astellas Company: Consultancy, Research Funding. Liu: Glycan Therapeutics: Current Employment.