As increasing numbers of people recover from and are vaccinated against COVID-19, tests are needed to measure levels of protective, neutralizing antibodies longitudinally to help determine duration of immunity. We developed a lateral flow assay (LFA) that measures levels of neutralizing antibodies in plasma, serum or whole blood. The LFA is based on the principle that neutralizing antibodies inhibit binding of the spike protein receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2). The test classifies high levels of neutralizing antibodies in sera that were titered using authentic SARS-CoV-2 and pseudotype neutralization assays with an accuracy of 98%. Sera obtained from patients with seasonal coronavirus did not prevent RBD from binding to ACE2. As a demonstration for convalescent plasma therapy, we measured conversion of non-immune plasma into strongly neutralizing plasma. This is the first report of a neutralizing antibody test that is rapid, highly portable and relatively inexpensive that might be useful in assessing COVID-19 vaccine immunity.
Background and purpose: Voltage-operated sodium channels constitute major target sites for local anaesthetic-like action. The clinical use of local anaesthetics is still limited by severe side effects, in particular, arrhythmias and convulsions. These side effects render the search for new local anaesthetics a matter of high interest. Experimental approach: We have investigated the effects of three halogenated structural analogues of propofol on voltageoperated human skeletal muscle sodium channels (Na V 1.4) and the effect of one compound (4-chloropropofol) on neuronal sodium channels (Na V 1.2) heterologously expressed in human embryonic kidney cell line 293. Key results: 4-Iodo-, 4-bromo-and 4-chloropropofol reversibly suppressed depolarization-induced whole-cell sodium inward currents with high potency. The IC 50 for block of resting channels at À150 mV was 2.3, 3.9 and 11.3 mM in Na V 1.4, respectively, and 29.2 mM for 4-chloropropofol in Na V 1.2. Membrane depolarization inducing inactivation strongly increased the blocking potency of all compounds. Estimated affinities for the fast-inactivated channel state were 81 nM, 312 nM and 227 nM for 4-iodopropofol, 4-bromopropofol and 4-chloropropofol in Na V 1.4, and 450 nM for 4-chloropropofol in Na V 1.2. Recovery from fast inactivation was prolonged in the presence of drug leading to an accumulation of block during repetitive stimulation at high frequencies (100 Hz). Conclusions and implications:Halogenated propofol analogues constitute a novel class of sodium channel-blocking drugs possessing almost 100-fold higher potency compared with the local anaesthetic and anti-arrhythmic drug lidocaine. Preferential drug binding to inactivated channel states suggests that halogenated propofol analogues might be especially effective in suppressing ectopic discharges in a variety of pathological conditions. (2008) 155, 265-275; doi:10.1038/bjp.2008 published online 23 June 2008 Keywords: sodium channels; voltage-operated; local anaesthetics; propofol analogues Abbreviations: ECI 50 , half-maximum effect in inactivated channels; ECR 50 , half-maximum effect on resting channels; k, Boltzmann parameter, slope factor of the availability curve; Na V 1.2, voltage-gated neuronal sodium channel (brain type IIA); Na V 1.4, voltage-gated skeletal muscle sodium channel; n H , Hill coefficient; V 0.5 , Boltzmann parameter, voltage of half-maximum channel availability; DV 0.5 , shift in the voltage dependence of channel availability British Journal of Pharmacology
Background While evaluating COVID-19 vaccine responses using a rapid neutralizing antibody (NAb) test, we observed that 25% of mRNA vaccine recipients did not neutralize >50%. We termed this group “vaccine poor responders” (VPRs). The objective of this study was to determine if individuals who neutralized <50% would remain VPRs, or if a third dose would elicit high levels of NAbs. Methods 269 healthy individuals ranging in age from 19 to 80 (Average age = 51; 165 females and 104 males) who received either BNT162b2 (Pfizer) or mRNA-1273 (Moderna) vaccines were evaluated. NAb levels were measured: (i) 2–4 weeks after a second vaccine dose, (ii) 2–4 months after the second dose, (iii) within 1–2 weeks prior to a third dose and (iv) 2–4 weeks after a third mRNA vaccine dose. Results Analysis of vaccine recipients reveals that 25% did not neutralize above 50% (Median neutralization = 21%, titers <1:80) within a month after their second dose. Twenty-three of these VPRs obtained a third dose of either BNT162b2 or mRNA-1273 vaccine 1–8 months (average = 5 months) after their second dose. Within a month after their third dose, VPRs show an average 5.4-fold increase in NAb levels (range: 46–99%). Conclusions The results suggest that VPRs are not permanently poor responders; they can generate high NAb levels with an additional vaccine dose. Although it is not known what levels of NAbs protect from infection or disease, those in high-risk professions may wish to keep peripheral NAb levels high, limiting infection, and potential transmission.
Background Coccidioidomycosis is often diagnosed with a collection of tests that rely on the patient’s ability to mount an immune response to the fungus (antibody-based diagnostics), making diagnosis of this infection challenging. Here we present an antigen-based assay that detects and quantifies coccidioidal chitinase-1 (CTS1) in human serum. Methods An inhibition-based enzyme-linked immunoassay (ELISA) was developed that utilizes a monoclonal antibody specific for coccidioidal CTS1. CTS1 was quantified in commercial antigen preparations using recombinant CTS1 as a standard. Sera from 192 individuals from an endemic area were tested which included 78 patients (40.6%) with proven or probable coccidioidomycosis. Results The quantity of CTS1 in diagnostic commercial antigen preparations from different suppliers varied. CTS1 antigenemia was detected in 87.2% of patients with proven or probable coccidioidomycosis. Specificity was determined to be 96.94% using serum from individuals who reside in the Phoenix, Arizona area who did not have coccidioidomycosis. Levels of CTS1 correlated with low- and high-titer serology from patients with a coccidioidomycosis diagnosis. Conclusions Since the CTS1 inhibition ELISA described in this report does not depend on the host immune response, it is a promising diagnostic tool to aid in diagnosis and disease monitoring of coccidioidomycosis.
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