Quantitative and Standardized Pseudovirus Neutralization Assay for COVID-19.

COVID-19 is a global pandemic caused by the highly infectious SARS-CoV-2 virus. Efforts to combat SARS-CoV-2 infection include mass vaccination and development of monoclonal and convalescent plasma therapeutics that require precise measurements of correlative, functional neutralizing antibodies that prevent virus infection. Developing rapid, safe, easy-to-use, and high-quality neutralization assays are essential for the success of the massive effort.

Monoclonal Antibodies as SARS-CoV-2 Serology Standards: Experimental Validation and Broader Implications for Correlates of Protection.

COVID-19 has highlighted challenges in the measurement quality and comparability of serological binding and neutralization assays. Due to many different assay formats and reagents, these measurements are known to be highly variable with large uncertainties. The development of the WHO international standard (WHO IS) and other pool standards have facilitated assay comparability through normalization to a common material but does not provide assay harmonization nor uncertainty quantification.

Development of a Cell-Based SARS-CoV-2 Pseudovirus Neutralization Assay Using Imaging and Flow Cytometry Analysis.

COVID-19 is an ongoing, global pandemic caused by the novel, highly infectious SARS-CoV-2 virus. Efforts to mitigate the effects of SARS-CoV-2, such as mass vaccination and development of monoclonal therapeutics, require precise measurements of correlative, functional neutralizing antibodies that block virus infection. The development of rapid, safe, and easy-to-use neutralization assays is essential for faster diagnosis and treatment.

Number Concentration Measurements of Polystyrene Submicrometer Particles.

The number of techniques to measure number concentrations and size distributions of submicrometer particles has recently increased. Submicrometer particle standards are needed to improve the accuracy and reproducibility of these techniques. The number concentrations of fluorescently labeled polystyrene submicrometer sphere suspensions with nominal 100 nm, 200 nm and 500 nm diameters were measured using seven different techniques.

Sources of Variability in the Response of Labeled Microspheres and B Cells during the Analysis by a Flow Cytometer.

A stochastic model of the flow cytometer measurement process was developed to assess the nature of the observed coefficient of variation (CV%) of the mean fluorescence intensity (MFI) from a population of labeled microspheres (beads). Several sources of variability were considered: the total number of labels on a bead, the path through the laser beam, the optical absorption cross-section, the quantum yield, the numerical aperture of the collection optics, and the photoelectron conversion efficiency of the photomultiplier (PMT) cathode. The variation in the number of labels on a bead had the largest effect on the CV% of the MFI of the bead population.

Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19.

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen.

Stochastic Reaction-Diffusion Model of the Binding of Monoclonal Antibodies to CD4 Receptors on the Surface of T Cells.

A stochastic reaction-diffusion model was developed to describe the binding of labeled monoclonal antibodies (mAbs) to CD4 receptors on the surface of T cells. The mAbs diffused to, adsorbed on, and underwent monovalent and bivalent binding to CD4 receptors on the cell surface. The model predicted the time-dependent nature of all populations involved in the labeling process.

Comparison of Volumetric and Bead-Based Counting of CD34 Cells by Single-Platform Flow Cytometry.

Background: Over 2,000 people a year in the United Kingdom need a bone marrow or blood stem cell transplant. It is important to accurately quantify the hematopoietic stem cells to predict whether the transplant will be successful in replenishing the immune system. However, they are present at low frequency, which complicates accurate quantification.

A Model for the Binding of Fluorescently Labeled Anti-Human CD4 Monoclonal Antibodies to CD4 Receptors on Human Lymphocytes.

The CD4 glycoprotein is a component of the T cell receptor complex which plays an important role in the human immune response. This manuscript describes the measurement and modeling of the binding of fluorescently labeled anti-human CD4 monoclonal antibodies (mAb; SK3 clone) to CD4 receptors on the surface of human peripheral blood mononuclear cells (PBMC). CD4 mAb fluorescein isothiocyanate (FITC) and CD4 mAb allophycoerythrin (APC) conjugates were obtained from commercial sources.

Quantitative Fluorescence Measurements with Multicolor Flow Cytometry.

Multicolor flow cytometer assays are routinely used in clinical laboratories for immunophenotyping, monitoring disease and treatment, and determining prognostic factors. However, existing methods for quantitative measurements have not yet produced satisfactory results independent of flow cytometers used. This chapter details a procedure for quantifying surface and intracellular protein biomarkers by calibrating the output of a multicolor flow cytometer in units of antibodies bound per cell (ABC).

Assignment of the Number of Equivalent Reference Fluorophores to Dyed Microspheres.

A procedure will be described to assign to each dyed microsphere a number called the Equivalent number of Reference Fluorophores (ERF). The ERF unit gives the number of reference fluorophores in solution which produce the same fluorescence signal as a single dyed microsphere. In the first step, fluorescence measurements were carried out on serial dilutions of a solution of reference fluorophores.

Quantitative Flow Cytometry Measurements in Antibodies Bound per Cell Based on a CD4 Reference.

Multicolor flow cytometer assays with fluorescently labeled antibodies are routinely used in clinical laboratories to measure the cell number of specific immunophenotypes and to estimate expression levels of specific receptors/antigens either on the cell surface or intracellularly. The cell number and specific receptors/antigens serve as biomarkers for pathological conditions at various stages of a disease. Existing methods and cell reference materials for quantitative expression measurements have not yet produced results that are of wide clinical interest or are instrument-independent across all fluorescence channels.

Consistent, multi-instrument single tube quantification of CD20 in antibody bound per cell based on CD4 reference.

Detecting changes in the expression levels of cell antigens could provide critical information for the diagnosis of many diseases, for example, leukemia, lymphoma, and immunodeficiency diseases, detecting minimal residual disease, monitoring immunotherapies and discovery of meaningful clinical disease markers. One of the most significant challenges in flow cytometry is how to best ensure measurement quality and generate consistent and reproducible inter-laboratory and intra-laboratory results across multiple cytometer platforms and locations longitudinally over time. In a previous study, we developed a procedure for instrument standardization across four different flow cytometer platforms from the same manufacturer.

Quantification of cells with specific phenotypes I: determination of CD4+ cell count per microliter in reconstituted lyophilized human PBMC prelabeled with anti-CD4 FITC antibody.

A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties.

Optical Properties of CdSe/ZnS Nanocrystals.

Measurements are presented of the absorbance, fluorescence emission, fluorescence quantum yield, and fluorescence lifetime of CdSe/ZnS nanocrystals, also known as quantum dots (QDs). The study included three groups of nanocrystals whose surfaces were either passivated with organic molecules, modified further with carboxyl groups, or conjugated with CD14 mouse anti-human antibodies. The surface modifications had observable effects on the optical properties of the nanocrystals.

Measurement of Scattering and Absorption Cross Sections of Dyed Microspheres.

Measurements of absorbance and fluorescence emission were carried out on aqueous suspensions of polystyrene (PS) microspheres with a diameter of 2.5 µm using a spectrophotometer with an integrating sphere detector. The apparatus and the principles of measurements were described in our earlier publications.

Human CD4+ lymphocytes for antigen quantification: characterization using conventional flow cytometry and mass cytometry.

To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC-National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples.

Development of Multicolor Flow Cytometry Calibration Standards: Assignment of Equivalent Reference Fluorophores (ERF) Unit.

A procedure is described for assigning the number of equivalent reference fluorophores (ERF) values to microspheres labeled with a fluorophore designed to produce a fluorescence response in a given fluorescence channel of a multicolor flow cytometer. A fluorimeter was calibrated by a series of solutions of the reference fluorophores. The fluorimeter was used to obtain the microsphere fluorescence intensity, and a multicolor flow cytometer was used to obtain the microsphere concentration.

Quantitative fluorescence measurements with multicolor flow cytometry.

Multicolor flow cytometer assays are routinely used in clinical laboratories for immunophenotyping, -monitoring disease and treatment, and determining prognostic factors. However, existing methods for quantitative measurements have not yet produced satisfactory results. This chapter details a procedure for quantifying surface and intracellular protein biomarkers by calibrating the output of a multicolor flow cytometer in units of antibodies bound per cell (ABC).

A model for harmonizing flow cytometry in clinical trials.

Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.

An international comparability study to determine the sources of uncertainty associated with a non-competitive sandwich fluorescent ELISA.

Background: Immunoassays allow the specific detection and quantitation of biological molecules in complex samples at physiologically relevant concentrations. However, there are concerns over the comparability of such techniques when the same assay is performed by different operators or laboratories. An international intercomparison study was performed to assess the uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA.

Process monitoring the inactivation of ricin and model proteins by disinfectants using fluorescence and biological activity.

It is important to develop rapid and reliable processes to monitor the decontamination of toxins released to the environment. The inactivation of the protein toxin ricin by the disinfectants bleach (sodium hypochlorite) and monochloramine was measured by the effect on mammalian cell cytotoxicity. The effect of the disinfectants on the native fluorescence (due mainly to tryptophan and to a lesser extent tyrosine) of ricin was also measured in parallel.

Modeling of Photochemical Reactions in a Focused Laser Beam, II.

A method is described for obtaining the rate constant of the photodegradation process of fluorophores illuminated by a focused laser beam. The explicit kinetic equations, describing the population dynamics of excited singlet and triplet states, are averaged over the illuminated volume to describe the resulting fluorescence signal. The illumination is modulated at frequencies from 1 Hz to 100 Hz.

Measurement of the Fluorescence Quantum Yield Using a Spectrometer With an Integrating Sphere Detector.

A method is proposed for measuring the fluorescence quantum yield (QY) using a commercial spectrophotometer with a 150 mm integrating sphere (IS) detector. The IS detector is equipped with an internal cuvette holder so that absorbance measurements can be performed with the cuvette inside the IS. In addition, the spectrophotometer has a cuvette holder outside the IS for performing conventional absorbance measurements.

Toward quantitative fluorescence measurements with multicolor flow cytometry.

A procedure is presented for calibrating the output of a multicolor flow cytometer in units of antibodies bound per cell (ABC). The procedure involves two steps. First, each of the fluorescence channels of the flow cytometer is calibrated using Ultra Rainbow beads with assigned values of equivalent number of reference fluorophores (ERF).