Biolayer interferometry for adeno-associated virus capsid titer measurement and applications to upstream and downstream process development.

Faster and more accurate analytical methods are needed to support the advancement of recombinant adeno-associated virus (rAAV) production systems. Recently, biolayer interferometry (BLI) has been developed for high-throughput AAV capsid titer measurement by functionalizing the AAVX ligand onto biosensor probes (AAVX-BLI). In this work, an AAVX-BLI method was evaluated using Octet AAVX biosensors across four rAAV serotypes (rAAV2, -5, -8, and -9) and applied in an upstream and downstream processing context. AAVX-BLI measured the capsid titer across a wide concentration range (1 × 1010-1 × 1012 capsids/mL) for different rAAV serotypes and sample backgrounds with reduced measurement variance and error compared to an enzyme-linked immunosorbent assay (ELISA) method. Biosensors were regenerated for repeated use, with lysate samples showing reduced regeneration capacity compared to purified and supernatant samples. The AAVX-BLI method was applied in a transfection optimization study where direct capsid titer measurement of culture supernatants generated a representative response surface for the total vector genome (VG) titer. For rAAV purification, AAVX-BLI was used to measure dynamic binding capacity with POROS CaptureSelect AAVX affinity chromatography, showing resin breakthrough dependence on the operating flow rate. Measurement accuracy, serotype and sample background flexibility, and high sample throughput make AAVX-BLI an attractive alternative to other capsid titer measurement techniques.

Evaluation of compressive damage in concrete using ultrasonic nonlinear coda wave interferometry.

This study investigates the feasibility of nonlinear coda wave interferometry (NCWI) for evaluating compressive damage in concrete, with a particular focus on the interference caused by the compressive stress-induced slow dynamics. Slow dynamics refers to a phenomenon in which the stiffness of concrete immediately decreases after moderate mechanical conditioning and then logarithmically evolves back to its initial value over time. A series of experiments were conducted to validate this concept. The experimental findings indicate that slow dynamics following the unloading of concrete specimen significantly interfere with NCWI testing. The changes in dv/v caused by the slow dynamics are opposite to those induced by the pump wave in NCWI. After the slow dynamics have been eliminated, an evaluation indicator, defined as the efficient nonlinear level αdv/v, demonstrates an excellent correlation with compressive damage. The value of the indicator decreases with increasing compressive stress. Furthermore, the coda wave interferometry (CWI) and direct wave interferometry (DWI) are performed as comparisons. In summary, the feasibility and superiority of NCWI are demonstrated in concrete compressive damage evaluation.

Impact of temperature and relative humidity variations on coda waves in concrete.

The microstructure of concrete can be affected by many factors, from non-destructive environmental factors through to destructive damage induced by transient stresses. Coda wave interferometry is a technique that is sensitive enough to detect weak changes within concrete by evaluating the ultrasonic signal perturbation compared to a reference state. As concrete microstructure is sensitive to many factors, it is important to separate their contributions to the observables. In this study, we characterize the relationships between the concrete elastic and inelastic properties, and temperature and relative humidity. We confirm previous theoretical studies that found a linear relationship between temperature changes and velocity variation of the ultrasonic waves for a given concrete mix, and provide scaling factors per Kelvin for multiple settings. We also confirm an anti-correlation with relative humidity using long-term conditioning. Furthermore, we explore beyond the existing studies to establish the relationship linking humidity and temperature changes to ultrasonic wave attenuation.

Application of bio-layer interferometry for the analysis of ribosome-protein interactions.

The ribosome, a ribonucleoprotein complex, performs the function of protein translation. While ribosomal RNA catalyzes polypeptide formation, several proteins assist the ribosome throughout the translation process. Studying the biochemical and kinetic properties of these proteins interacting with the ribosome is vital for elucidating their roles. Various techniques, such as zonal centrifugation, pull-down assays, dynamic light scattering (DLS), fluorescence polarization, and surface plasmon resonance (SPR) are employed for this purpose, each presenting unique advantages and limitations. We add to the repertoire of techniques by using Bio-Layer Interferometry (BLI) to examine interactions between the ribosome and translation factors. Our findings demonstrate that BLI can detect interactions of Escherichia coli ribosomes with two proteins: E. coli initiation factor 2 (IF2) and P. falciparum translation enhancing factor (PTEF). A protein (Green Fluorescent Protein; GFP) known not to bind to E. coli ribosomes, shows no binding in the BLI assay. We show that BLI could be used to study the ribosome-protein interactions as it has key advantages like label-free procedures, ease of assay performance, and ribosome sample reuse. Our results highlight the comprehensive use of BLI in studying the ribosome-protein interactions, in addition to studying protein-protein and protein-ligand interactions.

Real-time monitoring of peptic and tryptic digestions of immunoglobulin G and the impact of dietary hydrocolloids on digestion.

Immunoglobulin G (IgG) exhibits potent antiviral, antibacterial, and immunological activities. The digestion process and bioavailability of IgG are often a concern. Dietary hydrocolloids are crucial for regulating healthy digestion and the bioavailability of protein as functional components. Understanding the effects of dietary hydrocolloids on the digestive kinetics of IgG is requisite. Herein, the pepsin and trypsin digestion of IgG was investigated using ordered porous layer interferometry (OPLI). The real-time variation in the interference spectral shift reflected by OPLI can be converted into changes in the optical thickness (OT) to obtain a degradation kinetics curve. The impact of dietary hydrocolloids, including alginic acid sodium salt (ALG), polydextrose (PD), and konjac glucomannan (KG), on IgG degradation was evaluated using OPLI. The results demonstrated that ALG significantly inhibited the degradation of IgG by pepsin under acidic conditions, whereas the addition of PD increased the Michaelis-Menten constant for IgG degradation by trypsin. Notably, this dependence is not based on the hydrocolloid viscosity, but relies more on the electrical properties. The study enhances our understanding of how hydrocolloids affect IgG digestion and could provide valuable insights into preserving IgG activity and facilitating the development of oral drugs or health products related to IgG.

Methods for monitoring protein-membrane binding. Comparison based on the interactions between amyloidogenic protein human cystatin C and phospholipid liposomes.

A cell membrane is an essential cellular component providing protection against the outer environment. It is also a host for proteins and carbohydrates responsible for, e.g. transporter, receptor, or enzymatic functions. In parallel, the membrane may also be implicated in pathological processes leading, e.g. to the oligomerization of amyloid-forming proteins, a hallmark of i.a. Alzheimer's disease. The increasing need for detailed information on mechanisms driving the amyloid formation and the potential role of cell membranes in the process proves the research on protein-membrane interactions biologically relevant. Considering the potential and limitations of the relatively well established and newly developed methods, this study focused on selecting methods that allow a broad and comprehensive description of interactions between amyloidogenic protein human cystatin C and lipid bilayers. In the first step, dot-blot and ELISA tests were selected as techniques allowing fast screening for protein-ligand interactions. Next, surface plasmon resonance, spectral shift, biolayer interferometry, and switchSENSE® technology were used to determine kinetic parameters and binding constants for interactions between human cystatin C and the selected lipid bilayers. Based on the obtained results we have proposed the most promising candidates for monitoring of interactions and determining affinity between amyloidogenic proteins and membrane mimetics.

Intra- and interobserver reliability of ocular surface analyzer LacryDiag®.

AIM: To invastigate intra- and interobserver reliability of interferometry, tear meniscus height (TMH) measurement and meibography (MBG) of an ocular surface analyzer, LacryDiag (Quantel Medical, France). METHODS: Five consecutive measurements and subsequent analysis of interferometry, TMH, and MBG were recorded by two examiners using the LacryDiag. To assess intra- and interobserver reliability, we used Cohen's kappa for categorical variables (interferometry), or intraclass correlation coefficient for continuous variables (TMH, MBG). RESULTS: Thirty eyes of 30 examinees were included. For both observers, there was excellent intraobserver reliability for MBG (0.955 and 0.970 for observer 1 and 2, respectively). Intraobserver reliability for observer 1 was substantial for interferometry (0.799), and excellent for TMH (0.863). Reliability for observer 2 was moderate for interferometry (0.535) and fair to good for TMH (0.431). Interobserver reliability was poor for interferometry (0.074) and fair to good for TMH (0.680) and MBG (0.414). CONCLUSION: LacryDiag ocular surface analyzer in our study proves to be a reliable noninvasive tool for the evaluation of TMH and MBG. As for interferometry, poor interobserver reliability, fair to good intraobserver reliability for observer 1, and moderate for observer 2, leave room for improvement.

A Reflected-Light-Mode Multiwavelength Interferometer for Measurement of Step Height Standards.

The article is dedicated to measuring the thickness of step height standards using the author's version of the variable wavelength interferometer (VAWI) in the reflected-light mode, where the interference pattern is created by the combination of two Wollaston prisms. The element of novelty consists in replacing the traditional search for the coincidence of fringes in the object and background with a continuous measurement of their periods and phases relative to the zero-order fringe. The resulting system of sinusoids is then analyzed using two methods: the classical one and the second utilizing the criterion of uniform thickness. The theory is followed by simulation and experimental parts, providing insight to the metrological potential of the VAWI technology.

Correlation between meibomian gland dysfunction and sebaceous adenitis in dogs.

BACKGROUND: Sebaceous adenitis (SA) is an immune-mediated disease targeting the sebaceous glands. Meibomian gland dysfunction is a disease affecting meibomian glands with inflammatory features. Sebaceous and meibomian glands share anatomical, physiological and embryological similarities. The involvement of meibomian glands in dogs with SA is currently unknown. OBJECTIVES: To evaluate meibomian glands in dogs affected by SA and compare them with healthy dogs. ANIMALS: Eighteen dogs were enrolled. Nine dogs with SA were retrospectively identified from clinical records and represented the case group. Nine healthy, breed- and age-matched dogs were prospectively enrolled in the control group. MATERIALS AND METHODS: Both groups underwent dermatological examination, Schirmer tear test-1 (STT-1), tear meniscus height (TMH), slit-lamp biomicroscopy, interferometry (INT) and noncontact infrared meibography (NIM). RESULTS: One third of SA dogs presented subepithelial crystalline opacities. No significant difference between groups was observed in TMH (p = 0.944) and STT-1 values (p = 0.066). INT (p = 0.016) and NIM grades (p = 0.010) were significantly higher and lower in the SA group compared to the control group, respectively. INT values decreased with age (η = 0.930), while NIM scores (η = 0.935) increased. CONCLUSIONS: Clinical Relevance: Subepithelial crystalline opacities in SA dogs might reflect a reduced tear film quality. In the absence of standardised methods, INT and NIM proved to be noninvasive and useful methods to examine meibomian glands. Dogs with SA showed a thinner lacrimal lipid layer and more severe meibomian gland abnormalities than control dogs, which seemed to progress with age.

An Integrated Photonic Biosensing Platform for Pathogen Detection in Aquaculture.

Aquaculture is expected to play a vital role in solving the challenge of sustainably providing the growing world population with healthy and nutritious food. Pathogen outbreaks are a major risk for the sector, so early detection and a timely response are crucial. This can be enabled by monitoring the pathogen levels in aquaculture facilities. This paper describes a photonic biosensing platform based on silicon nitride waveguide technology with integrated active components, which could be used for such applications. Compared to the state of the art, the current system presents improvements in terms of miniaturization of the Photonic Integrated Circuit (PIC) and the development of wafer-level processes for hybrid integration of active components and for material-selective chemical and biological surface modification. Furthermore, scalable processes for integrating the PIC in a microfluidic cartridge were developed, as well as a prototype desktop readout instrument. Three bacterial aquaculture pathogens (Aeromonas salmonicida, Vagococcus salmoninarum, and Yersinia ruckeri) were selected for assay development. DNA biomarkers were identified, corresponding primer-probe sets designed, and qPCR assays developed. The biomarker for Aeromonas was also detected using the hybrid PIC platform. This is the first successful demonstration of biosensing on the hybrid PIC platform.

Comparison of Eye Axial Length Measurements Taken Using Partial Coherence Interferometry and OCT Biometry.

This study evaluates the inter-device measurement properties of partial coherence interferometry (PCI) and spectral domain optical coherence tomography (SD-OCT) in measuring axial length, particularly for myopia management. We recruited 82 eyes from 41 adult participants with a mean age of 31.0 ± 17.6 years and a mean spherical equivalent of -2.20 ± 2.28 D. Axial length was measured using SD-OCT and PCI for both the right and left eyes. Agreement between the two measurements was assessed using Bland-Altman analysis, and graphs and values were compared with linear mixed models. The results show a near-to-zero and non-significant bias between measurements. The 95% limits of agreement showed a value of 0.06 mm. Both devices can accurately measure the axial length. OCT biometry performed with SD-OCT can be successfully interchanged with partial coherence interferometry, but they should be cautiously interchanged when performing longitudinal comparisons.

The surface deformation of permafrost and active layer thickness in the upper reaches of the Black River basin, revealed by InSAR observations and independent component analysis.

The Heihe River Basin, located in the northeastern part of the Qinghai-Tibetan Plateau, is part of the perennial permafrost belt of the Qilian Mountains. Recent observations indicate ongoing permafrost degradation in this region. This study utilizes data from 255 observations provided by Sentinel-1 satellites, MODIS Land Surface Temperature, SMAP-L4 soil moisture data, GNSS measurements, and in situ measurement. We introduced Variational Bayesian independent Component Analysis (VB-ICA) in multi-temporal Interferometric Synthetic Aperture Radar (MT-InSAR) processing to investigate the spatial-temporal characteristics of surface deformation and permafrost active layer thickness (ALT) variations. The analysis demonstrates strong agreement with borehole data and offers improvements over traditional methodologies. The maximum value of ALT in the basin is found to be 5.7 m. VB-ICA effectively delineates seasonal deformations related to the freeze-thaw cycles, with a peak seasonal deformation amplitude of 60 mm. Moreover, the seasonal permafrost's lower boundary reaches an elevation of 3700 m, revealing that permafrost is experiencing widespread degradation and associated soil erosion in the high elevation region of The Heihe River Basin. The paper also explores the efficacy of reference point selection and baseline network establishment for employing the InSAR method in monitoring freeze-thaw deformations. The study underscores the InSAR method's adaptability and its importance for interpreting permafrost deformation and related parameters.

Investigation of ocular surface parameters in dogs with different cephalic conformations using veterinary ocular surface analyzer (OSA-VET).

OBJECTIVE: To compare ocular surface parameters in dogs with different cephalic conformations and evaluate correlations among tests. ANIMALS STUDIED: Sixty-eight privately owned dogs. PROCEDURES: The study categorized canine eyes into three groups based on the craniofacial ratio (CFR): brachycephaly (≤0.52), mesocephaly (>0.52 to <0.67), and dolichocephaly (≥0.67). All eyes were examined using an ocular surface analyzer (OSA-VET) to determine lipid layer thickness (LLT) of the tear film, tear meniscus height (TMH), non-invasive tear breakup time (NIBUT), and meibomian gland loss rate of the lower eyelids (MGLRL). Schirmer tear test 1 (STT-1) and tear film breakup time (TBUT) were also performed. Statistical analyses involved one-way ANOVA, Kruskal-Wallis H test, post hoc Holm-Sidak test, and Pearson correlation coefficient. RESULTS: While STT-1 showed no significant difference among dog groups, brachycephalic dogs had significantly lower values in TBUT, NIBUT, and LLT, and a higher TMH, compared to mesocephalic and dolichocephalic dogs. Additionally, brachycephalic dogs exhibited a significantly higher MGLRL than dolichocephalic dogs. Correlations among tests were generally weak to moderate (r < .6) except for a strong correlation between CFR and LLT (r = .641, p < .001), and between TBUT and NIBUT (r = .899, p < .001). CONCLUSIONS: Brachycephalic morphology predisposes dogs to a significantly thinner lipid layer and diminished tear film stability, likely due to factors such as impaired meibomian gland function and increased ocular exposure compared to other cephalic conformations, thereby increasing their risk of keratoconjunctivitis sicca (KCS). OSA-VET shows a valuable tool to provide more comprehensive and precise diagnosis for canine ocular surface disorders.

The utility of software-detected non-invasive tear break-up in comparison to fluorescein tear break-up measurements.

Purpose: The purpose of this study is to characterize and discuss the difference between software-detected non-invasive tear break-up time (NIBUT) and the traditional clinical method of fluorescein break-up time (FBUT). Methods: Tear interferometry with the KOWA DR-1α (Kowa, Japan) and a standardized comprehensive ocular surface/tear evaluation were performed in 307 eyes. Software-detected NIBUT in the KOWA DR-1α images and the investigator-detected FBUT were compared. Results: Software-detected NIBUT was significantly shorter than investigator-measured FBUT. NIBUT was 3.1 ± 2.5 s (mean ± SD), whereas FBUT was 4.8 ± 3.0 s. This difference was due to three different patterns or conditions: a spot break immediately after eyelid opening, moderate to severe keratitis sicca, and epithelial basement membrane corneal dystrophy (EBMD). In these cases, rapid tear film disruption was not captured by FBUT. A spot break immediately after eye opening that rapidly disappears was observed with conjunctivochalasis. This type of break-up may be difficult to detect using fluorescein because the human eye cannot catch such rapid blinks or post-blink events. In the second group with severe corneal epithelial disease, break-up may occur over the entire corneal surface upon eye opening, and distinct fluorescein tear break-up may not be identified because of poor dye dilution or spread over the corneal surface, whereas the non-invasive break-up is not solution-dependent, and the software can detect a distinct appearance. In the third group with EBMD, it is possible that focal break-up in the fluorescein pattern over the epithelial elevations, which might be missed visually, can be detected by software in video images. Conclusion: We found that software-detected NIBUT is more sensitive in detecting tear break-up, can identify certain tear film disruptions that are missed by traditional FBUT, and may be more useful in distinguishing certain tear disorders.

Evaluation of Meibomian Gland Structure and Function in Patients with Vernal Keratoconjunctivitis Using Non Contact Meibography and Lipid Layer Interferometry.

PURPOSE: This study evaluated the structural and functional impact of vernal keratoconjunctivitis (VKC) on meibomian glands (MGs) using a combination of noncontact meibography and lipid layer interferometry. METHODS: In this observational study 50 patients with moderate persistent or severe VKC and 50 age-matched controls underwent MG imaging and lipid layer thickness (LLT) measurements with Lipiview II. Image J software was used to assess MG loss (meibograde) in both lids. All patients underwent dry eye evaluation comprising tear break-up time (TBUT), ocular surface staining (OSS), Schirmer I scoring, and meiboscoring (expressibility and quality of meibum secreted). RESULTS: Meibograde, OSS score, and meiboscore was higher in cases (2.68 ± 0.96, 0.580 ± 1.07, and 0.56 ± 0.95 respectively) than controls (1.80 ± 0.67, 0.00 ± 0.00, 0.22 ± 0.47 respectively) (p < 0.001, 0.001, 0.025 respectively). LLT and TBUT was lower in cases (54.58 ± 9.43 nm and 4.92 ± 3.09 sec respectively) than controls (70.14 ± 22.50 nm and 12.02 ± 2.73 sec respectively) (both p's = 0.001). Both groups had comparable Schirmer I scores. CONCLUSION: Children with VKC have significant MG dropouts, deterioration in meibum quality and a thinner and less stable tear film. VKC patients are thus prone to a vicious cycle of inflammation attributable both to the allergic component and to deterioration in MG structure and function. Co-management of MG dysfunction warrants as much attention as the allergic component itself.

Antifoams in non-aqueous diesel fuels: Thin liquid film dynamics and antifoam mechanisms.

HypothesisFoaming in diesel fuels is not well understood and leads to operational challenges. To combat deleterious effects of foaming, diesel formulations can include additives called antifoams. Existing antifoams, unfortunately, are inherently ash-generating when combusted, with unknown environmental impacts. They are prohibited in certain countries, so identifying effective alternative ash-free antifoam chemistries is needed. ExperimentsWe conduct systematic characterization of foam stabilization and antifoaming mechanisms in diesel for two different antifoams (silicone-containing & ashless chemistries). Employing a custom technique combining single-bubble/single-antifoam-droplet manipulation with white light interferometry, we also obtain mechanistic insights into foam stability and antifoam dynamics. ResultsCoalescence times from both bulk foam and single bubble experiments confirm ashless antifoams are effective at reducing foaming, demonstrating the potential of ashless antifoams. Further, we perform single-antifoam-droplet experiments and obtain direct experimental evidence revealing the elusive antifoaming mechanisms. Interestingly, the silicone-containing and ashless antifoams seemingly function via two different mechanisms: spreading and dewetting respectively. This surprising finding refutes conventional wisdom that spreading is likely the only antifoam mechanism in diesels. These results and the reported experimental framework significantly enhance the scientific understanding of non-aqueous foams and will accelerate the engineering of alternative antifoam chemistries for non-aqueous systems.

Impact of Cyclic Error on Absolute Distance Measurement Based on Optical Frequency Combs.

Absolute distance measurements based on optical frequency combs (OFCs) have greatly promoted advances in both science and technology, owing to the high precision, large non-ambiguity range (NAR), and a high update rate. However, cyclic error, which is extremely difficult to eliminate, reduces the linearity of measurement results. In this study, we quantitatively investigated the impact of cyclic error on absolute distance measurement using OFCs based on two types of interferometry: synthetic wavelength interferometry and single-wavelength interferometry. The numerical calculations indicate that selecting a suitable reference path length can minimize the impact of cyclic error when combining the two types of interferometry. Recommendations for selecting an appropriate synthetic wavelength to address the tradeoff between achieving a large NAR and minimizing the risk of failure when combining the two methods are provided. The results of this study are applicable not only in absolute distance measurements but also in other applications based on OFCs, such as surface profile, vibration analysis, etc.

Displacement Sensing for Laser Self-Mixing Interferometry by Amplitude Modulation and Integral Reconstruction.

To robustly and adaptively reconstruct displacement, we propose the amplitude modulation integral reconstruction method (AM-IRM) for displacement sensing in a self-mixing interferometry (SMI) system. By algebraically multiplying the SMI signal with a high-frequency sinusoidal carrier, the frequency spectrum of the signal is shifted to that of the carrier. This operation overcomes the issue of frequency blurring in low-frequency signals associated with continuous wavelet transform (CWT), enabling the precise extraction of the Doppler frequency of the SMI signal. Furthermore, the synchrosqueezing wavelet transform (SSWT) is utilized to enhance the frequency resolution of the Doppler signal. Our experimental results demonstrate that the proposed method achieves a displacement reconstruction accuracy of 21.1 nm (0.89%). Additionally, our simulations demonstrated that this method can accurately reconstruct target displacement under the conditions of time-varying optical feedback intensity or a signal-to-noise ratio (SNR) of 0 dB, with a maximum root mean square (RMS) error of 22.2 nm. These results highlight its applicability in real-world environments. This method eliminates the need to manually determine the window length for time-frequency conversion, calculate the parameters of the SMI system, or add additional optical devices, making it easy to implement.

Identification of exfoliated monolayer hexagonal boron nitride films with a digital color camera under white light illumination.

Optical microscopy with white light illumination has been employed when obtaining exfoliated monolayer hexagonal boron nitride (1L hBN) films from a large number of randomly placed films on a substrate. However, real-time observation of 1L hBN using a color camera under white light illumination remains challenging since hBN is transparent in the visible wavelength range. The poor optical constant of 1L hBN films in microphotographs is significantly improved using a Si substrate coated with a SiNxthin-film (SiNx/Si). When observing hBN thin films on SiNx/Si using a color digital camera in an optical microscope under white light illumination, the clarity of the captured color images depends on the thickness of the SiNxfilm (d). For real-time direct observation, thedwas optimized based on quantitative chromatic studies tailored to Bayer filters of a color image sensor. Through image simulation, it was determined that the color difference between 1L hBN and the bare substrate is maximized atd= 59 or 70 nm, which was experimentally verified. The SiNx/Si with optimizeddvalues visualized 1L hBN films without requiring significant contrast enhancement via image processing under white light illumination in real-time. Furthermore, the captured color photographs facilitate the reliable determination of the number of layers in few-layer hBN films using the contrast of the green channel of the images.

Unique challenges required reassessment and alterations to critical reagents to rescue a neutralizing antibody assay.

Aim: To redevelop a neutralizing antibody (NAb) assay to be much more drug tolerant, have a large dynamic range and have high inhibition when using high levels of positive control (PC). Materials & methods: Early assay data suggested that typical biotin labeling of the capture reagent (Drug 1, produced in a human cell line) was blocking it from binding with the PC or the detection target, and that the detection target was out competing the PC. Methodical biotin labeling experiments were performed at several challenge ratios and an Fc linker was added to the detection target. Results & conclusion: A larger dynamic range, high inhibition and higher drug tolerance were achieved by adding an acid dissociation step to the assay, performing atypical biotin labeling of Drug 1 and switching to a detection target that contained an Fc linker to increase steric hinderance and decrease its binding affinity to Drug 1.

Many of the drugs available today are produced by a living organism and these are called biologics. Biologics are larger than chemical drugs and the human body can detect them as foreign and create antibodies against them. This is called immunogenicity. When the antibodies created against the biologic blocks the drug's ability to work correctly, they are called neutralizing antibodies (NAbs). Testing for NAbs is one of the requirements of regulatory agencies for biologics. Here we describe challenges encountered developing an assay to test for NAbs against a biologic.