Assessment of specificity of dermatopathologic criteria for IgG4-related skin disease.

BACKGROUND: IgG4-related disease (IgG4-RD) represents a recently characterized multisystemic fibroinflammatory condition that can manifest a spectrum of skin findings (IgG4-related skin disease; IgG4-RSD). Histopathologic and immunohistochemical criteria have been proposed; however, the specificity of these criteria merits scrutiny given the potential histopathologic overlap of IgG4-RSD and both neoplastic and inflammatory skin conditions featuring lymphoplasmacytic infiltrates (IgG4-RSD mimics). This study sought to assess the specificity of the criteria by quantifying the frequency by which an expanded spectrum of IgG4-RSD mimics meet proposed thresholds. METHODS: Following IRB approval, a total of 69 cases of IgG4-RD mimics, representing 14 different diagnoses featuring plasma cells, were reviewed and analyzed for the following histopathologic and immunohistochemical features: (i) maximum IgG4+ count/high-powered field (hpf) >200; (ii) IgG4/IgG ratio >0.4 averaged over 3 hpfs; (iii) IgG4+ count >10 per hpf. RESULTS: Screening for IgG4-RSD by histopathologic criteria demonstrated the high frequency of lymphoplasmacytic infiltrates, contrasted with the rarity of storiform fibrosis (only one case of erythema elevatum diutinum [EED]) and obliterative phlebitis (0 cases). By immunohistochemical criteria, the analysis revealed that no cases exceeded 200 IgG4+ cells; 13% (9/69) cases demonstrated an IgG4/IgG ratio of >0.4 averaged over 3 hpfs; and 23% (16/69) cases demonstrated a mean IgG4+ count of >10 per hpf. CONCLUSION: Application of proposed IgG4-RSD histopathologic criteria to an expanded spectrum of potential IgG4-RSD mimics (to include cutaneous marginal zone lymphoma, syphilis, necrobiosis lipoidica, lichen sclerosus, ALHE, psoriasis, lymphoplasmacytic plaque, EED, and erosive pustular dermatosis), highlights the relative nonspecificity of lymphoplasmacytic infiltrates contrasted with the stringency of storiform fibrosis and obliterative fibrosis. Furthermore, an IgG4+ cell count of >10 per hpf and an IgG4/IgG ratio of >0.4 are not specific to IgG4-RSD alone. In the appropriate clinical context for IgG4-RSD, histopathologic features still represent the entry threshold for diagnosis consideration, which then allows for further screening by immunohistochemical criteria.

Comparison of turbidometric immunoassay and brix refractometry to radial immunodiffusion for assessment of colostral immunoglobulin concentration in beef cattle.

BACKGROUND: Colostral immunoglobulin G (IgG) concentration is critical to the attainment of adequate transfer of passive immunity in cattle, however, studies comparing available tools for measurement of colostral IgG concentration in beef cattle are limited. OBJECTIVES: To report the agreement between 3 commercially available tests for evaluating IgG concentration in beef colostrum. ANIMALS: Two hundred six beef-breed cows hospitalized for calving management or dystocia. METHODS: Retrospective study to assess IgG of whole colostrum measured stall-side via turbidimetric immunoassay (TI) and brix refractometry (BRIX), compared to fat separated (FS) analysis via single radial-immunodiffusion (RID; reference standard), TI-FS and BRIX-FS. Test performance was assessed using Passing Bablock regression, Bland-Altman analysis, and area under the curve to determine optimal thresholds. RESULTS: Correlation between RID and TI-FS, BRIX-FS, or BRIX was similar (Spearman's ρ = 0.717, 0.715, 0.716, respectively) but correlation for TI was poor (ρ = 0.586). Regression analysis identified a substantial constant (-214.75 [CI: -272.03 to -178.07]) and proportional (13.24 [CI: 11.81-15.37]) bias between the RID and TI-FS which was similar for TI. TI-FS concentrations of 28.47, 38.75, and 50.62 g/L, BRIX-FS of ≤21.9%, ≤24.0%, and ≤27.4%, and BRIX of ≤21.3%, ≤23.8%, and ≤26.4% indicated IgG concentrations <50, <100, and <150 g/L, respectively; appropriate cutoffs for TI could not be generated. CONCLUSIONS AND CLINICAL IMPORTANCE: Both TI and TI-FS demonstrated a large constant and proportional bias compared to RID; BRIX and BRIX-FS were well correlated with RID and remain a reliable method for estimation of colostral IgG concentration in beef cattle.

Rapid COVID-19 Screening Based on Self-Reported Symptoms: Psychometric Assessment and Validation of the EPICOVID19 Short Diagnostic Scale.

BACKGROUND: Confirmed COVID-19 cases have been registered in more than 200 countries, and as of July 28, 2020, over 16 million cases have been reported to the World Health Organization. This study was conducted during the epidemic peak of COVID-19 in Italy. The early identification of individuals with suspected COVID-19 is critical in immediately quarantining such individuals. Although surveys are widely used for identifying COVID-19 cases, outcomes, and associated risks, no validated epidemiological tool exists for surveying SARS-CoV-2 infection in the general population. OBJECTIVE: We evaluated the capability of self-reported symptoms in discriminating COVID-19 to identify individuals who need to undergo instrumental measurements. We defined and validated a method for identifying a cutoff score. METHODS: Our study is phase II of the EPICOVID19 Italian national survey, which launched in April 2020 and included a convenience sample of 201,121 adults who completed the EPICOVID19 questionnaire. The Phase II questionnaire, which focused on the results of nasopharyngeal swab (NPS) and serological tests, was mailed to all subjects who previously underwent NPS tests. RESULTS: Of 2703 subjects who completed the Phase II questionnaire, 694 (25.7%) were NPS positive. Of the 472 subjects who underwent the immunoglobulin G (IgG) test and 421 who underwent the immunoglobulin M test, 22.9% (108/472) and 11.6% (49/421) tested positive, respectively. Compared to NPS-negative subjects, NPS-positive subjects had a higher incidence of fever (421/694, 60.7% vs 391/2009, 19.5%; P<.001), loss of taste and smell (365/694, 52.6% vs 239/2009, 11.9%; P<.001), and cough (352/694, 50.7% vs 580/2009, 28.9%; P<.001). With regard to subjects who underwent serological tests, IgG-positive subjects had a higher incidence of fever (65/108, 60.2% vs 43/364, 11.8%; P<.001) and pain in muscles/bones/joints (73/108, 67.6% vs 71/364, 19.5%; P<.001) than IgG-negative subjects. An analysis of self-reported COVID-19 symptom items revealed a 1-factor solution, the EPICOVID19 diagnostic scale. The following optimal scores were identified: 1.03 for respiratory problems, 1.07 for chest pain, 0.97 for loss of taste and smell 0.97, and 1.05 for tachycardia (ie, heart palpitations). These were the most important symptoms. For adults aged 18-84 years, the cutoff score was 2.56 (sensitivity: 76.56%; specificity: 68.24%) for NPS-positive subjects and 2.59 (sensitivity: 80.37%; specificity: 80.17%) for IgG-positive subjects. For subjects aged ≥60 years, the cutoff score was 1.28, and accuracy based on the presence of IgG antibodies improved (sensitivity: 88.00%; specificity: 89.58%). CONCLUSIONS: We developed a short diagnostic scale to detect subjects with symptoms that were potentially associated with COVID-19 from a wide population. Our results support the potential of self-reported symptoms in identifying individuals who require immediate clinical evaluations. Although these results come from the Italian pandemic period, this short diagnostic scale could be optimized and tested as a screening tool for future similar pandemics.

Developability Assessment of an Isolated CH2 Immunoglobulin Domain.

The IgG CH2 domain continues to hold promise for the development of new therapeutic entities because of its bifunctional role as a biomarker and effector protein. The need for further understanding of molecular stability and aggregation in therapeutic proteins has led to the development of a breakthrough quantum cascade laser microscope to allow for real-time comparability assessment of an array of related proteins in solution upon thermal perturbation. Our objective was to perform a comprehensive developability assessment of three similar monoclonal antibody (mAb) fragments: CH2, CH2s, and m01s. The CH2 construct consists of residues Pro238 to Lys340 of the IgG1 heavy chain sequence. CH2s has a 7-residue deletion at the N-terminus and a 16-residue C-terminal extension containing a histidine tag. The m01s construct is identical to CH2s, except for two cysteines introduced at positions 242 and 334. A series of hyperspectral images was acquired during thermal perturbation from 28 to 60 °C for all three proteins in an array. Co-distribution and two-dimensional infrared correlation spectroscopies yielded the mechanism of aggregation and stability for these three proteins. The level of detail is unprecedented, identifying the regions within CH2 and CH2s that are prone to self-association and establishing the differences in stability. Furthermore, CH2 helical segments, ß-sheets, ß-turns, and random coil regions were less stable than in CH2s and m01s because of the presence of the N-terminal 310-helix and ß-turn type III. The engineered disulfide bridge in m01s eliminated the self-association process and rendered this mAb fragment the most stable.

Developability assessment for monoclonal antibody drug candidates: a case study.

Various screening approaches are used by industry to evaluate development risks associated with discovery candidates. This process has become more complicated with biological therapeutics, a class dominated by monoclonal antibodies (mAb), and, increasingly, their derivative constructs. Effective early assessment for drug-like properties (DLP) can save time and costs by allowing a more complete consideration of issues that could impact the desired end result of a stable drug product. Here we report a case study of four IgG1 mAbs, with sequence variations in the variable domain region, screened as a set of possible drug candidates. Our comprehensive, tiered approach used a battery of analytical tools to assess molecular characteristics, conformational stability, colloidal stability, and short-term storage stability. While most DLP for the four candidates were developmentally acceptable and comparable, mAb-2 was associated with adverse colloidal properties. Further investigation of mAb-2 in an expanded pH range revealed a propensity for phase separation, indicating a need for the additional product development effort. Our results support that comprehensive DLP assessments in an expanded pH range are beneficial in identifying development options for promising molecules that show challenging stability trends. This adaptable approach may be especially useful in the development of increasingly complex antibody constructs.

A Dual-Antigen Enzyme-Linked Immunosorbent Assay Allows the Assessment of Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Seroprevalence in a Low-Transmission Setting.

Estimates of seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies have been hampered by inadequate assay sensitivity and specificity. Using an enzyme-linked immunosorbent assay-based approach that combines data about immunoglobulin G responses to both the nucleocapsid and spike receptor binding domain antigens, we show that excellent sensitivity and specificity can be achieved. We used this assay to assess the frequency of virus-specific antibodies in a cohort of elective surgery patients in Australia and estimated seroprevalence in Australia to be 0.28% (95% Confidence Interval, 0-1.15%). These data confirm the low level of transmission of SARS-CoV-2 in Australia before July 2020 and validate the specificity of our assay.

Prevention of SARS-CoV-2 Infection Among Police Officers in Poland-Implications for Public Health Policies.

BACKGROUND: This study aimed to characterize sources of knowledge on the means of prevention of SARS-CoV-2 infections as well as to assess the methods of preventing SARS-CoV-2 infection among police employees in Poland and their potential impact on the risk of SARS-CoV-2 infection. METHODS: The study consisted of two phases: questionnaire and laboratory tests for SARS-CoV-2 infection. The questionnaire included 30 questions related to risk factors, knowledge about SARS-CoV-2, and methods of infection prevention. RESULTS: Data were obtained from 5082 police employees. The most common source of knowledge for a daily update on SARS-CoV-2 infection prevention was the Internet (42.6%), television (40.3%), and radio (39.7%). The most commonly used methods of SARS-CoV-2 infection included washing one's hands for at least 20 seconds (95.8%), wearing facemasks (82.9%), and physical distancing (74.9%). Results of IgG tests were lower in police units where the overall compliance with the preventive measures was higher (p < 0.01). Women were more likely to exercise SARS-CoV-2 infection prevention behaviors compared to men. Compliance with the recommended protective measures increased with age. CONCLUSIONS: Lower anti-SARS-CoV-2 IgG seropositivity rates were observed in police units with better overall compliance with the preventive measures, suggesting the key importance of group rather than individual behaviors.

Subjective health complaints and exposure to tick-borne infections in southern Norway.

OBJECTIVES: Whether tick-borne infections can cause chronic subjective health complaints is heavily debated. If such a causal connection exists, one would expect to find more health complaints among individuals exposed to tick-borne infections than among non-exposed. In this study, we aimed to assess if exposure to tick-borne infections earlier in life, evaluated by examination of serum for IgG antibodies to tick-borne microbes, was associated with self-reported somatic symptom load. MATERIALS & METHODS: All individuals with residential address in Søgne municipality in southern Norway, aged 18-69 years, were invited to participate in the study. Blood samples were analyzed for IgG antibodies to different tick-borne microbes, and somatic symptom load was charted by the Patient Health Questionnaire-15 (PHQ-15). RESULTS: Out of 7424 invited individuals, 2968 (40.0%) were included in the study. We detected IgG antibodies to Borrelia burgdorferi sensu lato (Bb) in 22.9% (95% CI 21.4-24.4). Bb seropositive individuals reported less frequently moderate to severe somatic symptom load (ie, PHQ-15 sum score ≥ 10) than seronegative individuals (12.5% versus 17.7%, difference 5.2% [95% 2.1-8.0]). However, when adjusting for several other variables in a multivariable linear regression model, presence of serum IgG antibodies to Bb was not associated with somatic symptom load. Presence of IgG antibodies to other tick-borne microbes than Bb, or seropositivity to at least two microbes, was also not associated with somatic symptom load. CONCLUSION: Presence of serum IgG antibodies to tick-borne microbes was not associated with self-reported somatic symptom load.

Assessment of Brix refractometry to estimate immunoglobulin G concentration in beef cow colostrum.

BACKGROUND: Brix refractometry can be used to assess colostral immunoglobulin G (IgG) concentration, but studies identifying Brix percentages to detect high- and low-IgG colostrum are lacking for beef cows and interlaboratory agreement is unknown. OBJECTIVES: Evaluate Brix refractometer performance and interlaboratory agreement for assessing beef cow colostrum IgG concentration, including determination of thresholds to identify colostrum containing IgG concentrations <100 g/L and ≥150 g/L. ANIMALS: Beef cows (n = 416) from 11 cow-calf operations in Alberta, Canada. METHODS: Colostral IgG concentrations were measured using radial immunodiffusion (RID) and estimated by Brix refractometry for this retrospective study. Spearman correlation coefficients were assessed between RID and Brix refractometry. Likelihood ratios and misclassification cost-term analysis were used to determine optimal Brix percentages for detecting colostrum containing IgG concentrations <100 g/L and ≥150 g/L. Concordance correlation coefficient (CCC) and Bland-Altman analyses were performed for Brix percentages obtained at 3 different laboratories. RESULTS: Brix percentages obtained at 3 laboratories were positively correlated with IgG results (r = 0.72, 0.68, and 0.76, respectively). Colostrum Brix percentages of <24% and ≥30% were optimal for indicating IgG concentrations of <100 g/L and ≥150 g/L, respectively. Interlaboratory agreement was substantial, with CCC ranging from 0.89 to 0.96 and Bland-Altman analysis showing small mean differences (-1.2% to 0.09% Brix) and narrow limits of agreements (-4.8% to 2.4% Brix) among laboratories. CONCLUSIONS AND CLINICAL IMPORTANCE: Brix refractometry shows good potential for reliably estimating IgG concentrations in beef cow colostrum across multiple laboratories and can be recommended to aid colostrum management decisions on farms.

An application of Nano Differential Scanning Fluorimetry for Higher Order Structure assessment between mAb originator and biosimilars: Trastuzumab and Rituximab as case studies.

Differential scanning fluorimetry (DSF) or thermal shift has emerged in recent years as a high-throughput screening method in biotherapeutic formulation studies. The present article reports on a fast-track assessment platform for rapid investigation of therapeutic proteins such as monoclonal antibodies (mAb) with minimal sample concentration, volume, and preparation. The proposed nanoDSF platform has been demonstrated for rapid assessment of two commercial IgG 1 drug products (DP), trastuzumab and rituximab, and their biosimilars with respect to their conformational and colloidal stability. Domain specific differences for each of the IgGs have been elucidated with respect to onset of domain unfolding (Tonset) and melting temperatures. These thermal unfolding and transition midpoint (Tm) measurements are based on the intrinsic aromatic amino acid residue fluorescence of proteins. Moreover, to understand the possibility of nanoDSF as a predictive tool, data from nanoDSF has been correlated with accelerated stability studies. Melting temperatures across brands were found to be highly comparable to the rate of heating, thereby exhibiting a significant domain specific effect on melting temperatures for both trastuzumab and rituximab. Conservation of higher order structure (HOS) through reversible unfolding was also examined and both the mAbs were found to regain tertiary structure up till the first transition midpoint. No clear correlation was found between formation of higher molecular weight species (HMWS) and unfolding parameters (Tonset and Tagg) for accelerated stability studies. Finally, a discussion on the need for fast predictive assessment of conformation and colloidal stability as well as a comparison of advantages and limitations of the technique with routine/classical tools such as circular dichroism spectrophotometry and differential scanning calorimetry has been presented.

Virological assessment of hospitalized patients with COVID-2019.

Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6-8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.

Quantitative assessment of German Holstein dairy cattle colostrum and impact of thermal treatment on quality of colostrum viscosity and immunoglobulins.

OBJECTIVE: This study aimed to determine the color, fat, viscosity, IgG concentration, %Brix and refractive index of fresh postpartum colostrum of German Holstein dairy cattle and assess the impact of different thermal treatments on the visual and dynamic viscosity, in association to IgG concentration, of colostrum that can be used for pasteurization process. RESULTS: Of the total 40 fresh postpartum colostrum, the color of colostrum (ranging from white-pale yellow to yellow and dark-yellowish), fat (1.4-8.2 100 g-1), IgG (4-116 mg mL-1), %Brix (8.5-35.4%), refractive index (1.3454-1.3905 nD), visual (ranging from watery to liquid and thick) and dynamic (4.9-219 cp) viscosity, were recorded. Statistical analysis between visual and dynamic viscosity of fresh colostrum showed significant correlation coefficients (rs = 634). Moreover, a significant correlation between viscosity and three IgG concentrations was also observed. Heat-treated colostrum showed dynamic viscosity ranged from 25 to 3066 cP, where dynamic viscosity of colostrum before- and after heat-treatment showed no significant correlation. Treated colostrum at 60 °C/60 min and 63.5 °C/30 min containing IgG concentration ≤ 80 mg mL-1 and ≤ 68 mg mL-1 showed no significant change in the viscosity and can successfully be applied for pasteurization of first postpartum colostrum.

[Measurement of IgG concentration in bovine colostrum by immunoturbidimetric assay in comparison to ELISA-based assessment]. / Messung der IgG-Konzentration in Rinderkolostrum mit einem immunturbidimetrischen Schnelltest im Vergleich zum ELISA.

OBJECTIVE: The aim of the presented study was to compare the results of IgG measurements using a turbidimetric immunoassay (TIA), a newly developed laboratory-independent method for direct immunoglobulin determination in colostrum, with measurements obtained via enzyme-linked immunosorbent assay (ELISA). MATERIAL AND METHODS: In colostrum samples from 59 cows, IgG concentration was measured using TIA and ELISA. RESULTS: Correlation analysis according to Pearson revealed a correlation coefficient of r = 0.74 (p < 0.0001) between the 2 methods. The Bland-Altman analysis showed that measurement by TIA resulted in significantly lower mean IgG levels than the ELISA-based quantification. This difference was more pronounced in high IgG concentration ranges. By means of a double-logarithmized data transformation it was calculated that the TIA-determined IgG-values on average amounted to 66.4 % of the IgG-values measured by ELISA. Although colostrum with low IgG concentration could be quantified with satisfactory reliability (sensitivity 100 %), high-quality colostrum was not sufficiently assessed in the TIA-based IgG measurements (specificity 40.4 %). CONCLUSION AND CLINICAL RELEVANCE: Based on the results of the presented study, IgG measurement by TIA cannot be recommended. In comparison to ELISA-based assessment, this technique does not exhibit higher correlations than established indirect rapid evaluation methods (density and viscosity determination).

Performance evaluation of 14 specific proteins measurement checked by an External Quality Assessment Scheme.

AIMS OF THE STUDY: To evaluate the state-of-the-art of 14 specific proteins measurement; to evaluate the laboratories' performance and the degree of harmonization in reporting results of participants in the External Quality Assessment Program of the Centre of Biomedical Research (CRB). METHODS: Overall and system-related inter-laboratory analytical variability (mean CVs%) and between-system differences (mean bias%) were evaluated from data of six EQA cycles 2013-2018. Moreover, we evaluated the analytical performance of participants as well as the units used to express proteins results. RESULTS: Overall inter-laboratory variability ranged from 3.8% for haptoglobin (HPT) to 12.5% for α1-antitrypsin (AAT) and decreased for IgA, α2-macroglobulin (A2M) and transferrin (TRF). Mean CVs% were generally higher for Siemens BN and Beckman Immage immunonephelometric systems, but <7.0% for all proteins. Mean bias > 7.0% was observed for BN (IgA, C4, AAT, transthyretin TTR), Siemens Vista (IgA, C4) and Immage (C4), whereas mean bias < -7.0% was found for Immage (AAT), Beckman AU (IgM) and Roche Cobas (C4, TTR, C-reactive protein). The laboratories' performance within the limits ranged from 85.1% of albumin (ALB) to 97.2% of HPT. The census of units employed in 2018, demonstrated that ~ 70% of laboratories still express the results in mg/dL. CONCLUSIONS: Despite a reduction in inter-laboratory variability for some proteins, different analytical systems showed both proportional and constant bias between methods. Units used by participants have not been substantially changed and dL is still largely used. The CRB EQA Program, with its performance data sets, is a valuable resource for laboratories and IVD manufacturers and support the goals of harmonization.

[Quality Assessment of Bovine and Equine Colostrum - An Overview]. / Qualitätsbeurteilung von bovinen und equinen Kolostrum ­ Eine Übersicht.

INTRODUCTION: Good quality colostrum intake is essential for passive immunity in neonatal calf and foals. The quality of colostrum depends on its IgG content. Since in practice the possibilities for a direct determination of these parameter are limited, an estimation of colostrum quality is often based on its biophysical properties. The simple methodology allows to perform measurements directly on site and immediately after birth. The biophysical properties included in the current review are density, refractive index, relative density, viscosity, pH, and color. The determination of the density of bovine colostrum is a common method for the evaluation of the IgG content in practice since it correlates with IgG. Density of bovine colostrum should be 1047 g/l. Different colostrometers are available for the measurement. This method is also used in the horses. Density of equine colostrum should be 1060 g/l. Refractive index or the relative density by refractometer has been proven to correlate with the IgG and is an established method in cattle and horses. Good bovine colostrum should have an index of 22% brix and equine colostrum 23% brix. Although not yet established in practice an additional methodology would be the determination viscosity of bovine colostrum. Viscosity could be measured with an outlet cup. A relationship between viscosity and IgG has demonstrated. Sufficient data for this method is missing for equine colostrum. Color and pH are not suitable for estimating the IgG content.

INTRODUCTION: La prise de colostrum de bonne qualité est essentielle pour la construction d'une immunité passive chez le veau et le poulain nouveau-nés. La qualité du colostrum dépend de sa teneur en IgG. Comme dans la pratique les possibilités de détermination directe de ce paramètre sont limitées, une estimation de la qualité du colostrum est souvent basée sur ses propriétés biophysiques. Grâce à une méthodologie simple, la mesure peut généralement être effectuée directement sur place et immédiatement après la naissance. Les propriétés biophysiques incluses dans cette revue sont la densité, l'indice de réfraction, la densité relative, la viscosité, le pH et la couleur. La détermination de la densité du colostrum bovin est en pratique une méthode courante pour évaluer la teneur en IgG, car la densité est en corrélation avec la teneur en IgG. La densité du colostrum doit être d'au moins 1047 g/l. Différents colostromètres sont disponibles pour cette mesure. Cette méthode est également utilisée chez le cheval. La densité du colostrum équin doit être d'au moins 1060 g/l. La détermination de l'indice de réfraction ou de la densité relative au moyen d'un réfractomètre est une procédure établie chez les bovins et les chevaux, car une corrélation claire avec la teneur en IgG est prouvée. Pour être considéré comme bon, le colostrum bovin doit atteindre une valeur de 22% et le colostrum équin une valeur de 23% sur l'échelle de Brix. L'évaluation du colostrum bovin par viscosité est une autre approche qui doit encore être mise en pratique. Là encore, une relation entre la viscosité et la teneur en IgG pourrait être prouvée. La mesure de la viscosité peut être effectuée à l'aide d'une coupe d'écoulement. Chez le cheval, on ne dispose pas encore de données suffisantes sur cette méthode. La valeur du pH et la couleur ne conviennent pas pour estimer la teneur en IgG.

Assessment of CE-based baseline disturbances using simulation and targeted experimental evaluation-impact on the purity determination of therapeutic proteins.

The baseline instability for capillary electrophoretic analysis is an intrinsic feature of the technique, which has not been thoroughly examined for its impact on therapeutic protein purity analysis with the capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) applications. For the particular CE-SDS application, this phenomenon was manifested through peak migration time shifts and sliding of the superimposed baseline profile. These dual phenomena are closely associated so that experimental assessment alone may not shed enough light to the underlying drivers. In the current study, both experimental and simulation approaches were employed to assess the systematic drifts. Computer simulation was used to decipher the two underlying factors and test their contributions toward purity and impurity peak determination inaccuracies. The data generated in this study demonstrated that the electrophoretic baseline disturbance had more pronounced impact on the purity data than the migration time shift. In addition, the potential contributing factors to the baseline disturbances were assessed experimentally which indicated that the source is related to thermal disruption during a sample run and the unique baseline patterns came from the background electrolytes. To improve data reproducibility for drug purity testing in the industrial setting and quality control (QC) environment, it is recommended to run shorter injection sequences including fewer samples and closely monitor the baseline drift for accurate integration. Those methods would help reduce the impact of systematic drift and disturbances. Graphical abstract.

Quantitative Assessment of Individual Populations Present in Nanoparticle-Antibody Conjugate Mixtures Using AF4-ICP-MS/MS.

A current remaining challenge in nanotechnology is the fast and reliable determination of the ratios between engineered nanoparticles and the species attached to their surface after chemical functionalization. The approach proposed herein based on the online coupling of asymmetric flow field-flow fractionation (AF4) with inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS) allows for the first time the direct determination of such ratios in CdSe/ZnS core-shell quantum dot:rat monoclonal IgG2a antibody (QD:Ab) conjugate mixtures in a single run without any previous sample preparation (i.e., derivatization). AF4 provides full recovery and adequate resolution of the resulting bioconjugate from the excess of nanoparticles and proteins used in the different bioconjugation mixtures (1:1, 2:1, and 3:1 QD:Ab molar ratios were assessed). The point-by-point determination by ICP-MS/MS of the metal to sulfur ratios along the bioconjugate fractographic peak allowed disclosing the mixture of the different species in the bioconjugated sample, providing not only the limits of the range of QD:Ab ratios in the different bioconjugate species resulting after functionalization but also a good estimation of their individual relative abundance in the mixture. Interestingly, a wide variety of compositions were observed for the different bioconjugate mixtures studied (QD:Ab molar ratios ranging from 0.27 to 4.6). The resulting weighted QD:Ab ratio computed in this way for each bioconjugate peak matches well with both the global (average) QD:Ab ratio experimentally obtained by the simpler peak area ratio computation and the theoretical QD:Ab molar ratios assayed, which internally validates the procedure developed.

Overview of antibody-mediated immunity to S. pneumoniae: pneumococcal infections, pneumococcal immunity assessment, and recommendations for IG product evaluation.

Streptococcus pneumoniae (S. pneumoniae) strains colonize the nasopharynx and can cause mucosal infections in the upper airway and middle ear, pneumonias, and invasive infections like bacteremia, sepsis, and meningitis. Over 90 serotypes, defined by the structure of their capsular polysaccharides, are known. Twenty-three of these serotypes cause most infections and several of these serotypes can develop antibiotic resistance. Susceptibility factors that increase the susceptibility to S. pneumoniae mucosal and invasive infections include all forms of primary and secondary antibody deficiencies. Many patients affected by one of these deficiencies benefit from the regular administration of human gamma globulin (IgG) preparations. Donors of plasma units used to prepare human IgG have varying concentrations of IgG antibodies against relevant S. pneumoniae serotypes. These antibodies are developed in response to colonization and common subclinical infections and by routine vaccination with S. pneumoniae polysaccharide vaccines. The presence of an adequate concentration of these protective antibodies against all prevalent serotypes needs to be determined to assure the effectiveness of human IgG. All presently available methods to assess IgG antibodies against S. pneumoniae capsular polysaccharides have advantages and pitfalls that are analyzed in this review. In vitro testing does not provide a complete or necessarily accurate measurement of the effectiveness of antibodies in vivo. For regulatory purposes, caution needs to be used in the interpretation of currently available assays that measure pneumococcal antibody levels. Monitoring S. pneumoniae infections in patients treated with IgG and tracing information about IgG lots used to treat these patients should be encouraged.

Assessment of quality, safety, and pre-clinical toxicity of an equine polyvalent anti-snake venom (Pan Africa): Determination of immunological cross-reactivity of antivenom against venom samples of Elapidae and Viperidae snakes of Africa.

Snakebite causes a large amount of morbidities and mortalities in Africa. The safety, efficacy, and homogeneity of anti-snake venoms are crucial for snakebite treatments to be effective with minimal adverse effects. We assessed the homogeneity of preparations of three different batches of Combipack snake venom antiserums (Pan Africa) [CSVAPA] by quantitatively analysing F(ab')2, IgG, and other contaminating proteins of plasma. LC-MS/MS analysis showed that approximately 92.4% of the proteins from the CSVAPA samples was IgG/F(ab')2 and the percent composition of contaminating proteins in CSVAPA varied from 0.07 to 4.6%. Batch 1 of the CSVAPA also contained a minor amount of undigested IgG and F(ab')2 aggregates. CSVAPA contained more than 60% venom-specific antibodies, showed moderate complement activation, no IgE contamination, safe level of endotoxin, and also showed pre-clinical safety. The immuno cross-reactivity of CSVAPA against 14 Viperidae and Elapidae snake venoms of Africa was tested by ELISA and immunoblotting, and the neutralization of major enzymatic venom activities, demonstrating that high molecular weight (>50 kDa) venom proteins are better recognized/neutralized compared to relatively low molecular weight (<20 kDa) venom proteins. CSVAPA at a dose of 3-12 times higher than the clinical dose did not cause deaths or adverse reaction of treated rabbits. The results suggest the satisfactory quality, safety, and efficacy of CSVAPA.

Label-free real-time detection of biotinylated bovine serum albumin using a low-cost optical cavity-based biosensor.

We have developed a low-cost optical cavity-based biosensor with a differential detection method for point-of-care medical diagnostics. To experimentally demonstrate its label-free real-time biosensing capability, we performed the detection of biotinylated bovine serum albumin (BSA). Streptavidin is introduced into the optical cavity structure and immobilized on 3-aminopropyltriethoxysilane (APTES) coated surface. After rinsing out unbound streptavidin with DI water, biotinylated BSA without any labeling is introduced. A CMOS camera captures the transmitted light of two different wavelengths passing through the optical cavity sensing area in real-time. Then, the differential values are calculated to enhance the responsivity. We successfully demonstrated the label-free real-time detection of biotinylated BSA, and the measurement results matched well with the simulation results. The limit of detection of the optical cavity-based biosensor for the biotinylated BSA detection with the sensing area of 180 µm × 180 µm is estimated to be 2.82 pM, which could be reduced further for a smaller sensing area with the tradeoff of a longer sensing time.