Subcutaneous administration of natalizumab can lead to lower drug concentrations compared to intravenous administration.

BACKGROUND: Several studies reported lower drug concentrations with subcutaneous natalizumab compared to intravenous natalizumab. With the emergence of extended interval dosing, gaining more insight into lower concentrations after subcutaneous administration is essential. METHODS: We compared serum trough concentrations between subcutaneous and intravenous administration within a matched cohort (n = 50). RESULTS: Subcutaneous administration (n = 25) was associated with lower concentrations compared to intravenous administration (n = 25) (log-B=-0.28, p = 0.01). In an exploratory group of 11 patients receiving extended interval dosing of subcutaneous natalizumab, the median trough concentration was even lower. CONCLUSION: Subcutaneous natalizumab can lead to lower drug concentrations, potentially limiting extended interval dosing.

Belimumab concentration measurements using a homologous anti-idiotype immunoassay.

Monitoring belimumab concentrations in patients can be a valuable tool for assessing treatment response and for personalizing drug doses. Various assay formats may be used to measure concentrations of therapeutic monoclonal antibodies. A particularly useful format involves the use of anti-idiotype monoclonal antibodies, selected to be highly specific to the antibody of interest. Here, we describe the development of a specific, high-affinity anti-idiotype antibody to belimumab, and the application of this antibody in a homologous sandwich ELISA to measure belimumab concentrations.

Helping athletes to select botanical supplements for the right reasons: A comparison of self-reported reasons for use versus evidence-based supplement claims.

Use of food supplements (FS) by athletes is well characterised but there is little information on 'herbal' or 'botanical' FS beyond 'natural'. This study determined, by questionnaire, whether athletes' main self-reported reason for using FS was reflective of what was written on product labels or, when these claims were unavailable, was in accordance with the scientific literature. In 217 elite (n = 55) and amateur (n = 162) athletes living on the island of Ireland, 71% (n = 153) consumed any kind of FS, with 16% (n = 34) of the entire cohort deemed botanical consumers. 'Protein' (21%, n = 46), 'vitamin D' (17%, n = 37) and 'vitamin C' (15% n = 32) were most consumed with the top reasons for use being 'to support health', 'to prevent illness/for immunity purposes' and 'recovery'. There was generally good agreement between approved nutrition and health claims for such products and athletes' main reported reasons for use. Only the amateur athletes in our pool described using botanical supplements, with reasons for use stated as 'sleep improvement' (21%), 'recovery' (14%), 'supporting health' (12%) and 'energy' (12%), resulting in poor agreement with either approved claims or scientific evidence. Only half of amateur athletes knew if their botanical FS were third-party tested. Athletes and practitioners require guidance to avoid consuming supplements for which there is little scientific evidence, and which may risk being contaminated/fraudulent.

Collegiate student-athletes use and knowledge of third-party tested nutritional supplements: An exploratory study.

CONTEXT: Nutritional supplement use in athletes is common, accompanied by potential doping risk. OBJECTIVE: Determine athletes' nutritional supplement and third-party tested (TPT) supplement use, supplement knowledge as well as factors influencing their behavior. DESIGN: Cross-Sectional Study. SETTING: NCAA DI athletic departments. PARTICIPANTS: Student-athletes (n=410, 53% female, age 21.4±1.6 years). MAIN OUTCOME MEASURES: Survey questions addressed topics including nutritional supplement knowledge and use, TPT supplement knowledge, use and logo recognition and data were 11 stratified for sex differences. RESULTS: Athletes (91%) report the use of supplements, but the total number of supplements used (median and interquartile range, IQR) is lower in females (7, 4-11), than males (9, 4-12), with U=17960, p=0.01. A total of 48% (n=191, out of 402 responses) reported purchasing supplements outside of their athletic department, with significantly fewer females (40%, n=84) than males (56%, n=107) reporting this behavior (χ2=11.20, p<0.001). No association between TPT logo recognition and TPT use was seen (χ2=0.238, p=0.63). Of all athletes using supplements, 38% (n=140) reported "consistent TPT use", while females (36%, n=70) reported this less often than males (41%, n=70, χ2=0.952, p<0.32). No sex differences were seen for receiving nutritional counseling (89%, p=0.37), or the low nutritional supplement knowledge (<50%, p=0.38), however, males had 2.5 times greater odds at recognizing a TPT organization logo than females (OR=2.45, with 95% CI=1.58-3.79). CONCLUSIONS: Most athletes use nutritional supplements. Females report slightly fewer supplements than males, while also less frequently purchasing them outside their athletic department, potentially explaining the lower TPT logo recognition in female athletes.

Explorative Characterization of GI Complaints, General Physical and Mental Wellbeing, and Gut Microbiota in Trained Recreative and Competitive Athletes with or without Self-Reported Gastrointestinal Symptoms.

The current state of the literature lacks a clear characterization of gastrointestinal (GI) symptoms, gut microbiota composition, and general physical and mental wellbeing in well-trained athletes. Therefore, this study aimed to characterize differences in self-reported symptoms, gut microbiota composition, and wellbeing (i.e., sleep quality, mood, and physical (PHQ) and mental wellbeing) between athletes with and without GI symptoms. In addition, we assessed the potential impact of a 3-week multi-ingredient fermented whey supplement in the GI complaints group, without a control group, on the gut microbiota and self-reported GI symptoms and wellbeing. A total of 50 athletes (24.7 ± 4.5 years) with GI issues (GI group at baseline, GI-B) and 21 athletes (25.4 ± 5.3 years) without GI issues (non-GI group, NGI) were included. At baseline, there was a significant difference in the total gastrointestinal symptom rating scale (GSRS) score (24.1 ± 8.48 vs. 30.3 ± 8.82, p = 0.008) and a trend difference in PHQ (33.9 ± 10.7 vs. 30.3 ± 8.82, p = 0.081), but no differences (p > 0.05) were seen for other outcomes, including gut microbiota metrics, between groups. After 3-week supplementation, the GI group (GI-S) showed increased Bifidobacterium relative abundance (p < 0.05), reported a lower number of severe GI complaints (from 72% to 54%, p < 0.001), and PHQ declined (p = 0.010). In conclusion, well-trained athletes with GI complaints reported more severe GI symptoms than an athletic reference group, without showing clear differences in wellbeing or microbiota composition. Future controlled research should further investigate the impact of such multi-ingredient supplements on GI complaints and the associated changes in gut health-related markers.

Development of a screener to assess athlete risk behavior of not using third-party tested nutritional supplements.

Introduction: The aim of this cross-sectional study was to develop an algorithm to predict athletes use of third-party tested (TPT) supplements. Therefore, a nutritional supplement questionnaire was used with a section about self-reported TPT supplement use. Methods: Outcomes were randomly assigned to a training dataset to identify predictors using logistic regression models, or a cross-validation dataset. Training data were used to develop an algorithm with a score from 0 to 100 predicting use or non-use of TPT nutritional supplements. Results: A total of n = 410 NCAA Division I student-athletes (age: 21.4 ± 1.6 years, 53% female, from >20 sports) were included. Then n = 320 were randomly selected, of which 34% (n = 109) of users consistently reported that all supplements they used were TPT. Analyses resulted in a 10-item algorithm associated with use or non-use of TPT. Risk quadrants provided the best fit for classifying low vs. high risk toward inconsistent TPT-use resulting in a cut-off ≥60% (χ2(4) = 61.26, P < 0.001), with reasonable AUC 0.78. There was a significant association for TPT use (yes/no) and risk behavior (low vs. high) defined from the algorithm (χ2(1)=58.6, P < 0.001). The algorithm had a high sensitivity, classifying 89% of non-TPT users correctly, while having a low specificity, classifying 49% of TPT-users correctly. This was confirmed by cross-validation (n = 34), reporting a high sensitivity (83%), despite a lower AUC (0.61). Discussion: The algorithm classifies high-risk inconsistent TPT-users with reasonable accuracy, but lacks the specificity to classify consistent users at low risk. This approach should be useful in identifying athletes that would benefit from additional counseling.

Energy expenditure and physiological markers during a city mountain hike in the heat: A case study.

Background: Energy expenditure may be difficult to assess when hiking difficult trails. Case presentation: We measured physical activity exercise energy expenditure (PAEE) directly from oxygen uptake using a mobile device (cardiopulmonary exercise testing, CPET), and by using a formula based on heart rate (HR), or metabolic equivalent values from the Compendium of Physical Activity, and other physiological outcomes. Outcomes and implications: Total PAEE (1342 kcal) using CPET showed a two-fold difference between ascending and descending (887 vs. 455 kcal) during a 124-min hike. For HR, PAEE was 1893kcal (+551 kcal overreporting), while compendium-based scenarios ranged from 1179 to 1446 kcal, which was in closer range (-163 to +104 kcal/min) compared to the CPET data. Fluid consumption was 1300 mL/hour, with 1.1% bodyweight loss, peak skin temperature of 35.2°C and core body temperature of 39.2°C. Recommendations: Tables reasonably predict energy expenditure while not precisely reflecting the actual situation.

Patient-perspective and feasibility of home finger-prick testing to complement and facilitate large-scale research in rheumatology.

BACKGROUND: During the COVID-19 pandemic, we developed a digital research platform to longitudinally investigate COVID-19-related outcomes in patients with rheumatic diseases and healthy controls. We used home finger-prick testing in order to collect serum samples remotely and increase the overall efficiency of the platform. The aim of the present study was to evaluate the success rate of the finger prick and patients' perspective towards the finger prick. METHODS: Serum samples were collected up to five times during follow-up, either via a venepuncture at the research institute or a finger prick from participants' home. Participants were asked to complete a digital evaluation questionnaire of the finger prick after their attempts. RESULTS: A total of 2135 patients and 899 controls performed at least one finger prick and were included in this study. The first finger prick was successfully done by 92% (95% CI: 90% to 93%) of patients, 94% (95% CI: 92% to 95%) of controls, 93% (95% CI: 92% to 94%) of all participants aged ≤70 years and 89% (95% CI: 86% to 92%) of all participants aged >70 years. Sex did not impact these success rates. Repeated failure occurred in 11/439 (0.8%) patients and 4/712 (0.6%) controls. Both patients and controls were less willing to perform a finger prick for individual healthcare compared with scientific research. CONCLUSION: The vast majority of participants, among which elderly and patients with rheumatic diseases, were able to successfully draw the required amount of blood for serological analyses. This shows that finger-prick testing is suitable for a high-throughput implementation to monitor patients remotely.

Detectability and intra-fraction motion of individual elective lymph nodes in head and neck cancer patients on the Magnetic Resonance Image guided linear accelerator.

Background and purpose: Individual elective lymph node irradiation instead of elective neck irradiation is a new concept for head-and-neck cancer (HNC) patients developed for the Magnetic Resonance Image guided linear accelerator (MR-linac). To prepare this, the detectability, volume changes and intra-fraction motion of elective lymph nodes on the MR-linac was assessed. Materials and methods: A total of 15 HNC patients underwent diagnostic pre-treatment MRI. Additionally, two MR-linac scans were obtained with a 10-minute time difference in the first week of radiation treatment. Elective lymph node contours inside lymph node levels (Ib-V) were segmented on the pre-treatment MRI and the MR-linac scans and compared on number and maximal transversal diameter. Intra-fraction motion of elective lymph nodes on the MR-linac was estimated using Center of Mass (COM) distances and incremental isotropic expansion of lymph node segmentations. Results: Of all 679 detected lymph nodes on the pre-treatment MRI, eight lymph nodes were not detectable on the first MR-linac scan and 16 new lymph nodes were detected. Lymph node diameters between the pre-treatment MRI scan and the MR-linac scan varied from -0.19 to + 0.13 mm. COM distances varied from 1.2 to 1.7 mm and lymph node contours had to be expanded with 3 mm. Conclusions: Nearly all elective lymph nodes were detectable on the 1.5T MR-linac scan with no major changes in target volumes compared to the pre-treatment MRI. Simulated intra-fraction motion during the MR-linac scans was smaller than the 5-mm margin that will be used in the first elective lymph node radiation treatment.

Screening of representative rainfall event series for long-term hydrological performance evaluation of grassed swales.

Evaluation of the hydrological performance of grassed swales usually needs long-term monitoring data. At present, suitable techniques for simulating the hydrological performance using limited monitoring data are not available. Therefore, current study aims to investigate the relationship between saturated hydraulic conductivity (Ks) fitting results and rainfall characteristics of various events series length. Data from a full-scale grassed swale (Enschede, the Netherlands) were utilized as long-term rainfall event series length (95 rainfall events) on the fitting outcomes. Short-term rainfall event series were extracted from these long-term series and used as input in fitting into a multivariate nonlinear model between Ks and its influencing rainfall indicators (antecedent dry days, temperature, rainfall, rainfall duration, total rainfall, and seasonal factor (spring, summer, autumn, and winter, herein refer as 1, 2, 3, and 4). Comparison of short-term and long-term rainfall event series fitting results allowed to obtain a representative short-term series that leads to similar results with those using long-term series. A cluster analysis was conducted based on the fitting results of the representative rainfall event series with their rainfall event characteristics using average values of influencing rainfall indicators. The seasonal index (average value of seasonal factors) was found to be the most representative short rainfall event series indicator. Furthermore, a Bayesian network was proposed in the current study to predict if a given short-term rainfall event series is representative. It was validated by a data series (58 rainfall events) from another full-scale grassed swale located in Utrecht, the Netherlands. Results revealed that it is quite promising and useful to evaluate the representativeness of short-term rainfall event series used for long-term hydrological performance evaluation of grassed swales.

High School Athletes' Use and Knowledge of (Safe) Nutritional Supplement Use: An Exploratory Study.

The use of nutritional supplements can lead to doping risk and no data exist on high school athletes' use of certified third-party tested supplements. A cross-sectional cohort design was developed using an anonymous survey. Descriptive data for supplement use, use of third-party tested supplements, and knowledge in high school athletes were reported. A total of 225 high school athletes, ranging from 14-19 years of age, from a private high school in the western US, were included in the analysis of the results. A total of 94% (n=211) of athletes reported nutritional supplement use within the past year with an average of six (interquartile range: 3-9) individual supplements, ranging from 0-20 supplements per person. Most frequently reported were sports drinks (72%), vitamins (65%), sports bars (60%), protein powder (58%), caffeine (37%, as part of normal beverages, or 13% as a supplement), followed by creatine (23%). A total of 24% claimed to know for sure that all their supplements were third-party tested. In addition, the recognition of third-party testing organization icons was low (46% in supplement users vs. 14% in nonusers). Athletes also scored low in reporting how to find (22%) and how to order (25%) third-party tested supplements. In conclusion, almost all athletes in this study reported the use of multiple nutritional supplements annually. Only one-fourth of the athletes reported consistently using third-party tested supplements. Knowledge of where and how tested supplements could be purchased was limited in this high school athlete population.

Clinical Validation of a Capillary Blood Home-Based Self-Sampling Technique for Monitoring of Infliximab, Vedolizumab, and C-Reactive Protein Concentrations in Patients With Inflammatory Bowel Disease.

BACKGROUND: Therapeutic drug monitoring provides important guidance for treatment of patients with inflammatory bowel disease (IBD) and could help to early identify treatment failure. This study aimed to validate a finger prick-based capillary blood sampling technique to measure biological trough levels and C-reactive protein (CRP) and evaluate patient performance and -support. METHODS: In this prospective cohort study, patients with IBD receiving infliximab (IFX) or vedolizumab (VEDO) therapy performed finger prick-based capillary blood sampling at home. Additionally, blood was collected through routinely performed in-hospital venepuncture prior to biological infusion. IFX, VEDO, and CRP concentrations were measured by enzyme-linked immunosorbent assay. The concordance between methods was statistically evaluated and a survey was conducted to assess practicality and patient support. RESULTS: In total, 81 patients (46 IFX, 35 VEDO) were enrolled. Mean differences between both methods were 0.42 (95% confidence interval, -1.74 to 2.58) µg/mL for IFX and 0.72 (95% confidence interval, -5.50 to 6.94) µg/mL for VEDO. Passing-Bablok regressions demonstrated no evidence for systematic or proportional biases. Venous and capillary IFX (ρ = 0.96, P < .001) and VEDO (ρ = 0.97, P < .001) levels strongly correlated and showed high intermethod agreement (Cohen's kappa: IFX = 0.82; VEDO = 0.94). Similarly, venous and capillary CRP levels were strongly correlated (ρ = 0.99, P < .001). Most patients (>95%) were able to successfully perform the self-sampling at home without prior instructions. CONCLUSIONS: This study clinically validated a finger prick-based capillary blood self-sampling technique allowing concomitant home monitoring of biological levels and CRP for patients with IBD, who reported substantial support, tolerability, and practicality.

Validity of combined hydration self-assessment measurements to estimate a low vs. high urine concentration in a small sample of (tactical) athletes.

PURPOSE: Relationships between body weight, urine color (Uc), and thirst level (WUT) have been proposed as a simple and inexpensive self-assessment method to predict dehydration. This study aimed to determine if this method also allowed us to accurately identify a low vs. high urine concentration in (tactical) athletes. METHODS: A total of n = 19 Army Reserve Officer Training Corps cadets and club sports athletes (22.7 ± 3.8 years old, of which 13 male) were included in the analysis, providing morning body weight, thirst sensation, and Uc for five consecutive days. Each item received a score 0 or 1, resulting in a WUT score ranging from 0 (likely hydrated) to 3 (very likely dehydrated). WUT model and individual item outcomes were then compared with a ≥ 1.020 urine specific gravity (USG) cut-off indicating a high urine concentration, using descriptive comparisons, generalized linear mixed models, and logistic regression (to calculate the area under the curve (AUC)). RESULTS: WUT score was not significantly predictive of urine concentration, z = 1.59, p = 0.11. The AUC ranged from 0.54 to 0.77 for test days, suggesting a fair AUC on most days. Only Uc was significantly related to urine concentration, z = 2.49, p = 0.01. The accuracy of the WUT model for correctly classifying urine samples with a high concentration was 68% vs. 51% of samples with a low concentration, resulting in an average accuracy of 61%. CONCLUSION: This study shows that WUT scores were not predictive of urine concentration, and the method did not substantially outperform the accuracy of Uc scoring alone.

Time-varying characteristics of saturated hydraulic conductivity in grassed swales based on the ensemble Kalman filter algorithm -A case study of two long-running swales in Netherlands.

Saturated hydraulic conductivity (Ks) of the filler layer in grassed swales are varying in the changing environment. In most of the hydrological models, Ks is assumed as constant or decrease with a clogging factor. However, the Ks measured on site cannot be the input of the hydrological model directly. Therefore, in this study, an Ensemble Kalman Filter (EnKF) based approach was carried out to estimate the Ks of the whole systems in two monitored grassed swales at Enschede and Utrecht, the Netherlands. The relationship between Ks and possible influencing factors (antecedent dry period, temperature, rainfall, rainfall duration, total rainfall and seasonal factors) were studied and a Multivariate nonlinear function was established to optimize the hydrological model. The results revealed that the EnKF method was satisfying in the Ks estimation, which showed a notable decrease after long-term operation, but revealed a recovery in summer and winter. After the addition of Multivariate nonlinear function of the Ks into hydrological model, 63.8% of the predicted results were optimized among the validation events, and compared with constant Ks. A sensitivity analysis revealed that the effect of each influencing factors on the Ks varies depending on the type of grassed swale. However, these findings require further investigation and data support.

The Development and Efficacy of a High School Athlete Education Program for Safe Nutritional Supplement Use.

Many high school athletes report using nutritional supplements. Due to a lack of education at the high school level, the use of safe for sports third-party tested nutritional supplements may be limited. To determine the impact of a short online nutritional supplement education program on safe dietary supplement behavior a cross-sectional repeated measures design was used. Therefore, a convenience sample of 106 high school athletes (14-19 years old) was recruited to measure pre-post education difference for nutritional supplement use and third-party tested (TPT) supplements. Additionally, it was analyzed if nutritional supplement related Theory of Planned Behavior (TPB) constructs were associated with athlete choices. The most popular supplements included protein powder (65%), caffeine from beverages (45%), and different types of vitamins (ranging from 38-44%). Consistent use of (safe) third-party tested individual supplements was low, ranging from 35-77% for the most frequently reported supplements. The combined TPB determinants explained 26% of the variance of the intention to use safe supplements (F3, 102 = 13.03, p < 0.001, Adj R2 = 0.26). The self-reported intention to use third-party tested supplements increased significantly (+7%-36% per individual supplement) after following the education program (Z = -3.288, p = 0.001) resulting in an intentional use of 54-94% TPT supplements. In conclusion, education resulted in more high school athletes reporting future third-party tested supplements use, and TPB construct scores did not change over time but could explain a substantial part of the variance of safe supplement use intentions.

An Exploratory Study Investigating the Prevalence of Gastrointestinal Symptoms in Collegiate Division I American Football Athletes.

Gastrointestinal (GI) symptoms may limit performance, but their prevalence and impact among team sports athletes is not well-documented. The objective of this study was to examine the prevalence of GI symptoms in a small sample of collegiate DI American football athletes, using a survey including the Gastrointestinal Symptoms Ratings Scale (GSRS). Forty-six athletes responded to the survey and reported scores for the 15-question GSRS with additional questions about dietary habits and supplement use. A total of 44 athletes were included in the study (45% of the current roster, age: 20.7 ± 1.7 years, 50% Afro-American or black, 39% skill position, 18% NSAIDs use, and 41% reporting protein supplement use); approximately half of the athletes (52%) reported experiencing GI complaints during exercise. Two-thirds of the athletes (61%) reported at least one or more GI symptoms in general, and 50% reported at least four moderate complaints. Seven athletes (16%) reported ≥2 severe GI symptoms with 5-13 moderate complaints. The most reported symptom was stomach pain (39%, n = 17), followed by hunger pain (36%, n = 16). Athletes reporting the use of protein supplements reported a higher GSRS score (22.0 and interquartile range (IQR) 17.0-31.8) vs. athletes not reporting protein use (15.0 and IQR 15.0-19.3), p = 0.001. Most athletes surveyed reported experiencing GI symptoms. A small group of these athletes reported multiple, varied, and severe symptoms that were associated with self-reported protein supplement use. In conclusion, the number of complaints varied among athletes, confirming the value of integrating the GSRS for screening purposes, and the expected need for individual dietary treatment approaches.

Athletes Perceived Level of Risk Associated with Botanical Food Supplement Use and Their Sources of Information.

Athletes should carefully consider the use of botanical food supplements (BFSs) given the current lack of substantiation for botanical nutrition and health claims under EU and UK food laws. In addition, athletes may be at an increased risk of doping violations and other adverse outcomes potentially associated with BFS use; however, little is known about athletes' intake, knowledge, or perceptions in relation to BFS use. An online cross-sectional survey of n = 217 elite and amateur athletes living on the island of Ireland was conducted using Qualtrics XM to assess intake, knowledge, attitudes, and perceptions. General food supplements (FSs) were reported by approximately 60% of the study cohort, and 16% of the supplements reported were categorized as BFS. The most frequently consumed BFSs were turmeric/curcumin (14%), Ashwagandha (10%), and Beetroot extract (8%). A higher proportion of amateur athletes would source information about BFSs from less credible sources, such as fellow athletes, or from internet sources or their coach, compared to elite athletes. Those who sourced information about botanicals from fellow athletes (p = 0.03) or the internet (p = 0.02) reported a lower perceived level of risks associated with BFS use. This study therefore suggests that amateur athletes may be more likely to source information from less credible sources compared to elite athletes who may have more access to nutrition professionals and their knowledge/advice. This may have potential adverse implications for amateur athletes, e.g., Gaelic games players, who are included within the doping testing pool but who may not have access to evidence-based nutrition advice.

Lumpy Skin Disease Virus Genome Sequence Analysis: Putative Spatio-Temporal Epidemiology, Single Gene versus Whole Genome Phylogeny and Genomic Evolution.

Lumpy Skin Disease virus is a poxvirus from the genus Capripox that mainly affects bovines and it causes severe economic losses to livestock holders. The Lumpy Skin Disease virus is currently dispersing in Asia, but little is known about detailed phylogenetic relations between the strains and genome evolution. We reconstructed a whole-genome-sequence (WGS)-based phylogeny and compared it with single-gene-based phylogenies. To study population and spatiotemporal patterns in greater detail, we reconstructed networks. We determined that there are strains from multiple clades within the previously defined cluster 1.2 that correspond with recorded outbreaks across Eurasia and South Asia (Indian subcontinent), while strains from cluster 2.5 spread in Southeast Asia. We concluded that using only a single gene (cheap, fast and easy to routinely use) for sequencing lacks phylogenetic and spatiotemporal resolution and we recommend to create at least one WGS whenever possible. We also found that there are three gene regions, highly variable, across the genome of LSDV. These gene regions are located in the 5' and 3' flanking regions of the LSDV genome and they encode genes that are involved in immune evasion strategies of the virus. These may provide a starting point to further investigate the evolution of the virus.

Optimization of a Quantitative Anti-Drug Antibodies against Infliximab Assay with the Liquid Chromatography-Tandem Mass Spectrometry: A Method Validation Study and Future Perspectives.

Monoclonal antibodies (mAbs), such as infliximab, are important treatment options for different diseases. Immunogenicity is a major risk, resulting in anti-drug antibodies (ADAs), being associated with adverse events and loss of response, influencing long-term outcomes. The development of ADAs against infliximab is primarily measured by immunoassays like radioimmunoassay (RIA). Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly utilized across different fields, this technique is currently not used for ADAs against infliximab measurements. Therefore, we developed the first LC-MS/MS method. Stable isotopically labeled infliximab antigen-binding fragments (SIL IFX F(ab')2) were used to bind and measure ADAs indirectly. Protein A magnetic beads were used to capture IgG, including ADAs, whereafter SIL IFX F(ab')2 was added for labeling. After washing, internal standard addition, elution, denaturation and digestion samples were measured by LC-MS/MS. Internal validation showed good linearity between 0.1 and 16 mg/L (R2 > 0.998). Sixty samples were used for cross-validation with RIA, and no significant difference between ADA concentrations was found. The methods had high correlation (R = 0.94, p < 0.001) and excellent agreement, intraclass correlation coefficient = 0.912 (95% confidence interval 0.858-0.947, p < 0.001). We present the first ADA against the infliximab LC-MS/MS method. The method is amendable for quantifying other ADAs, making it applicable as a template for future ADA methods.