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Non-specific binding can be induced in NHS. Non-specific binding in NHS with elevated IgG concentration is dependent on IgG concentration. IVIg was added to NHS to elevate total IgG concentration to the indicated concentration and incubated at 40°C for 24h. Nonspecific IgG deposition was determined using the ELISA setup described in Materials and methods.
Source publication
Enzyme-linked immunosorbent assay (ELISA) is a validated and sensitive method for detection of human autoantibodies, but may have problems with specificity. Non-specific binding is a well-known problem often observed in tests for autoantibodies, when sera are incubated on plastic surfaces, e.g. an ELISA plate. To understand the mechanisms underlyin...
Citations
... Currently, conventional TDM methods primarily focus on the use of Enzyme-Linked Immunosorbent Assay (ELISA) and other immunological tests to determine drug concentrations in the blood [2,3]. However, these methods face challenges, including limited sensitivity and specificity, the risk of cross-reactivity with similar molecules, and the time-consuming and costly need to develop and conduct separate tests for each medication [4,5]. In particular, anti-drug antibodies (ADA) can influence the results of immunological tests, such as ELISA, by blocking respective epitopes. ...
Background
Presently, antibody concentration measurements for patients undergoing treatment are predominantly determined by ELISA, which still comes with known disadvantages. Therefore, our aim was to establish a targeted mass-spectrometric assay enabling the reproducible absolute quantification of peptides from the hypervariable and interaction regions of infliximab.
Methods
Peptides of infliximab were measured post-trypsin digestion and subsequent separation on a Vanquish Horizon UHPLC coupled to a TSQ Altis Triple-Quad mass spectrometer. Normalization and absolute quantification were conducted using stable isotope-synthesized peptides. Calibration curves covering a range of 0.25-50 µg/ml were employed for quantitation.
Results
We demonstrated the substantial influence of peptide selection, choice of hydrolase for digestion, and digestion time on absolute peptide yield (28–44% for peptide 1 and 64–97% for peptide 2). Furthermore, we showed that the generated calibration curves for absolute quantification were highly reproducible and robust (LLOQ1 0.72 µg/ml and LLOQ2 1.00 µg/ml) over several months. In comparison to ELISA values, the absolute values obtained by mass spectrometry often yielded lower results for both targeted peptides.
Conclusions
In this study, a semi-automated workflow was employed and tested with 8 patients and corresponding replicates ( n = 3–4). We demonstrated the robust implementation of calibration curves for the absolute quantification of infliximab in patient samples, with coefficients of variation ranging from 0.5 to 9%. Taken together, we have developed a platform enabling the rapid (2 days of sample preparation and 30 min of measurement time per sample) and robust quantification of Infliximab antibody concentration in patients. The use of mass spectrometry also facilitates the straightforward expansion of the method to include additional antibody peptides.
... In contrast, a false positive only occurred in the plasma matrices tested with the running buffer with Tergitol (SI Fig. S3b). Researchers have reported that testing sera can result in non-specific binding, possibly due to the presence of naturally occurring, poly-specific, and low-affinity heterophile antibodies 42,43 . The sample matrix (i.e., serum) might require further dilution in the running buffer to reduce such non-specific binding. ...
... ,42 . ...
Bothrops and Lachesis are two of Brazil’s medically most relevant snake genera, causing tens of thousands of bites annually. Fortunately, Brazil has good accessibility to high-quality antivenoms at the genus and inter-genus level, enabling the treatment of many of these envenomings. However, the optimal use of these treatments requires that the snake species responsible for the bite is determined. Currently, physicians use a syndromic approach to diagnose snakebite, which can be difficult for medical personnel with limited training in clinical snakebite management. In this work, we have developed a novel monoclonal antibody-based multiplex lateral flow assay for differentiating Bothrops and Lachesis venoms within 15 min. The test can be read by the naked eye or (semi)-quantitatively by a smartphone supported by a 3D-printed attachment for controlling lighting conditions. The LFA can detect Bothrops and Lachesis venoms in spiked plasma and urine matrices at concentrations spanning six orders of magnitude. The LFA has detection limits of 10–50 ng/mL in spiked plasma and urine, and 50–500 ng/mL in spiked sera, for B. atrox and L. muta venoms. This test could potentially support medical personnel in correctly diagnosing snakebite envenomings at the point-of-care in Brazil, which may help improve patient outcomes and save lives.
... If polyreactivity of IgG caused the anti-PTM reactivity, a significant correlation of the different anti-PTM reactivities should be found. We and others have shown that pIgG can be associated with false positive results even in commercially available autoantibody ELISAs for autoimmune liver diseases (4,5). ...
... Polyreactivity of IgG is not limited to autoimmune liver diseases, but is often found in diseases with high inflammation and even more often in diseases with polyclonal hypergammaglobulinemia (5). PIgG appears to be generated during class switching from IgM to IgG and during somatic hypermutation in the transition to IgG+ memory B cells (6, 7). ...
... Additionally, proteomics is inherently specific as it measures the mass and fragmentation spectra of peptides derived from sequence specific digestion of peptides [9]. This is an advantage, for example, compared to immunoassays, which may suffer from non-specific binding [26]. We demonstrated that the serum proteome of VA-ECMO patients on day 1 differed strongly from the control group, as expected. ...
Background
Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is applied in patients with refractory hemodynamic failure. Exposure of blood components to high shear stress and the large extracorporeal surfaces in the ECMO circuit trigger a complex inflammatory response syndrome and coagulopathy which are believed to worsen the already poor prognosis of these patients. Mass spectrometry-based proteomics allow a detailed characterization of the serum proteome as it provides the identity and concentration of large numbers of individual proteins at the same time. In this study, we aimed to characterize the serum proteome of patients receiving VA-ECMO.
Methods
Serum samples were collected on day 1 and day 3 after initiation of VA-ECMO. Samples underwent immunoaffinity based depletion for the 14 most abundant serum proteins, in-solution digestion and PreOmics clean-up. A spectral library was built with multiple measurements of a master-mix sample using variable mass windows. Individual samples were measured in data independent acquisition (DIA) mode. Raw files were analyzed by DIA-neural network. Unique proteins were log transformed and quantile normalized. Differential expression analysis was conducted with the LIMMA—R package. ROAST was applied to generate gene ontology enrichment analyses.
Results
Fourteen VA-ECMO patients and six healthy controls were recruited. Seven patients survived. Three hundred and fifty-one unique proteins were identified. One hundred and thirty-seven proteins were differentially expressed between VA-ECMO patients and controls. One hundred and forty-five proteins were differentially expressed on day 3 compared to day 1. Many of the differentially expressed proteins were involved in coagulation and the inflammatory response. The serum proteomes of survivors and non-survivors on day 3 differed from each other according to partial least-squares discriminant analysis (PLS-DA) and 48 proteins were differentially expressed. Many of these proteins have also been ascribed to processes in coagulation and inflammation (e.g., Factor IX, Protein-C, Kallikrein, SERPINA10, SEMA4B, Complement C3, Complement Factor D and MASP-1).
Conclusion
The serum proteome of VA-ECMO patients displays major changes compared to controls and changes from day 1 until day 3. Many changes in the serum proteome are related to inflammation and coagulation. Survivors and non-survivors can be differentiated according to their serum proteomes using PLS-DA analysis on day 3. Our results build the basis for future studies using mass-spectrometry based serum proteomics as a tool to identify novel prognostic biomarkers.
Trial registration: DRKS00011106.
... There is also a need for standardization of antibody detection techniques. Currently, enzyme-linked immunosorbent assay (ELISA) is mostly used to detect human autoantibodies; however, non-specific binding is a well-recognized problem leading to frequent false positive results [86]. In a recent study, there was no difference in concentration of circulating antibodies against the most common cardiovascular GPCRs between POTS patients and controls using standard ELISA methodology [87]. ...
... This observation questions the use of ELISA for future explorative studies on POTS and Long Covid-associated dysautonomias and more studies are required for definitive conclusions. Non-specific binding sera contain increased concentrations of IgG and other inflammatory mediators, suggesting that this non-specific finding correlates with the IgG concentration and is therefore indicative of elevated IgG and inflammation [86]. ...
Orthostatic intolerance and other autonomic dysfunction syndromes are emerging as distinct symptom clusters in Long Covid. Often accompanying these are common, multi-system constitutional features such as fatigue, malaise and skin rashes which can signify generalized immune dysregulation. At the same time, multiple autoantibodies are identified in both Covid-related autonomic disorders and non-Covid autonomic disorders, implying a possible underlying autoimmune pathology. The lack of specificity of these findings precludes direct interpretations of cause and association, but their prevalence with its supporting evidence is compelling.
... Non-specific binding sera contain 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 10 increased concentrations of IgG and other inflammatory mediators, suggesting that this nonspecific finding correlates with the IgG concentration and is therefore indicative of elevated IgG and inflammation. 88 Immunoprecipitation (IP) is also commonly used, but this has been difficult to develop for GPCRs. Equally, the requirement for radioisotopes limits its widespread application. ...
Orthostatic intolerance and other autonomic dysfunction syndromes are emerging as distinct symptom clusters in Long Covid. Often accompanying these are common, multi-system constitutional features such as fatigue, malaise and skin rashes which can signify generalised immune dysregulation. At the same time, multiple autoantibodies are identified in both Covid-related autonomic disorders and non-Covid autonomic disorders, implying a possible underlying autoimmune pathology. The lack of specificity of these findings precludes direct interpretations of cause and association, but prevalence with its supporting evidence is compelling.
... Thus higher ACET effects can be obtained in a more conductive fluid, but on the other hand the maximum allowable temperature is limited: a high temperature can lead not only to electrolysis and bubbles formation that can affect the experiments and lead to the electrodes deterioration, but can also affect the bioreagents' stability and functionality. 31,[39][40][41] On the contrary, some applications might benefit directly from the coupled effects of microfluidic mixing and the high temperature generated, such as thermal cycling for DNA amplification for PCR applications. 42 Another disadvantage might be that the microelectrodes required for the ACET actuation can hinder the optical readout for different assays, but an alternative solution would be the usage of transparent conductors such as indium tin oxide (ITO). ...
... 39 Moreover, the stability and functionality of the bioreagents can be sensitive to high temperatures. 40,41 Here we show the ability of the new in-plane vortex design to enhance the analyte concentration replenishment by obtaining a large in-plane volumetric mixing. We have carried out a comparison of the performance of the in-plane design versus the vertical vortex design. ...
Biochemical reaction rates in microfluidic systems are known to be limited by the diffusional transport of reagents, leading often to lowered sensitivity and/or longer detection times in immunoassays. Several methods, including electrically powering electrodes to generate AC electrothermal flow (ACET) on-chip, have been adopted to enhance the mass transport of the reagents and improve microfluidic mixing. Here, we report a novel ACET electrode design concept for generating in-plane microfluidic mixing vortices that act over a large volume close to the reaction surface of interest. This is different from the traditional ACET parallel electrode design that provides rather local vertical mixing vortices directly above the electrodes. Both numerical simulation and experimental studies were performed to validate the new design. Moreover, numerical simulation was carried out to show the effects of experimental factors such as the reaction kinetics (association constant) and the reagent concentration on the ACET-enhanced surface-based assays. As a proof of concept, the new design for the ACET-enhanced immunoassays was used to improve the immunostaining signal of the HER2 (human epidermal growth factor receptor 2) cancer biomarker on breast cancer cells. Finally, the concept of scaling up the design has been validated by experiments (immunoassays on breast cancer cells for different ACET power and different assay times). In particular, we show that larger ACET in-plane designs can agitate and mix the fluid over large microfluidic volumes, which further enhances the immunoassay's output. We have achieved a 6-times enhancement in the assay signal with a 75% reduction in assay time.
... There has been a study using monoclonal antibodies where sVNT was able to differentiate between neutralizing antibodies and binding antibodies, while ELISA using the identical RBD antigen failed to distinguish neutralizing antibodies . Although a number of serological assays utilize the RBD as the antigenic target, which is the same as for GenScript cPass sVNT, the protein coating process can cause exposure of hidden epitopes and changes in epitopes that do not exist in the natural state, which occur due to conformational changes (Lee and Belfort, 1989;Sethuraman and Belfort, 2005;Raffaini and Ganazzoli, 2010;Guven et al., 2014;de Thier et al., 2015). This phenomenon could result in lower specificity due to antibodies binding to newly appearing epitopes (Mannik et al., 1997;Guven et al., 2014). ...
... Although a number of serological assays utilize the RBD as the antigenic target, which is the same as for GenScript cPass sVNT, the protein coating process can cause exposure of hidden epitopes and changes in epitopes that do not exist in the natural state, which occur due to conformational changes (Lee and Belfort, 1989;Sethuraman and Belfort, 2005;Raffaini and Ganazzoli, 2010;Guven et al., 2014;de Thier et al., 2015). This phenomenon could result in lower specificity due to antibodies binding to newly appearing epitopes (Mannik et al., 1997;Guven et al., 2014). However, despite this limitation of serological assays, a high correlation between anti-RBD IgG and neutralizing antibodies has been shown in previous studies (Dispinseri et al., 2021;Dogan et al., 2021), and our study shows the feasibility of using semiquantitative serologic assays targeting the RBD in predicting the neutralization titer with Abbott AdviseDx SARS-CoV2 IgG II, which measures IgG against the RBD showing the highest performance. ...
... However, these solutions entail trade-offs in terms of assay performance. For example, overly stringent washing can undermine assay sensitivity by causing loss of signal, while the use of insufficient dAb concentrations will undermine the assay's ability to accurately resolve target concentrations 6,7 . ...
... It is well known that the use of excess concentrations of dAb in an ELISA can contribute greatly to non-specific binding 6,19 . Decreasing the level of dAb employed can mitigate this problem, but at the cost of reduced sensitivity due to loss of signal. ...
... The source data underlying Figs. 2b, d, 3b, c, d, 4a and Supplementary Figs 5,6,7,8,9,10,13,14,15b, c, and 16 are provided as a Source Data file. Source data are provided with this paper. ...
Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still faces notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background. Here, we developed a Single-Molecule Colocalization Assay (SiMCA) that overcomes this problem by employing total internal reflection fluorescence microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized signals, we can eliminate the effects of background produced by non-specific binding of detection antibodies. Using TNF-α, we show that SiMCA achieves a three-fold lower limit of detection compared to conventional single-color assays and exhibits consistent performance for assays performed in complex specimens such as serum and blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects. A major challenge of enzyme-linked immunosorbent assays is discriminating true signal from non-specific binding. Here the authors present a Single-Molecule Colocalization Assay (SiMCA) which eliminates such effects, enabling reproducible detection of picomolar protein concentrations.
... Nonspecific binding is a common phenomenon in immunoassays for the detection of AABs, and high levels of inflammatory parameters such as C-reactive protein (CRP) correlate with non-specific binding of (auto-) antibodies [22]. Therefore, we probed the specificity of all samples exceeding the 97.5th percentile of the HC sera in the IFN-AAB ELISAs and 117 and 118 samples that scored below this cut-off, respectively, from all five cohorts in a competition assay (Fig. 1b, Table S2). ...
... In several studies, detection and quantification of IFN-AABs in sera from COVID-19 patients relies on ELISA and multiplex particle-based assay. While these assays are amenable to high-throughput and are highly sensitive, they result in a small proportion of false-positive results [22,24], highlighting the ongoing need to reanalyze positivetested patient material in functional assays demonstrating the neutralization activity. However, such assays are sophisticated and time-consuming. ...
Purpose
Six to 19% of critically ill COVID-19 patients display circulating auto-antibodies against type I interferons (IFN-AABs). Here, we establish a clinically applicable strategy for early identification of IFN-AAB-positive patients for potential subsequent clinical interventions.
Methods
We analyzed sera of 430 COVID-19 patients from four hospitals for presence of IFN-AABs by ELISA. Binding specificity and neutralizing activity were evaluated via competition assay and virus-infection-based neutralization assay. We defined clinical parameters associated with IFN-AAB positivity. In a subgroup of critically ill patients, we analyzed effects of therapeutic plasma exchange (TPE) on the levels of IFN-AABs, SARS-CoV-2 antibodies and clinical outcome.
Results
The prevalence of neutralizing AABs to IFN-α and IFN-ω in COVID-19 patients from all cohorts was 4.2% (18/430), while being undetectable in an uninfected control cohort. Neutralizing IFN-AABs were detectable exclusively in critically affected (max. WHO score 6–8), predominantly male (83%) patients (7.6%, 18/237 for IFN-α-AABs and 4.6%, 11/237 for IFN-ω-AABs in 237 patients with critical COVID-19). IFN-AABs were present early post-symptom onset and at the peak of disease. Fever and oxygen requirement at hospital admission co-presented with neutralizing IFN-AAB positivity. IFN-AABs were associated with lower probability of survival (7.7% versus 80.9% in patients without IFN-AABs). TPE reduced levels of IFN-AABs in three of five patients and may increase survival of IFN-AAB-positive patients compared to those not undergoing TPE.
Conclusion
IFN-AABs may serve as early biomarker for the development of severe COVID-19. We propose to implement routine screening of hospitalized COVID-19 patients for rapid identification of patients with IFN-AABs who most likely benefit from specific therapies.
