Antibody mimetic drug conjugate manufactured by high-yield Escherichia coli expression and non-covalent binding system.

Antibody-drug conjugates (ADCs) are a major therapeutic tool for the treatment of advanced cancer. Malignant cells in advanced cancer often display multiple genetic mutations and become resistant to monotherapy. Therefore, a therapeutic regimen that simultaneously targets multiple molecules with multiple payloads is desirable. However, the development of ADCs is hampered by issues in biopharmaceutical manufacturing and the complexity of the conjugation process of low-molecular-weight payloads to biologicals. Here, we report antibody mimetic-drug conjugates (AMDCs) developed by exploiting the non-covalent binding property of payloads based on high-affinity binding of mutated streptavidin and modified iminobiotin. Miniprotein antibodies were fused to a low immunogenic streptavidin variant, which was then expressed in Escherichia coli inclusion bodies, solubilized, and refolded into functional tetramers. The AMDC developed against human epidermal growth factor receptor 2 (HER2) effectively killed cultured cancer cells using bis-iminobiotin conjugated to photo-activating silicon phthalocyanine. The HER2-targeting AMDC was also effective in vivo against a mouse KPL-4 xenograft model. This AMDC platform provides rapid, stable, and high-yield therapeutics against multiple targets.

Interference of anti-streptavidin antibodies: More common than we thought? In relation to six confirmed cases.

Automated immunoassays are extensively used in routine laboratory diagnostics of endocrine disorders because of their advantages, such as high sensitivity, precision, and specificity. However, these methods are limited by the susceptibility of the immunochemical reaction to various interferences. They may present interferences related to the assay's design, for example, the endogenous presence of anti-streptavidin antibodies (ASA) in platforms that use the biotin-streptavidin interaction. To date, there have been few reports in the literature of interference from endogenous ASA. However, such antibodies would potentially lead to falsely decreased or increased results of hormones that can lead to incorrect diagnoses. We report six patients with unusual thyroid function tests, incongruent to their clinical findings. They present elevated concentrations of total T3 and T4 and TSH values within the reference range when measured at Cobas 8000® e801 module (Roche Diagnostics®). Neither patient had been taking biotin; however, all demonstrated the presence of ASA causing falsely high results on competitive assays and also falsely low results on sandwich assays. The hormone panel was also analyzed in the same samples using a different platform available in our laboratory: Cobas 6000® e601 module (Roche Diagnostics®). Nine samples were sent to an external laboratory to be measured with the chemiluminescent method: ADVIA Centaur® (Siemens® Healthcare Diagnostics). The interference seems to affect e801 module and competitive assays the most without affecting results obtained by this chemiluminescent method. This interference could potentially affect other assays performed on the same platform, such as ATPO and estradiol. Finally, laboratories should suspect the presence of interference when there is no correlation between the hormone profile and the patient's clinic. The biotin neutralization protocol demonstrated its effectiveness to eliminate ASA interference.

Endogenous anti-streptavidin antibodies causing erroneous laboratory results more common than anticipated.

All immunological methods are vulnerable to different kinds of interference. The purpose of this work was to study the cause and frequency of method-dependent interference in the Roche thyroid immunoassays. Serum samples with discordant thyroid function tests (TFT) were selected from samples sent to the Hormone Laboratory, Oslo University Hospital from June 2013 to September 2018. We identified 93 patients with discordant pathological TFT when analysed with the Roche methods and normal results when analysed with alternative methods. 42 of these samples were sent to Roche Diagnostics (Germany) for investigation of the interfering substance. Roche found interference to be caused by the presence of endogenous anti-streptavidin antibodies (ASA) (34 of 42 patients), ruthenium or the idiotype of the ruthenium labelled antibody (3 of 42 patients) and mouse antigens (1 of 42 patients). Method-dependent interference was estimated to affect 0.37% of the patients investigated in our laboratory. Interference due to the presence of endogenous ASA were further explored in other (non-thyroid) immunoassays by comparing analyte levels before and after pre-adsorption of the patients' sera with streptavidin-coated paramagnetic beads. An underestimation of hormone levels was observed in sandwich immunoassays, while an overestimation was found in competitive assays. Method-dependent interference in Roche thyroid immunoassays is caused mainly by ASA and is not a very rare phenomenon. Misleading results may lead to misdiagnosis and inappropriate medical treatment. The supplier of the assay should be alerted when the available alternative methodology reveals method-dependent errors.

Interference of anti-streptavidin antibodies in immunoassays: a very rare phenomenon or a more common finding?

Background Anti-streptavidin antibodies (ASA) may cause analytical interference on certain immunoassay platforms. Streptavidin is purified from the non-pathogenic Streptomyces avidinii soil bacterium. In contrast to interference with biotin, ASA interference is supposed to be much rarer. In-depth studies on this topic are lacking. Therefore, we carried out an analysis toward the prevalence and the possible underlying cause of this interference. Methods Anti-streptavidin (AS)-immunoglobulin G (IgG) and AS-IgM concentrations were determined on multiple samples from two patients with ASA interference and on 500 random samples. On a subset of 100 samples, thyroid-stimulating hormone (TSH) was measured on a Cobas analyzer before and after performing a neutralization protocol which removes ASA. The relationship between the ratio of TSH after neutralization/TSH before neutralization and the ASA concentration was evaluated. Subsequently, an extract of S. avidinii colonies was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Results A positive correlation between AS-IgM concentrations and TSH ratio was obtained. Eight samples out of 500 exceeded the calculated AS-IgM cut-off value. In comparison to the AS-IgM concentrations in the population, titers from the two described cases clearly stood out. The isolated cases represent the end of a broader spectrum as there is a continuum of AS-IgM reactivity in the general population. We could not observe any differences in the immunoblot patterns between the cases and controls, which may indicate the general presence of ASA in the population. Conclusions Interference due to ASA is more prevalent than initially thought and is caused by IgM antibodies.

Hydrogel based protein biochip for parallel detection of biomarkers for diagnosis of a Systemic Inflammatory Response Syndrome (SIRS) in human serum.

The Systemic Inflammatory Response Syndrome (SIRS), a sepsis related inflammatory state, is a self-defense mechanism against specific and nonspecific stimuli. The six most extensively studied inflammatory biomarkers for the clinical diagnosis of SIRS are interleukin 4 (hIL-4), interleukin 6 (hIL-6), interleukin 10 (hIL-10), tumor necrosis factor alpha (hTNF-α), interferon gamma (hIFN-γ) and procalcitonin (hPCT). These biomarkers are naturally present (but usually only at low concentration) in SIRS infected patients [1, 2] and thus the development of a highly sensitive detection method is of major clinical interest. However, the existing analytical techniques are lacking in required analytical sensitivity and parallel determination of these biomarkers. We developed a fast, easy and cost-efficient protein microarray biochip where the capture molecules are attached on hydrogel spots, enabling SIRS diagnosis by parallel detection of these six clinically relevant biomarkers with a sample volume of 25 µl. With our hydrogel based protein microarray biochip we achieved a limit of detection for hIL-4 of 75.2 pg/ml, for hIL-6 of 45.1 pg/ml, for hIL-10 of 71.5 pg/ml, for hTNF-α of 56.7 pg/ml, for IFN-γ of 46.4 pg/ml and for hPCT of 1.1 ng/ml in spiked human serum demonstrating sufficient sensitivity for clinical usage. Additionally, we demonstrated successful detection of two relevant SIRS biomarkers in clinical patient samples with a turnaround time of the complete analysis from sample-to-answer in less than 200 minutes.

[Anti-estreptavidin antibodies. Diagnostic confusion by biochemical interference]. / Anticuerpos anti-estreptavidina. Confusión diagnóstica por interferencia bioquímica.

We present the case of a patient who, during studies for fertility and subsequent pregnancy, showed an altered thyroid profile with elevated levels of free T4 and normal TSH. After ruling out a thyrotropic adenoma and in the absence of clinical symptoms of hyperthyroidism, the possibility of analytical interference in the immunoassays used to measure hormones was investigated. Interferences caused by heterophile antibodies, macro TSH, anti-thyroid antibodies, biotin, and to a lesser extent anti-streptavidin and anti-ruthenium antibodies have been described. The analysis of the patient was carried out in a self-analyzer whose platform uses the streptavidin-biotin system that is very susceptible to several interferents. A proposed algorithm includes a series of simple tests to perform and interpret that allow detecting or ruling out the presence of interferents. Accordingly, a comparison was made with a different analytical platform (which does not use the streptavidin-biotin system), serial dilutions, precipitation with polyethylene glycol 6000 and treatment with microparticles coated with streptavidin. Results obtained confirmed the presence of anti-streptavidin antibodies in the patient's serum. In the case of disagreements between clinical manifestations and laboratory results, the possibility of methodological interferences should be investigated in order to avoid the potential iatrogenic risk involved in an erroneous biochemical interpretation.

Se presenta el caso de una paciente que, durante los estudios por búsqueda de fertilidad y posterior embarazo, mostraba un perfil tiroideo alterado con niveles elevados de T4 libre y TSH normal. Luego de descartar un adenoma tirotropo y ante la ausencia de sintomatología clínica de hipertiroidismo, se investigó la posibilidad de interferencias analíticas en los inmunoensayos utilizados para la medición de las hormonas. Se han descrito interferencias causadas por anticuerpos heterófilos, macro TSH, anticuerpos anti-tiroideos, biotina, y en menor medida anticuerpos anti-estreptavidina y anti-rutenio. Los análisis de la paciente se realizaron en autoanalizador cuya plataforma emplea el sistema estreptavidina-biotina que es muy susceptible a varios interferentes. Un algoritmo propuesto incluye una serie de pruebas simples de realizar e interpretar que permiten detectar o descartar la presencia de interferentes. De acuerdo al mismo, se efectuó la comparación con una plataforma analítica diferente (que no utiliza el sistema estreptavidina-biotina), diluciones seriadas, precipitación con polietilenglicol 6000 y tratamiento con micropartículas recubiertas con estreptavidina. Los resultados obtenidos confirmaron la presencia de anticuerpos anti-estreptavidina en el suero de la paciente. Ante discordancias entre las manifestaciones clínicas y los resultados de laboratorio, se debe investigar la posibilidad de interferencias metodológicas para evitar el riesgo iatrogénico potencial que implica una interpretación bioquímica errónea.

Anticuerpos anti-estreptavidina: Confusión diagnóstica por interferencia bioquímica / Anti-estreptavidin antibodies. Diagnostic confusion by biochemical interference

Se presenta el caso de una paciente que, durante los estudios por búsqueda de fertilidad y posterior embarazo, mostraba un perfil tiroideo alterado con niveles elevados de T4 libre y TSH normal. Luego de descartar un adenoma tirotropo y ante la ausencia de sintomatología clínica de hipertiroidismo, se investigó la posibilidad de interferencias analíticas en los inmunoensayos utilizados para la medición de las hormonas. Se han descrito interferencias causadas por anticuerpos heterófilos, macro TSH, anticuerpos anti-tiroideos, biotina, y en menor medida anticuerpos anti-estreptavidina y anti-rutenio. Los análisis de la paciente se realizaron en autoanalizador cuya plataforma emplea el sistema estreptavidina-biotina que es muy susceptible a varios interferentes. Un algoritmo propuesto incluye una serie de pruebas simples de realizar e interpretar que permiten detectar o descartar la presencia de interferentes. De acuerdo al mismo, se efectuó la comparación con una plataforma analítica diferente (que no utiliza el sistema estreptavidina-biotina), diluciones seriadas, precipitación con polietilenglicol 6000 y tratamiento con micropartículas recubiertas con estreptavidina. Los resultados obtenidos confirmaron la presencia de anticuerpos anti-estreptavidina en el suero de la paciente. Ante discordancias entre las manifestaciones clínicas y los resultados de laboratorio, se debe investigar la posibilidad de interferencias metodológicas para evitar el riesgo iatrogénico potencial que implica una interpretación bioquímica errónea.

We present the case of a patient who, during studies for fertility and subsequent pregnancy, showed an altered thyroid profile with elevated levels of free T4 and normal TSH. After ruling out a thyrotropic adenoma and in the absence of clinical symptoms of hyperthyroidism, the possibility of analytical interference in the immunoassays used to measure hormones was investigated. Interferences caused by heterophile antibodies, macro TSH, anti-thyroid antibodies, biotin, and to a lesser extent anti-streptavidin and anti-ruthenium antibodies have been described. The analysis of the patient was carried out in a self-analyzer whose platform uses the streptavidin-biotin system that is very susceptible to several interferents. A proposed algorithm includes a series of simple tests to perform and interpret that allow detecting or ruling out the presence of interferents. Accordingly, a comparison was made with a different analytical platform (which does not use the streptavidin-biotin system), serial dilutions, precipitation with polyethylene glycol 6000 and treatment with microparticles coated with streptavidin. Results obtained confirmed the presence of anti-streptavidin antibodies in the patient's serum. In the case of disagreements between clinical manifestations and laboratory results, the possibility of methodological interferences should be investigated in order to avoid the potential iatrogenic risk involved in an erroneous biochemical interpretation.

Real-Time Profiling of Anti-(Epithelial Cell Adhesion Molecule)-Based Immune Capture from Molecules to Cells Using Multiparameter Surface Plasmon Resonance.

Antibodies of epithelial cell-adhesion molecule (anti-EpCAM)-based interfaces have proven to be highly efficient at capturing circulating tumor cells (CTCs). To achieve the bonding of anti-EpCAM to the interface, biotin and streptavidin are used to modify the surface. These processes are critical to subsequent cell-capture efficiencies. However, quantitative research on the interactions between biotin, streptavidin, and biotinylated anti-EpCAM on the interface is lacking. In this work, the thermodynamics and kinetics of biomolecular interactions were determined by using surface plasmon resonance. The equilibrium binding affinities for biotinylated anti-EpCAM to streptavidin and streptavidin to biotin (illustrated by biotin-PEG400-thiol) were found to be 2.75 × 106 and 8.82 × 106 M-1, respectively. Each streptavidin can bind up to 2.30 biotinylated anti-EpCAM under thermodynamic equilibrium. The findings provide useful information to optimize the modification of anti-EpCAM and improve the capture efficiency of CTCs.

Competitive light-initiated chemiluminescent assay: using 5-α-dihydrotestosterone-BSA as competitive antigen for quantitation of total testosterone in human sera.

This paper described a homogeneous method, light-initiated chemiluminescent assay (LICA), for quantitation of total testosterone in human sera. The assay was bead based and built on a competitive-binding reaction format, in which 5-α-dihydrotestosterone (5-α-DHT) competed with the testosterone in serum samples in binding with biotinylated anti-testosterone antibody. The more testosterone in the serum sample, the less 5-α-DHT that bonded with biotinylated anti-testosterone antibodies. 5-α-DHT was coupled with emission beads (doped with thioxene derivatives and Eu(III) as a chemiluminescence emitter) via bovine serum albumin as a linker. Once streptavidin-coated sensitizer beads (modified with phthalocyanine as a photosensitizer) were added, the streptavidin/biotin reaction between 5-α-DHT-bound anti-testosterone antibody and sensitizer beads could bring emission and sensitizer beads together, which allowed energy transfer from sensitizer bead to emission bead. As such, an exciting light (680 nm) impinging on the sensitizer beads led to light emission at 520-620 nm by emission beads. The strength of the emitted light was inversely proportional to the testosterone in serum sample. The detection range of this assay was from 13.3 to 1200 ng/dL. The coefficient variation for intra- and inter-assay was lower than 15%. The recovery of this method ranged from 95.5 to 105.9% for different samples. Moreover, the LICA assay was highly specific with low cross-reactivity and interference. The concentration of testosterone from 58 serum samples analyzed by the LICA method significantly correlated (y = 0.97x + 1.87, R2 = 0.970, p < 0.001) with those obtained with the SIEMENS Centaur Xp System. Graphical abstract ᅟ.

Localized immune tolerance from FasL-functionalized PLG scaffolds.

Intraportal allogeneic islet transplantation has been demonstrated as a potential therapy for type 1 diabetes (T1D). The placement of islets into the liver and chronic immunosuppression to control rejection are two major limitations of islet transplantation. We hypothesize that localized immunomodulation with a novel form of FasL chimeric with streptavidin, SA-FasL, can provide protection and long-term function of islets at an extrahepatic site in the absence of chronic immunosuppression. Allogeneic islets modified with biotin and engineered to transiently display SA-FasL on their surface showed sustained survival following transplantation on microporous scaffolds into the peritoneal fat in combination with a short course (15 days) of rapamycin treatment. The challenges with modifying islets for clinical translation motivated the modification of scaffolds with SA-FasL as an off-the-shelf product. Poly (lactide-co-glycolide) (PLG) was conjugated with biotin and fabricated into particles and subsequently formed into microporous scaffolds to allow for rapid and efficient conjugation with SA-FasL. Biotinylated particles and scaffolds efficiently bound SA-FasL and induced apoptosis in cells expressing Fas receptor (FasR). Scaffolds functionalized with SA-FasL were subsequently seeded with allogeneic islets and transplanted into the peritoneal fat under the short-course of rapamycin treatment. Scaffolds modified with SA-FasL had robust engraftment of the transplanted islets that restored normoglycemia for 200 days. Transplantation without rapamycin or without SA-FasL did not support long-term survival and function. This work demonstrates that scaffolds functionalized with SA-FasL support allogeneic islet engraftment and long-term survival and function in an extrahepatic site in the absence of chronic immunosuppression with significant potential for clinical translation.

Development of a Nanobody-AviTag Fusion Protein and Its Application in a Streptavidin-Biotin-Amplified Enzyme-Linked Immunosorbent Assay for Ochratoxin A in Cereal.

Ochratoxin A (OTA) is a common food contaminant that threatens consumers' safety and health. A sensitive and selective biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) for OTA using a nanobody-AviTag fusion protein (Nb-AviTag) was developed in this study. The prokaryotic expression vector Nb28-AviTag-pAC6 for Nb-AviTag was constructed, followed by transformation to the AVB101 cells for antibody expression and in vivo biotinylation. The purified Nb28-AviTag was used to establish the BA-ELISA and the procedures for this Nb-AviTag-based BA-ELISA were optimized. The Nb-AviTag-based BA-ELISA exhibited the half maximal inhibitory concentration (IC50) of 0.14 ng mL-1 and the limit of detection (LOD = IC10) of 0.028 ng mL-1 for OTA basing on the optimized experiment parameters. The assay sensitivity was improved 4.6 times and 4.3 times compared to Nb-based ELISA, respectively. This method had LODs of 1.4 µg kg-1 in barley, 0.56 µg kg-1 in oats, and 0.84 µg kg-1 in rice for OTA. The average recovery percent was in a range of 84-137%, and the relative standard derivation percent ranged from 0.64% to 7.8%. The content of OTA in contaminated cereal samples was determined by both the developed Nb-AviTag-based method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results demonstrated that the Nb-AviTag was a robust and promising bioreceptor in highly sensitive detection of OTA and other low molecular weight compounds using BA system.

Apparent Hyperthyroidism Caused by Biotin-Like Interference from IgM Anti-Streptavidin Antibodies.

BACKGROUND: Exclusion of analytical interference is important when there is discrepancy between clinical and laboratory findings. However, interferences on immunoassays are often mistaken as isolated laboratory artefacts. The mechanism of a rare cause of interference in two patients that caused erroneous thyroid function tests, and also affects many other biotin dependent immunoassays, was characterized and reported. PATIENT FINDINGS: Patient 1 was a 77-year-old female with worsening fatigue while taking carbimazole over several years. Her thyroid function tests, however, were not suggestive of hypothyroidism. Patient 2 was a 25-year-old female also prescribed carbimazole for apparent primary hyperthyroidism. Despite an elevated free thyroxine, the lowest thyrotropin on record was 0.17 mIU/L. In both cases, thyroid function tests performed by an alternative method were markedly different. Further characterization of both patients' serum demonstrated analytical interference on many immunoassays using the biotin-streptavidin interaction. Sandwich assays (e.g., thyrotropin, follicle-stimulating hormone, troponin T, beta-human chorionic gonadotropin) were falsely low, while competitive assays (e.g., free thyroxine, free triiodothyronine, TSH binding inhibitory immunoglobulin) were falsely high. Pre-incubation of serum with streptavidin microparticles removed the analytical interference, initially suggesting the cause of interference was biotin. However, neither patient had been taking biotin. Instead, a ∼100 kDa immunoglobulin M (IgM) immunoglobulin with high affinity to streptavidin was isolated from each patient's serum. The findings confirm IgM anti-streptavidin antibodies as the cause of analytical interference. SUMMARY: Two patients with apparent hyperthyroidism as a result of analytical interference caused by IgM anti-streptavidin antibodies are described. CONCLUSION: Analytical interference identified on one immunoassay should raise the possibility of other affected results. Characterization of interference may help to identify other potentially affected immunoassays. In the case of anti-streptavidin antibodies, the pattern of interference mimics that due to biotin ingestion. However, the degree of interference varies between individual assays and between patients.

Anti-streptavidin IgG antibody interference in anti-cyclic citrullinated peptide (CCP) IgG antibody assays is a rare but important cause of false-positive anti-CCP results.

BACKGROUND: The detection of anti-cyclic citrullinated peptide (anti-CCP) IgG antibodies in blood is mainly used for the diagnosis of rheumatoid arthritis. Falsely elevated anti-CCP IgG antibodies due to anti-streptavidin IgG antibodies were suspected in our laboratory. METHODS: In this study, we evaluated, in a standardized approach, the prevalence of anti-streptavidin IgG antibodies in a primary care setting and the effect of anti-streptavidin IgG antibodies on anti-CCP IgG assays from three different important commercial manufacturers (Abbott, Roche Diagnostics, Thermo Fisher Scientific). Three different populations were consecutively and prospectively studied: serum samples from 1000 ambulatory patients, 286 serum samples from patients for which anti-CCP was requested and 89 serum samples from patients which had previously given a positive anti-CCP result on Architect® i2000. RESULTS: The frequency of confirmed anti-streptavidin IgG-positive samples detected in this study was 0.6% (8/1375). Anti-CCP IgG was determined on the eight samples with confirmed anti-streptavidin IgG antibodies: with the Cobas® method, seven positive anti-CCP results were observed and five positive anti-CCP results with the Architect® method. No positive anti-CCP IgG results were obtained with the EliA™ method. Rheumatoid factor was negative in these eight samples. CONCLUSIONS: Anti-streptavidin IgG antibodies rarely cause false-positive results in some anti-CCP assays. However, despite being an infrequent assay problem, it could possibly lead to diagnostic confusion or even an incorrect diagnosis of rheumatoid arthritis.

Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction.

Single-chain variable fragment antibodies (scFvs) are attractive for use in applications that require high specificity and binding to a target, such as biosensors. Previously, we demonstrated that a variety of scFvs can be immobilized onto a streptavidin surface through in vivo biotinylation of the biotin carboxyl carrier protein (BCCP) or smaller AviTag fused to the scFvs. However, the BCCP constructs showed better immobilization than the AviTag constructs. In this work, we investigated whether the discrepancy between the biotinylation tags could be alleviated by incorporating a flexible (G4 S)n linker of varying lengths or a rigid (EA3 K)3 linker between the biotinylation tags and the scFvs scFv13R4 and scFv5. Fusion of the (G4 S)5 linker or the (G4 S)3 linker to the AviTag construct of scFv13R4 or scFv5, respectively, and fusion of the (EA3 K)3 linkers to the AviTag constructs of both scFvs enhanced immobilization. Meanwhile, the robust immobilization of the BCCP construct of the scFv constructs remained unaffected. The positive to neutral effects of the linkers, with no adverse effects, make them beneficial tools to incorporate into fusion proteins that show poor immobilization without a linker.