MALDI-TOF-MS-Based Identification of Monoclonal Murine Anti-SARS-CoV-2 Antibodies within One Hour.

During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used 35 monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied to the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 60 min and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 °C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0. This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.

A broadly cross-reactive monoclonal antibody against hepatitis E virus capsid antigen.

To generate a hepatitis E virus (HEV) genotype 3 (HEV-3)-specific monoclonal antibody (mAb), the Escherichia coli-expressed carboxy-terminal part of its capsid protein was used to immunise BALB/c mice. The immunisation resulted in the induction of HEV-specific antibodies of high titre. The mAb G117-AA4 of IgG1 isotype was obtained showing a strong reactivity with the homologous E. coli, but also yeast-expressed capsid protein of HEV-3. The mAb strongly cross-reacted with ratHEV capsid protein derivatives produced in both expression systems and weaker with an E. coli-expressed batHEV capsid protein fragment. In addition, the mAb reacted with capsid protein derivatives of genotypes HEV-2 and HEV-4 and common vole hepatitis E virus (cvHEV), produced by the cell-free synthesis in Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cell lysates. Western blot and line blot reactivity of the mAb with capsid protein derivatives of HEV-1 to HEV-4, cvHEV, ratHEV and batHEV suggested a linear epitope. Use of truncated derivatives of ratHEV capsid protein in ELISA, Western blot, and a Pepscan analysis allowed to map the epitope within a partially surface-exposed region with the amino acid sequence LYTSV. The mAb was also shown to bind to human patient-derived HEV-3 from infected cell culture and to hare HEV-3 and camel HEV-7 capsid proteins from transfected cells by immunofluorescence assay. The novel mAb may serve as a useful tool for further investigations on the pathogenesis of HEV infections and might be used for diagnostic purposes. KEY POINTS: • The antibody showed cross-reactivity with capsid proteins of different hepeviruses. • The linear epitope of the antibody was mapped in a partially surface-exposed region. • The antibody detected native HEV-3 antigen in infected mammalian cells.

Antibody Binding at the Liposome-Water Interface: A FRET Investigation toward a Liposome-Based Assay.

Different signal amplification strategies to improve the detection sensitivity of immunoassays have been applied which utilize enzymatic reactions, nanomaterials, or liposomes. The latter are very attractive materials for signal amplification because liposomes can be loaded with a large amount of signaling molecules, leading to a high sensitivity. In addition, liposomes can be used as a cell-like "bioscaffold" to directly test recognition schemes aiming at cell-related processes. This study demonstrates an easy and fast approach to link the novel hydrophobic optical probe based on [1,3]dioxolo[4,5-f]-[1,3]benzodioxole (DBD dye mm239) with tunable optical properties to hydrophilic recognition elements (e.g., antibodies) using liposomes for signal amplification and as carrier of the hydrophobic dye. The fluorescence properties of mm239 (e.g., long fluorescence lifetime, large Stokes shift, high photostability, and high quantum yield), its high hydrophobicity for efficient anchoring in liposomes, and a maleimide bioreactive group were applied in a unique combination to build a concept for the coupling of antibodies or other protein markers to liposomes (coupling to membranes can be envisaged). The concept further allowed us to avoid multiple dye labeling of the antibody. Here, anti-TAMRA-antibody (DC7-Ab) was attached to the liposomes. In proof-of-concept, steady-state as well as time-resolved fluorescence measurements (e.g., fluorescence depolarization) in combination with single molecule detection (fluorescence correlation spectroscopy, FCS) were used to analyze the binding interaction between DC7-Ab and liposomes as well as the binding of the antigen rhodamine 6G (R6G) to the antibody. Here, the Förster resonance energy transfer (FRET) between mm239 and R6G was monitored. In addition to ensemble FRET data, single-molecule FRET (PIE-FRET) experiments using pulsed interleaved excitation were used to characterize in detail the binding on a single-molecule level to avoid averaging out effects.

Dye Tool Box for a Fluorescence Enhancement Immunoassay.

Immunochemical analytical methods are very successful in clinical diagnostics and are nowadays also emerging in the control of food as well as monitoring of environmental issues. Among the different immunoassays, luminescence based formats are characterized by their outstanding sensitivity making this format especially attractive for future applications. The need for multiparameter detection capabilities calls for a tool box of dye labels in order to transduce the biochemical reaction into an optically detectable signal. Here, in a multiparameter approach each analyte may be detected by a different dye with a unique emission color (covering the blue to red spectral range) or a unique luminescence decay kinetics. In the case of a competitive immunoassay format for each of the different dye labels an individual antibody would be needed. In the present paper a slightly modified approach is presented using a 7-aminocoumarin unit as the basic antigen against which highly specific antibodies were generated. Leaving the epitope region in the dyes unchanged but introducing a side group in positon 3 of the coumarin system allowed us to tune the optical properties of the coumarin dyes without the necessity of new antibody generation. Upon modification of the parent coumarin unit the full spectral range from blue to deep red was accessed. In the manuscript the photophysical characterization of the coumarin derivatives and their corresponding immunocomplexes with two highly specific antibodies is presented. The coumarin dyes and their immunocomplexes were characterized by steady-state and time-resolved absorption as well as emission spectroscopy. Moreover, fluorescence depolarization measurements were carried out to complement the data stressing the different binding modes of the two antibodies. The binding modes were evaluated using the photophysics of 7-aminocoumarins and how it was affected in the respective immunocomplexes, namely, the formation of the intramolecular charge transfer (ICT) as well as the twisted intramolecular charge transfer (TICT). In contrast to other antibody-dye pairs reported a distinct fluorescence enhancement upon formation of the antibody-dye complex up to a factor of 50 was found. Because of the easy emission color tuning by tailoring the coumarin substitution for the antigen binding in nonrelevant position 3 of the parent molecule, a dye tool box is on hand which can be used in the construction of competitive multiparameter fluorescence enhancement immunoassays (FenIA).

Hapten-Specific Single-Cell Selection of Hybridoma Clones by Fluorescence-Activated Cell Sorting for the Generation of Monoclonal Antibodies.

The conventional hybridoma screening and subcloning process is generally considered to be one of the most critical steps in hapten-specific antibody production. It is time-consuming, monoclonality is not guaranteed, and the number of clones that can be screened is limited. Our approach employs a novel hapten-specific labeling technique of hybridoma cells. This allows for fluorescence-activated cell sorting (FACS) and single-cell deposition and thereby eliminates the above-mentioned problems. A two-step staining approach is used to detect antigen specificity and antibody expression: in order to detect antigen specificity, hybridoma cells are incubated with a hapten-horseradish peroxidase conjugate (hapten-HRP), which is subsequently incubated with a fluorophore-labeled polyclonal anti-peroxidase antibody (anti-HRP-Alexa Fluor 488). To characterize the expression of membrane-bound immunoglobulin G (IgG), a fluorophore-labeled anti-mouse IgG antibody (anti-IgG-Alexa Fluor 647) is used. Hundreds of labeled hybridoma cells producing monoclonal antibodies (mAbs) specific for a hapten were rapidly isolated and deposited from a fusion mixture as single-cell clones via FACS. Enzyme-linked immunosorbent assay (ELISA) measurements of the supernatants of the sorted hybridoma clones revealed that all hapten-specific hybridoma clones secrete antibodies against the target. There are significant improvements using this high-throughput technique for the generation of mAbs including increased yield of antibody-producing hybridoma clones, ensured monoclonality of sorted cells, and reduced development times.

Bright or dark immune complexes of anti-TAMRA antibodies for adapted fluorescence-based bioanalysis.

Fluorescence labels, for example fluorescein or rhodamin derivatives, are widely used in bioanalysis applications including lateral-flow assays, PCR, and fluorescence microscopy. Depending on the layout of the particular application, fluorescence quenching or enhancement may be desired as the detection principle. Especially for multiplexed applications or high-brightness requirements, a tunable fluorescence probe can be beneficial. The alterations in the photophysics of rhodamine derivatives upon binding to two different anti-TAMRA antibodies were investigated by absorption and fluorescence-spectroscopy techniques, especially determining the fluorescence decay time and steady-state and time-resolved fluorescence anisotropy. Two monoclonal anti-TAMRA antibodies were generated by the hybridoma technique. Although surface-plasmon-resonance measurements clearly proved the high affinity of both antibodies towards 5-TAMRA, the observed effects on the fluorescence of rhodamine derivatives were very different. Depending on the anti-TAMRA antibody either a strong fluorescence quenching (G71-DC7) or a distinct fluorescence enhancement (G71-BE11) upon formation of the immune complex was observed. Additional rhodamine derivatives were used to gain further information on the binding interaction. The data reveal that such haptens as 5-TAMRA could generate different paratopes with equal binding affinities but different binding interactions, which provide the opportunity to adapt bioanalysis methods including immunoassays for optimized detection principles for the same hapten depending on the specific requirements.

A new one-step antigen heterologous homogeneous fluorescence immunoassay for progesterone detection in serum.

A new homogeneous immunoassay for the detection of progesterone was developed to measure its concentration in human serum. We utilized the weak cross-reactivity of a monoclonal anti-progesterone antibody to an analog molecule (in this case ß-estradiol) to create a mixture, in which the fluorescence-labeled antibody (AbF) and quencher-labeled BSA-estradiol (eBSAq) were at optimized equilibrium. At this stage, most antibodies were bound to eBSAq and the fluorescence of AbF was quenched. After adding samples containing free progesterone to the system, these would replace the eBSAq at the antigen-binding site. The fluorescence would be released. In contrast to conventional competitive immunoassays, the fluorescence signal increases with increasing progesterone concentration, greatly simplifying detection and calibration. The performance of the assay was very simple; there was only one mixing step; and other hormones like testosterone, estradiol or dehydroepiandrosterone (DHEA) do not interfere the assay. A wide linear range from 0.1 µg/L to 100 µg/L was achieved in buffer, with a LOD of 0.1 µg/L. In human serum the LOD was 5 µg/L, and the linear range was 5-500 µg/L. For this assay it is important to find the right combination of antibody and cross-reactive antigen. If such a combination could be defined, it is conceivable to apply this assay to a wide range of analytes.

Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA.

A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity.

Antibody mediated fluorescence enhancement of nucleoside analogue 1,3-diaza-2-oxophenoxazine (tC°).

We report on the generation and analytical application of the monoclonal antibody G93-ED2 raised against the tricyclic fluorescent nucleoside analogue 1,3-diaza-2-oxophenoxazine (tC°). G93-ED2 is specifically binding this deoxycytidine analogue and was found to raise its fluorescence intensity by a factor of 5. This unique feature makes it a valuable tool in fluorescence dependent immunoassays. G93-ED2 was successfully applied in a homogeneous fluorescence quenching immunoassay (DNA-Q) for the sequence specific determination of DNA.

A water soluble fluorescent polymer as a dual colour sensor for temperature and a specific protein.

We present two thermoresponsive water soluble copolymers prepared via free radical statistical copolymerization of N-isopropylacrylamide (NIPAm) and of oligo(ethylene glycol) methacrylates (OEGMAs), respectively, with a solvatochromic 7-(diethylamino)-3-carboxy-coumarin (DEAC)-functionalized monomer. In aqueous solutions, the NIPAm-based copolymer exhibits characteristic changes in its fluorescence profile in response to a change in solution temperature as well as to the presence of a specific protein, namely an anti-DEAC antibody. This polymer emits only weakly at low temperatures, but exhibits a marked fluorescence enhancement accompanied by a change in its emission colour when heated above its cloud point. Such drastic changes in the fluorescence and absorbance spectra are observed also upon injection of the anti-DEAC antibody, attributed to the specific binding of the antibody to DEAC moieties. Importantly, protein binding occurs exclusively when the polymer is in the well hydrated state below the cloud point, enabling a temperature control on the molecular recognition event. On the other hand, heating of the polymer-antibody complexes releases a fraction of the bound antibody. In the presence of the DEAC-functionalized monomer in this mixture, the released antibody competitively binds to the monomer and the antibody-free chains of the polymer undergo a more effective collapse and inter-aggregation. In contrast, the emission properties of the OEGMA-based analogous copolymer are rather insensitive to the thermally induced phase transition or to antibody binding. These opposite behaviours underline the need for a carefully tailored molecular design of responsive polymers aimed at specific applications, such as biosensing.

Specific DNA detection using antibody mediated fluorescence quenching.

First homogenous immunoassay for sequence-specific nucleic acid detection is developed. The assay bases on our finding that a fluorophore inserted into a DNA probe instead of one of the internal nucleotides may get protected from fluorescence quenching caused by an anti-fluorophore antibody, if the probe is hybridized with the target sequence. This ensures a positive signal in the antibody presence. The assay enables quantitative detection and may have potential for development of homogenous high-throughput platforms.

Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system.

Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner.

Non-invasive monitoring of immunization progress in mice via IgG from feces.

A non-invasive method to monitor the humoral immune response in mice after immunization is described. From fecal pellets of an individual mouse, a sufficient amount of active immunoglobulins or their fragments can be extracted to perform a regular examination of the status of the immune response by immunoassay. Hapten-specific antibodies from the feces of mice from three immunization trials showed very similar characteristics to those obtained from serum at a given date. Therefore, it can be suspected that some serum IgG enters the intestinal lumen and ends up in the feces, where they appear to be considerably stable. Hapten-specific IgAs were not found in the feces. Being able to analyze antibody titers in feces could be an interesting animal welfare refinement to standard practice that does not entail repeated blood sampling.

Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G.

The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem.

Generation of an antibody against the protein phosphatase 1 inhibitor KEPI and characterization of the epitope.

A monoclonal antibody against the potential tumor suppressor kinase-enhanced protein phosphatase 1 (PP1) inhibitor KEPI (PPP1R14C) was generated and characterized. Human KEPI was expressed in Escherichia coli and used to immunize Balb/c mice. Using hybridoma technology, one clone, G18AF8, was isolated producing antibodies which bound specifically to the KEPI protein in ELISA, immunoblotting and flow cytometry. The antibody was also successfully applied to stain KEPI protein in paraffin sections of human brain. The epitope was mapped using peptide array technology and confirmed as GARVFFQSPR. This corresponds to the N-terminal region of KEPI. Amino acid substitution analysis revealed that two residues, F and Q, are essential for binding. Affinity of binding was determined by competitive ELISA as 1 microM. In Western blot assays testing G18AF8 antibody on brain samples of several species, reactivity with hamster, rat and chicken samples was found, suggesting a broad homology of this KEPI epitope in vertebrates. This antibody could be used in expression studies at the protein level e.g. in tumor tissues.

The sarcomeric Z-disc component myopodin is a multiadapter protein that interacts with filamin and alpha-actinin.

Here we introduce myopodin as a novel filamin C binding partner. Corroborative yeast two-hybrid and biochemical analyses indicate that the central part of myopodin that shows high homology to the closely related protein synaptopodin and that is common to all its currently known or predicted variants interacts with filamin C immunoglobulin-like domains 20-21. A detailed characterization of the previously described interaction between myopodin and alpha-actinin demonstrates for the first time that myopodin contains three independent alpha-actinin-binding sites. Newly developed myopodin-specific antibodies reveal expression at the earliest stages of in vitro differentiation of human skeletal muscle cells preceding the expression of sarcomeric alpha-actinin. Myopodin colocalizes with filamin and alpha-actinin during all stages of muscle development. By contrast, colocalization with its previously identified binding partner zyxin is restricted to early developmental stages. Genetic and cellular analyses of skeletal muscle provided direct evidence for an alternative transcriptional start site in exon three, corroborating the expression of a myopodin variant lacking the PDZ domain encoded by exons 1 and 2 in skeletal muscle. We conclude that myopodin is a multiadapter protein of the sarcomeric Z-disc that links nascent myofibrils to the sarcolemma via zyxin, and might play a role in early assembly and stabilization of the Z-disc. Mutations in FLNC, ACTN2 and several other genes encoding Z-disc-related proteins cause myopathy and cardiomyopathy. Its localization and its association with the myopathy-associated proteins filamin C and alpha-actinin make myopodin an interesting candidate for a muscle disease gene.

Detection and characterization of gamete-specific molecules in Mytilus edulis using selective antibody production.

The mussel Mytilus edulis can be used as model to study the molecular basis of reproductive isolation because this species maintains its species integrity, despite of hybridizing in zones of contact with the closely related species M. trossulus or M. galloprovincialis. This study uses selective antibody production by means of hybridoma technology to identify molecules which are involved in sperm function of M. edulis. Fragmented sperm were injected into mice and 25 hybridoma cell clones were established to obtain monoclonal antibodies (mAb). Five clones were identified producing mAb targeting molecules putatively involved in sperm function based on enzyme immunoassays, dot and Western blotting as well as immunostaining of tissue sections. Specific localization of these mAb targets on sperm and partly also in somatic tissue suggests that all five antibodies bind to different molecules. The targets of the mAb obtained from clone G26-AG8 were identified using mass spectrometry (nano-LC-ESI-MS/MS) as M6 and M7 lysin. These acrosomal proteins have egg vitelline lyses function and are highly similar (76%) which explains the cross reactivity of mAb G26-AG8. Furthermore, M7 lysin was recently shown to be under strong positive selection suggesting a role in interspecific reproductive isolation. This study shows that M6 and M7 lysin are not only found in the sperm acrosome but also in male somatic tissue of the mantle and the posterior adductor muscle, while being completely absent in females. The monoclonal antibody G26-AG8 described here will allow elucidating M7/M6 lysin function in somatic and gonad tissue of adult and developing animals.

Epitope mapping of antibodies against S-tagged fusion proteins and molecular weight markers.

Monoclonal antibodies against S-tagged fusion proteins expressed in pET vectors were generated and further characterized. Most pET vectors contain a 15-meric S-tag as a fusion tag for the detection of recombinant proteins. Two antibodies, G18BA3 and G18BE8, recognized this S-tag in enzyme immunoassay and Western blot. Their epitopes were mapped using peptide array technology and were confirmed to be AAKFERQHMDSPD. This corresponds to the C-terminal region of the S-tag plus additional amino acids P and D, which are also present in most available pET vectors. Amino acid substitution analysis revealed several essential residues for binding. The binding motif was therefore FExxHxDxxD for G18BA3 and AxxFExxH for G18BE8. Since some commercially available protein standards are expressed in pET vectors, G18BA3 and G18BE8 were also found to detect the ladder bands of a molecular weight marker on immunoblot analysis. Both antibodies should be highly useful for the simultaneous detection of recombinant pET vector-expressed fusion proteins and protein molecular weight standards in Western blotting, especially when chemoluminescent detection systems are used.

Cloning and characterization of a single chain antibody to glucose oxidase from a murine hybridoma.

Glucose oxidase (GOD) is an oxidoreductase catalyzing the reaction of glucose and oxygen to peroxide and gluconolacton (EC 1.1.3.4.). GOD is a widely used enzyme in biotechnology. Therefore the production of monoclonal antibodies and antibody fragments to GOD are of interest in bioanalytics and even tumor therapy. We describe here the generation of a panel of monoclonal antibodies to native and heat inactivated GOD. One of the hybridomas, E13BC8, was used for cloning of a single chain antibody (scFv). This scFv was expressed in Escherichia coli XL1-blue with the help of the vector system pOPE101. The scFv was isolated from the periplasmic fraction and detected by western blotting. It reacts specifically with soluble active GOD but does not recognize denatured GOD adsorbed to the solid phase. The same binding properties were also found for the monoclonal antibody E13BC8.

Chimeric polyomavirus-derived virus-like particles: the immunogenicity of an inserted peptide applied without adjuvant to mice depends on its insertion site and its flanking linker sequence.

We inserted the sequence of the carcinoembryonic antigen-derived T cell epitope CAP-1-6D (CEA) into different positions of the hamster polyomavirus major capsid protein VP1. Independently from additional flanking linkers, yeast-expressed VP1 proteins harboring the CEA insertion between VP1 amino acid residues 80 and 89 (site 1) or 288 and 295 (site 4) or simultaneously at both positions assembled to chimeric virus-like particles (VLPs). BALB/c mice immunized with adjuvant-free VLPs developed VP1- and epitope-specific antibodies. The level of the CEA-specific antibody response was determined by the insertion site, the number of inserts, and the flanking linker. The strongest CEA-specific antibody response was observed in mice immunized with VP1 proteins harboring the CEA insert at site 1. Moreover, the CEA-specific antibodies in these mice were still detectable 6 mo after the final booster immunization. Our results indicate that hamster polyomavirus-derived VLPs represent a highly immunogenic carrier for foreign insertions that might be useful for clinical and therapeutic applications.