6 research outputs found
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
A new one-step antigen heterologous homogeneous fluorescence immunoassay for progesterone detection in serum
Antibody Binding at the Liposome–Water Interface: A FRET Investigation toward a Liposome-Based Assay
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)
Bright or dark immune complexes of anti-TAMRA antibodies for adapted fluorescence-based bioanalysis
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