| 1. |
- Mravinacová, Sára, et al.
(författare)
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A cell-free high throughput assay for assessment of SARS-CoV-2 neutralizing antibodies
- 2022
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 66, s. 46-52
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Tidskriftsartikel (refereegranskat)abstract
- Highly accurate serological tests are key to assessing the prevalence of SARS-CoV-2 antibodies and the level of immunity in the population. This is important to predict the current and future status of the pandemic. With the recent emergence of new and more infectious SARS-CoV-2 variants, assays allowing for high throughput analysis of antibodies able to neutralize SARS-CoV-2 become even more important. Here, we report the development and validation of a robust, high throughput method, which enables the assessment of antibodies inhibiting the binding between the SARS-CoV-2 spike protein and angiotensin converting enzyme 2 (ACE2). The assay uses recombinantly produced spike-f and ACE2 and is performed in a bead array format, which allows analysis of up to 384 samples in parallel per instrument over seven hours, demanding only one hour of manual handling. The method is compared to a microneutralization assay utilising live SARS-CoV-2 and is shown to deliver highly correlating data. Further, a comparison with a serological method that measures all antibodies recognizing the spike protein shows that this type of assessment provides important insights into the neutralizing efficiency of the antibodies, especially for individuals with low antibody levels. This method can be an important and valuable tool for large-scale assessment of antibody-based neutralization, including neutralization of new spike variants that might emerge.
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| 2. |
- Zhao, Hongxing, et al.
(författare)
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Detection of SARS-CoV-2 antibodies in serum and dried blood spot samples of vaccinated individuals using a sensitive homogeneous proximity extension assay.
- 2022
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Ingår i: New biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 72, s. 139-148
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Tidskriftsartikel (refereegranskat)abstract
- A homogeneous PCR-based assay for sensitive and specific detection of antibodies in serum or dried blood spots (DBS) is presented and the method is used to monitor individuals infected with or vaccinated against SARS-CoV-2. Detection probes were prepared by conjugating the recombinant spike protein subunit 1 (S1), containing the receptor binding domain (RBD) of SARS-CoV-2, to each of a pair of specific oligonucleotides. The same was done for the nucleocapsid protein (NP). Upon incubation with serum or DBS samples, the bi- or multivalency of the antibodies (IgG, IgA or IgM) brings pairs of viral proteins with their conjugated oligonucleotides in proximity, allowing the antibodies to be detected by a modified proximity extension assay (PEA). Anti-S1 and anti-NP antibodies could be detected simultaneously from one incubation reaction. This Antibody PEA (AbPEA) test uses only 1µl of neat or up to 100,000-fold diluted serum or one ø1.2mm disc cut from a DBS. All 100 investigated sera and 21 DBS collected prior to the COVID-19 outbreak were negative, demonstrating a 100% specificity. The area under the curve, as evaluated by Receiver Operating Characteristic (ROC) analysis reached 0.998 (95%CI: 0.993-1) for samples taken from 11 days after symptoms onset. The kinetics of antibody responses were monitored after a first and second vaccination using serially collected DBS from 14 individuals. AbPEA offers highly specific and sensitive solution-phase antibody detection without requirement for secondary antibodies, no elution step when using DBS sample in a simple procedure that lends itself to multiplex survey of antibody responses.
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| 3. |
- de Oliveira, Felipe Marques Souza, et al.
(författare)
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Detection of post-translational modifications using solid-phase proximity ligation assay.
- 2018
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Ingår i: New biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 45:October, s. 51-59
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Tidskriftsartikel (refereegranskat)abstract
- Post-translational modifications (PTMs) regulate protein activities to help orchestrate and fine-tune cellular processes. Dysregulation of PTMs is often related with disorders and malignancies, and may serve as a precise biomarker of disease. Developing sensitive tools to measure and monitor low-abundant PTMs in tissue lysates or serum will be instrumental for opening up new PTM-based diagnostic avenues. Here, we investigate the use of solid-phase proximity ligation assay (SP-PLA) for detection of different PTMs. The assay depends on the recognition of the target protein molecule and its modification by three affinity binders. Using antibodies and lectins, we applied the method for detection of glycosylated CD44 and E-Cadherin, and phosphorylated p53 and EGFR. The assay was found to have superior dynamic range and limit of detection compared to standard ELISAs. In summary, we have established the use of SP-PLA as an appropriate method for sensitive detection of PTMs in lysates and sera, which may provide a basis for future PTM-based diagnostic and prognostic biomarkers.
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| 4. |
- McGinn, Steven, et al.
(författare)
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New Technologies for DNA analysis-A review of the READNA Project.
- 2016
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784.
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Forskningsöversikt (refereegranskat)abstract
- The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 4 1/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.
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| 5. |
- Mebrahtu, Aman, et al.
(författare)
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Co-culture platform for tuning of cancer receptor density allows for evaluation of bispecific immune cell engagers
- 2024
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 79, s. 120-126
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Tidskriftsartikel (refereegranskat)abstract
- Cancer immunotherapy, where a patient's immune system is harnessed to eradicate cancer cells selectively, is a leading strategy for cancer treatment. However, successes with immune checkpoint inhibitors (ICI) are hampered by reported systemic and organ-specific toxicities and by two-thirds of the patients being non-responders or subsequently acquiring resistance to approved ICIs. Hence substantial efforts are invested in discovering novel targeted immunotherapies aimed at reduced side-effects and improved potency. One way is utilizing the dual targeting feature of bispecific antibodies, which have made them increasingly popular for cancer immunotherapy. Easy and predictive screening methods for activation ranking of candidate drugs in tumor contra non-tumor environments are however lacking. Herein, we present a cell-based assay mimicking the tumor microenvironment by co-culturing B cells with engineered human embryonic kidney 293 T cells (HEK293T), presenting a controllable density of platelet-derived growth factor receptor β (PDGFRβ). A target density panel with three different surface protein levels on HEK293T cells was established by genetic constructs carrying regulatory elements limiting RNA translation of PDGFRβ. We employed a bispecific antibody-affibody construct called an AffiMab capable of binding PDGFRβ on cancer cells and CD40 expressed by B cells as a model. Specific activation of CD40-mediated signaling of immune cells was demonstrated with the two highest receptor-expressing cell lines, Level 2/3 and Level 4, while low-to-none in the low-expressing cell lines. The concept of receptor tuning and the presented co-culture protocol may be of general utility for assessing and developing novel bi-specific antibodies for immuno-oncology applications.
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| 6. |
- Häggmark, Anna, et al.
(författare)
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Classification of protein profiles from antibody microarrays using heat and detergent treatment.
- 2011
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 29:5, s. 564-570
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Tidskriftsartikel (refereegranskat)abstract
- Antibody microarrays offer new opportunities for exploring the proteome and to identify biomarker candidates in human serum and plasma. Here, we have investigated the effect of heat and detergents on an antibody-based suspension bead array (SBA) assay using polyclonal antibodies and biotinylated plasma samples. With protein profiles from more than 2300 antibodies generated in 384-plex antibody SBAs, three major classes of heat and detergent susceptibility could be described. The results show that washing of the beads with SDS (rather than Tween) after target binding lowered intensity levels of basically all profiles and that about 50% of the profiles appeared to be lowered to a similar extent by heating of the sample. About 33% of the profiles appeared to be insensitive to heat treatment while another 17% showed a positive influence of heat to yield elevated profiles. The results suggest that the classification of antibodies is driven by the molecular properties of the antibody-antigen interaction and can generally not be predicted based on protein class or Western blot data. The experimental scheme presented here can be used to systematically categorize antibodies and thereby combine antibodies with similar properties into targeted arrays for analysis of plasma and serum.
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| 7. |
- Jakobsen, L., et al.
(författare)
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Functional proteomics of the human centrosome
- 2010
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Ingår i: New Biotechnology. - : ELSEVIER SCIENCE BV. - 1871-6784 .- 1876-4347. ; 27, s. S82-S82
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 8. |
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| 9. |
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| 10. |
- Carlsson, Fredrika, et al.
(författare)
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Novel antibody specificities targeting glycoprotein B of cytomegalovirus identified by molecular library technology
- 2009
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 25:6, s. 429-436
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies provide some protection against cytomegalovirus-mediated disease. All aspects of antibody recognition of important viral antigens are however not fully appreciated. Glycoprotein B (gB), a key protein in the viral membrane, participates in viral infection and it is a component of prototype vaccines. By using combinatorial antibody library and selection technology, novel antibody specificities targeting gB have now been isolated. We define a monoclonal antibody fragment able to recognize site I of antigenic domain (AD) 2, a poorly immunogenic epitope targeted by potent virus-neutralizing antibodies, in a way that is different from the binding of antibodies induced by infection but similar to those induced by vaccination. We also describe the existence of a novel epitope overlapping site I of AD-2 and AD-1, the immunodominant epitope of gB. These specificities, derived by molecular engineering, will be useful for the future assessment of humoral immune responses against this opportunistic viral infection.
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