免疫学

The COVID-19 pandemic led to the rapid development of antibody-based therapeutics and vaccines targeting the SARS-CoV-2 spike protein. Several antibodies have been instrumental in protecting vulnerable populations, but their utility was limited by the emergence of spike variants with diminished susceptibility to antibody binding and neutralization. Moreover, these spike variants exhibited reduced neutralization by polyclonal antibodies in vaccinated individuals. Accordingly, the characterization of antibody binding to spike variants is critical to define antibody potency and understand the impact of amino acid changes. A key challenge in this effort is poor spike stability, with most current methods assessing antibody binding using individual domains instead of the intact spike or variants with stabilizing amino acid changes in the ectodomain (e.g., 2P or HexaPro). The use of non-native spike may not accurately predict antibody binding if changes lie within the epitope or alter epitope accessibility by altering spike dynamics. Here, we present methods to characterize antibody affinity for and activity against unmodified SARS-CoV-2 spike protein variants displayed on a mammalian cell membrane that recapitulates the native spike environment on infected cells. These include a flow cytometry–based method to determine the effective antibody binding affinity (K_D) and an antibody-dependent cellular cytotoxicity (ADCC) assay to assess Fc-mediated activities. These methods can readily evaluate antibody activity across a panel of spike variants and contribute to our understanding of spike/antibody co-evolution.

PD-1 is an immune checkpoint on T cells. Antibodies to PD-1 or its ligand PD-L1 are gaining popularity as a leading immunotherapy approach. In the US, 40% of all cancer patients will be treated with anti-PD-1 or anti-PD-L1 antibodies but, unfortunately, only 30% will respond, and many will develop immune-related adverse events. There are nine FDA-approved anti-PD-1/PD-L1 antibodies, and approximately 100 are in different stages of clinical development. It is a clinical challenge to choose the correct antibody for a given patient, and this is critical in advanced malignancies, which often do not permit a second-line intervention. To resolve that, an in vitro assay to compare the performance of the different anti-PD-1/PD-L1 antibodies is not only a critical tool for research purposes but also a possible tool for personalized medicine. There are some assays describing the binding affinity and function of anti-PD-1/PD-L1 antibodies. However, a significant limitation of existing assays is that they need to consider the location of PD-1 in the immune synapse, the interface between the T cell and tumor cells, and, therefore, ignore a critical component in its biology. To address this, we developed and validated an imaging-based assay to quantify and compare the ability of different anti-PD-1/PD-L1 antibodies to remove PD-1 from the immune synapse. We correlated that with the same antibodies' ability to increase cytokine secretion from the targeted cells. The strong correlation between PD-1 location and its function in vitro and in vivo within the antibody treatment setting validates this assay's usability, which is easily recordable and straightforward.

Surface Plasmon Resonance(SPR) is a label-free optical technique to assess protein–protein interaction kinetics and affinities in a real-time setting. Traditionally, Biacore SPR employs a continuous film of gold to detect any change in the angle of re-emitted light when the refractive index of a ligand conjugated to the flat gold surface is altered by its interaction with a local analyte. In contrast, the Nicoya Lifesciences’ OpenSPR technology uses gold nanoparticles to detect small changes in the absorbance peak wavelength of a conjugated ligand after its engagement by an analyte. Specifically, when broadband white light is shone onto the gold nanoparticles, it produces a strong resonance absorbance peak corresponding to the refractive index of a ligand conjugated to the surface of gold nanoparticles. Upon its interaction with an analyte, however, the absorbance wavelength peak of the conjugated ligand will be changed and timely recorded as sensorgrams of dynamic ligand–analyte interactions. Thus, the improvement in the detection method (from traditional detection of changes in the angle of re-emitted light to the contemporary detection of changes in the wavelength of the absorbance peak) features OpenSPR as a cost-effective and user-friendly technique for in-depth characterization of protein–protein interactions. Here, we describe the detailed method that we used to characterize procathepsin L (pCTS-L) interactions with two putative pattern recognition receptors (TLR4 and RAGE) using the 1st generation of Nicoya Lifesciences’ OpenSPR instrument with a 1-channel detection.

Key features

• Nicoya OpenSPR is a benchtop small-size equipment that provides in-depth label-free binding kinetics and affinity measurement for protein–protein interactions in real-time fashion.

• This technology is relatively intuitive and user-friendly for scientists at any skill level.

• OpenSPR sensors employ nanotechnology to reduce the cost of manufacturing complex optical hardware and Sensor Chips, and similarly reduce the consumption of precious analyte samples.

• The manufacturer provides online training for OpenSPR (Catalog: TRAIN-REMOTE) and TraceDrawer (Catalog: TRAIN-TD) to customer scientists.

The recombinant receptor-binding domain (RBD) of the viral spike protein from SARS-CoV-1 and 2 are reliable antigens for detecting viral-specific antibodies in humans. We and others have shown that the levels of RBD-binding antibodies and SARS-CoV-2 neutralizing antibodies in patients are correlated. Here, we report the expression and purification of properly folded RBD proteins from SARS and common-cold HCoVs in mammalian cells. RBD proteins were produced with cleavable tags for affinity purification from the cell culture medium and to support multiple immunoassay platforms and drug discovery efforts.

Graphic abstract:

High-Yield Production of Viral Spike RBDs for Diagnostics and Drug Discovery

Human leukocyte antigen class I (HLA-I) molecules are a group of structurally-related cell surface proteins with a high degree of variability within the population. While only up to six variants are expressed in an individual person, the whole population contains thousands of different variants. The ability to distinguish specific variants is important in the clinic to determine compatibility during organ and bone marrow transplantation and in the laboratory to study the biological properties of individual variants. Solid phase bead arrays contain purified, individually identifiable HLA-I molecules that can be used to determine antibody specificity for individual HLA-I proteins. This method is high-throughput, highly specific, and allows for simultaneous screening of antibodies against multiple HLA-I allotypes. The beads are particularly useful for screening patient sera for the presence of donor-specific antibodies against individual HLA-I variants (which can arise during pregnancy, blood transfusion, or organ transplantation). Alternate approaches, such as the use of individual HLA-I-expressing cell lines, are more time consuming, and such cell lines are difficult to procure and standardize. The HLA-I beads are also useful to study HLA-I specificity and selectivity for other receptors and binding partners.

Non-infectious virus-like particles (VLPs) containing dengue virus (DENV) pre-membrane (prM) and envelope (E) proteins have been demonstrated to be highly immunogenic and can be used as a potential vaccine candidate as well as a tool for serodiagnostic assays. Successful application of VLPs requires abundant, and high-purity production methods. Here, we describe a robust protocol for producing DENV VLPs from transiently-transformed or stable COS-1 cells and further provide an easily adaptable antigen purification method by sucrose gradient centrifugation.

Membrane proteins such as cytokine receptors and G protein-coupled receptors can be drug targets. Recently, we have generated specific monoclonal antibodies (mAbs) against the mouse IL-9 receptor (IL-9R) and found that IL-9R on memory B cells have critical roles in T-dependent immune response. So far, most antibodies against cell surface proteins have been generated by immunization of animals with recombinant proteins produced in Escherichia coli (E. coli) or peptides derived from the protein. However, such antibodies often fail to recognize native proteins on cell surfaces because these antigens lack posttranslational modification and natural protein conformations. To circumvent such problems, we have developed a mouse immunization method, the DNA-immunization utilizing hyaluronidase and E. coli GroEL. Herein, we report an application of the original mouse immunization method in rats to generate anti-mouse IL-9R mAbs which could react with the native form of mouse IL-9R on cell surfaces. Thus, we suggest that the DNA-immunization method is feasible for generating monoclonal antibodies against cell surface proteins in rats.

Kinetic analysis of antibodies is one of the important studies for characterization of antibodies and screening of ligands. In our recent study (Ingale et al., 2014), we compared the antigenic profiles and binding characteristics of four HIV-1 envelope glycoprotein (Env) core immunogens using multiple monoclonal antibodies by Bio-Layer Light Interferometry (BLI). This technology enables real-time analysis of interactions on the surface of a fiber optic biosensor by accurately measuring kinetic constants such as K_a, K_d, and K_D in a 96-well format.

The fluorescence-linked antigen quantification (FLAQ) assay allows a fast quantification of HIV-1 p24^Gag antigen. Viral supernatant are lysed and incubated with polystyrene microspheres coated with polyclonal antibodies against HIV-1 p24^Gag and detector antibodies conjugated to fluorochromes (Figure 1). After washes, the fluorescence of microspheres is measured by flow cytometry and reflects the abundance of the antigen in the lysate. The speed, simplicity, and wide dynamic range of the FLAQ assay are optimum for many applications performed in HIV-1 research laboratories.

Surface Plasmon Resonance (SPR) is widely used to generate kinetic and affinity information on specific interactions between biomolecules. This technique is label-free and monitors the binding event in real-time. It is generally used for characterization of monoclonal antibody - antigen interactions. This protocol describes specifically the use of SPR with a Biacore T100 instrument to measure the affinity of crude hybridoma samples to a protein. For that purpose an anti-IgG antibody was firstly covalently immobilized onto a CM5 chip by amide coupling (Canziani et al., 2004; Schraml and Biehl, 2012). Then the antibodies from hybridoma supernatants were captured non-covalently onto the surface via their Fc region providing an optimal analyte-binding orientation. Finally, the resulting complex was stabilized by crosslinking with EDC/NHS to avoid baseline drift during measurement and regeneration (Pope et al., 2009). Then the interaction with the protein was monitored at several concentrations and its affinity towards the immobilized antibodies was determined with the corresponding K_D obtained from classical kinetics analysis. This set-up avoids the avidity effects of the bivalent antibodies, allows the use of non-purified analytes with unknown concentrations and the specific capture of the antibodies in a similar stable covalent-orientated manner.