Journal: Viruses

Article Title: Urine and Free Immunoglobulin Light Chains as Analytes for Serodiagnosis of Hantavirus Infection

doi: 10.3390/v11090809

Figure Lengend Snippet: Western blot analysis of urine samples. Urine samples (30 µL/lane) separated in SDS-PAGE under non-reducing conditions were blotted onto nitrocellulose and sequentially immunoblotted with anti-lambda and anti-kappa light chain antibodies. Patients #1–#4 are included in . The panels on left show the results for anti-lambda light chain staining (probed first, detected using IR800-conjugated secondary antibody) and the panels on right show anti-kappa light chain staining (detected using AF680-conjugated secondary antibody). Molecular weight markers (Bio-Rad, precision plus protein dual color standards) are always the leftmost lane. FLC (free light chain) indicates monomeric and FLC 2 dimeric FLCs. All detections were performed using an Odyssey Infrared Imaging System (LI-COR Biosciences).

Article Snippet: We coupled mouse monoclonal anti-kappa (clone 4C11) and anti-lambda (clone 3D12) free light chain antibodies (both from HyTest Ltd., Turku, Finland) to Pierce NHS-activated Magnetic Beads (Thermo Fisher Scientific, Vantaa, Finland) following the manufacturer’s protocol with 400 μg of antibody per 500 μL of activated bead slurry.

Techniques: Western Blot, SDS Page, Staining, Molecular Weight, Imaging

Journal: Viruses

Article Title: Urine and Free Immunoglobulin Light Chains as Analytes for Serodiagnosis of Hantavirus Infection

doi: 10.3390/v11090809

Figure Lengend Snippet: Immunoprecipitation (IP) of PUUV N protein using FLCs and purification of free kappa light chains from urine: ( A ) Monoclonal antibodies against free kappa (clone 4C11) and lambda (3D12) light chains were conjugated to Pierce NHS-activated magnetic beads (Thermo Fisher Scientific) and used for IP of FLCs and PUUV N protein. The left lanes show anti-kappa IP and the right lanes anti-lambda IP results of AF647-labeled PUUV N protein. The samples are indicated above each lane (u stands for urine and p for plasma); the PUUV+ pools were represented by samples collected during hospitalization. The bound PUUV N protein was visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences) at IR700 channel after SDS-PAGE separation; M represents the molecular weight marker (Bio-Rad, precision plus protein dual color standards); ( B ) The experimental setup described in panel A was used for IP of AF647-labeled PUUV with FLCs from the urine and plasma of healthy volunteers and PUUV patients (three time points). The left panel shows the results of IP with anti-kappa coated beads and the right panel IP with anti-lambda coated beads. The samples are indicated above each lane (u stands for urine and p for plasma) ( C ) Eluates (20 μL/lane) from monoclonal (clone 4C11) free kappa light chain antibody coupled CNBr-activated Sepharose 4B columns after passing through urine from patients with acute PUUV infection (PUUV+, 2 mL) and healthy volunteers (PUUV−, 15 mL) were analyzed by western blotting using a polyclonal anti-kappa light chain antibody. Detection was performed used an Odyssey Infrared Imaging System (LI-COR Biosciences), M represents the molecular weight marker (Bio-Rad, precision plus protein dual color standards).

Article Snippet: We coupled mouse monoclonal anti-kappa (clone 4C11) and anti-lambda (clone 3D12) free light chain antibodies (both from HyTest Ltd., Turku, Finland) to Pierce NHS-activated Magnetic Beads (Thermo Fisher Scientific, Vantaa, Finland) following the manufacturer’s protocol with 400 μg of antibody per 500 μL of activated bead slurry.

Techniques: Immunoprecipitation, Purification, Bioprocessing, Magnetic Beads, Labeling, Clinical Proteomics, Imaging, SDS Page, Molecular Weight, Marker, Infection, Western Blot