Generation of Anti-Boa Immunoglobulin Antibodies for Serodiagnostic Applications, and Their Use to Detect Anti-Reptarenavirus Antibodies in Boa Constrictor

Abstract

Immunoglobulins (Igs), the key effectors of the adaptive immune system, mediate the specific recognition of foreign structures, i.e. antigens. In mammals, IgM production commonly precedes the production of IgG in the response to an infection. The reptilian counterpart of IgG is IgY, but the exact kinetics of the reptilian immune response are less well known. Boid inclusion body disease (BIBD), an often fatal disease of captive boas and pythons has been linked to reptarenavirus infection, and BIBD is believed to be immunosuppressive. However, so far, the study of the serological response towards reptarenaviruses in BIBD has been hampered by the lack of reagents. Thus we set up a purification protocol for boa constrictor IgY and IgM, which should also be applicable for other snake species. We used centrifugal filter units, poly ethylene glycol precipitation and gel permeation chromatography to purify and separate the IgM and IgY fractions from boa constrictor serum, which we further used to immunise rabbits. We affinity purified IgM and IgY specific reagents from the produced antiserum, and labelled the reagents with horseradish peroxidase. Finally, using the sera of snakes with known exposure to reptarenaviruses we demonstrated that the newly generated reagents can be utilised for serodiagnostic purposes, such as immunoblotting and immunofluorescent staining. To our knowledge, this is the first report to show reptarenavirus-specific antibodies in boa constrictors.

Snake deltavirus utilizes envelope proteins of different viruses to generate infectious particles

“…To generate affinity purified anti-UHV NP and anti-SDAg antibodies, we coupled baculovirus expressed recombinant UHV NP 53 and E. coli expressed SDAg 6 to CNBr-Activated Sepharose 4 Fast Flow (GE Healthcare Life Sciences) following the manufacturer’s protocol. We used a protocol described in Korzyukov et al, 2016 54 to affinity purify the antibodies. After dialysis and concentration of the antibodies we labelled the affinity purified fractions using Alexa Fluor 488 TFP ester (ThermoFisher Scientific) or Alexa Fluor 594 NHS Ester (ThermoFisher Scientific) following the manufacturer’s recommendation.…”

“…We performed SDS-PAGE (self-prepared gels and 4–20% Mini-PROTEAN® TGX gels from Bio-Rad) and immunoblotting using methods described in Korzyukov et al, 2016 54 . The antibody dilutions used were 1:10,000 to 1:20,000 for the rabbit anti-SDAg pAb 6 , 1:4,000 for the mouse anti HA-tag mAb (AE008, ABclonal), 1:10,000 for AlexaFluor 680 donkey anti-rabbit (IgG) and anti-mouse (IgG) (ThermoFisher Scientific), and 1:10,000 for IRDye 800CW donkey anti-rabbit (IgG) and anti-mouse (IgG) (LI-COR Biosciences).…”

Serological and molecular findings during SARS-CoV-2 infection: the first case study in Finland, January to February 2020

“…Timeline of events around the first COVID-19 case imported to Finland, January-February 2020 buffer, and Western blotting (WB) of lysates was performed as described previously [4]. At 1:200 dilution, the convalescent serum on Day 20 identified SARS-CoV2 N, S and E protein bands (Figure 3).…”

Nidovirus-Associated Proliferative Pneumonia in the Green Tree Python (Morelia viridis)

“…The purified recombinant protein was used to immunize a rabbit by applying the following scheme: initial immunization on day 0, first booster on day 7, second booster on day 14, third booster on day 42, and a final bleeding on day 49 (BioGenes GmbH, Berlin, Germany; BioGenes adheres to EU and global animal welfare regulations). The generated antiserum was cleaned by affinity purification by a method similar to the methods described previously (68,69). Briefly, 500 g of purified recombinant N protein was dialyzed in PBS, followed by coupling to CNBr-activated Sepharose 4B (GE Healthcare Life Sciences) following the manufacturer's protocol.…”