In vitro neutralization of viral hemorrhagic septicemia virus by plasma from immunized zebrafish.

We studied humoral long-term adaptive viral neutralization responses in zebrafish (Danio rerio), an increasingly useful vertebrate model for viral diseases actually limited by the absence of standardized anti-zebrafish immunoglobulin M (IgM) antibodies. We established an alternative method, similar to those used in other fish, to achieve a first estimation of zebrafish anti-viral antibody-like responses. We used the viral hemorrhagic septicemia virus (VHSV) model because, although protection after this non-natural infection was demonstrated in cold-acclimatized zebrafish, little is known about their induced anti-VHSV antibody-like responses. Therefore, we first optimized a micro-neutralization method based on immunostaining VHSV-infected fish cell monolayers to detect zebrafish neutralizing activity in plasma samples in one day. We then used the method to measure the specific anti-VHSV neutralization in plasma obtained from individual zebrafish under various VHSV challenges or immunization protocols. The neutralizing activity was inhibited by protein A-sepharose and rabbit anti-zebrafish IgM antibodies, suggesting the implication of IgM zebrafish antibodies in such responses. To our knowledge, this is the first report to demonstrate detectable and significant VHSV neutralization titers in zebrafish surviving VHSV infections. This micro-method might be useful, not only for the follow-up of infection/vaccine development in the zebrafish/VHSV model in particular, but also for similar work involving other in vitro neutralizable zebrafish pathogens. This technique might also further the development of alternative ELISA assay methods to measure specific immunoglobulins in zebrafish.

Biocompatibility of a novel avidin-agarose adsorbent for extracorporeal removal of redundant radiopharmaceutical from the blood.

The use of monoclonal antibodies (MAbs) in cytotoxic conjugates (radionuclides, toxins, or drugs) for targeting tumor cells is restricted due to toxicity in vital organs. Through improved tumor targeting, it is possible to administer larger amounts of such labeled MAbs, thus improving the ability to eradicate tumor cells without increased normal organ toxicity. Extracorporeal affinity adsorption treatment (ECAT) has therefore been developed using an avidin-agarose (AA) adsorbent with high binding affinity for the biotinylated radiolabeled MAb, rituximab. During ECAT, excess radioimmunoconjugates, not bound to the tumor cells, can be removed improving tumor targeting. The present study was performed to estimate the biocompatibility of the AA adsorber. Seven patients with B-cell lymphoma not responding to conventional treatment were studied. During the ECAT procedure, blood (B) components, plasma (P) complement fragments C3a, C5a, and P-bradykinin were analyzed, and other laboratory tests were carried out. Slight decreases in B-hemoglobin (8.3%), B-thrombocytes (11.4%), and P-albumin (14.3%) were observed, and could be explained by the dilution of the blood with normal saline and acid citrate dextrose. The AA adsorbent had no effect on the blood cells, immunological status or P-bradykinin level. The AA adsorber demonstrated good hemocompatibility and biocompatibility, without any side effects in the patients.