Highly sensitive immunoassay for direct diagnosis of viral hemorrhagic septicemia which uses antinucleocapsid monoclonal antibodies.

An antigen capture enzyme-linked immunosorbent assay (ELISA) based on the detection of the viral nucleocapsid (anti-N system) was developed for the diagnosis of viral hemorrhagic septicemia. Four monoclonal antibodies directed against the viral nucleocapsid were produced; they all recognized the four viral hemorrhagic septicemia virus (VHSV) serotypes. Three of these monoclonal antibodies were used in a new antigen capture ELISA. The efficiency of the anti-N system in detecting purified and crude viruses as well as the virus in infected-organ extracts and infected blood was compared with that of a recently described antigen capture ELISA based on the detection of viral envelope glycoprotein Gp (anti-G system). For the detection of purified virus, the anti-N system was found to be as sensitive as the anti-G system (detection limit, 1 ng of total viral protein per ml), but the anti-N system was much more sensitive than the anti-G system for the detection of crude VHSV I (detection limits, 1 x 10(4) PFU/ml versus 5 x 10(5) PFU/ml). In organ extracts, VHSV I could be detected by both systems 3 days postinfection. The signal for the assay of VHSV I in blood 24 h postinfection was higher with the anti-N system than the anti-G system. Furthermore, VHSV I could be detected in 80% of the brain samples of surviving trout by the anti-N system and also by the anti-G system, but with a lower signal. In conclusion, we have developed a highly sensitive immunoassay for VHSV I that is more rapid and easier to perform than the currently used plaque assay.

Novel anti-digoxin monoclonal antibodies with different binding specificities for digoxin metabolites and other glycosides.

Spleen cells of BALB/c mice immunized with a digoxin-bovine serum albumin conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Seven monoclonal antibodies (MAb) selected by indirect ELISA were produced, purified and characterized. All the MAb were IgG1 isotypes. The apparent equilibrium association constants (Ka) of four of the MAb, determined by Scatchard analysis of the RIA data, ranged from 1 x 10(9) M-1 to 5.9 x 10(9) M-1. The estimated Ka values of the three other MAb were found to be between 4.8 x 10(7) M-1 and 5.9 x 10(8) M-1. Using digoxin and eighteen structurally-related compounds, the seven MAb could be divided into five groups based on their binding specificities assessed by an inhibition immunoenzymatic test. The MAb in Groups I and II, in particular, showed very different specificity profiles: the two MAb in Group I had low cross-reactivity with cardioinactive digoxin metabolites, whereas the high affinity MAb in Group II recognized all the digoxin metabolites tested. The MAb in Group I might be useful in a digoxin immunoassay and the Group II MAb in therapeutic reversal of digoxin intoxication.

Antigen-capture ELISA for viral haemorrhagic septicaemia virus serotype I.

An antigen-capture ELISA for viral haemorrhagic septicaemia virus serotype I (VHSV I) was developed. The assay employs two monoclonal antibodies (mAb) directed against distinct epitopes of the viral envelope glycoprotein (Gp). The antigen bound by the capture mAb (A17) was detected by addition of a second mAb (L7) conjugated to horseradish peroxidase, followed by addition of the enzyme substrate. The technique is highly sensitive, enabling detection of the virus at a protein concentration as low as 1 ng/ml of total proteins (1.5 fmol of envelope Gp per ml) in purified preparations. VHSV I was also detected in culture supernatants (5 x 10(5) PFU/ml) and in extracts of kidney and spleen of rainbow trout infected experimentally (5 x 10(5) PFU/ml). The assay was highly specific: infectious haematopoietic necrosis virus, infectious pancreatic necrosis virus, spring viraemia of carp virus, pike fry rhabdovirus, eel rhabdovirus and perch rhabdovirus could not be detected by the antigen-capture ELISA for VHSV I.