Use of monoclonal antibodies against Hendra and Nipah viruses in an antigen capture ELISA

Abstract

Background: Outbreaks of Hendra (HeV) and Nipah (NiV) viruses have been reported starting in 1994 and 1998, respectively. Both viruses are capable of causing fatal disease in humans and effecting great economical loss in the livestock industry.

Results: Through screening of hybridomas derived from mice immunized with gamma-irradiated Nipah virus, we identified two secreted antibodies; one reactive with the nucleocapsid (N) protein and the other, the phosphoprotein (P) of henipaviruses. Epitope mapping and protein sequence alignments between NiV and HeV suggest the last 14 amino acids of the carboxyl terminus of the N protein is the target of the anti-N antibody. The anti-P antibody recognizes an epitope in the amino-terminal half of P protein. These monoclonal antibodies were used to develop two antigen capture ELISAs, one for virus detection and the other for differentiation between NiV and HeV. The lower limit of detection of the capture assay with both monoclonal antibodies was 400 pfu. The anti-N antibody was used to successfully detect NiV in a lung tissue suspension from an infected pig.

Conclusion: The antigen capture ELISA developed is potentially affordable tool to provide rapid detection and differentiation between the henipaviruses.

Figure 1

Characterizations of antibodies produced by hybridomas. (A) The 4-12% gradient gels were loaded with cell lysate equivalent to 2 μg of protein in each lane as follows, lane 1 and 2 (also lane 6 and 7) represent 2 preparations of NiV Malaysia infected Vero cell lysates; lane 3 and 8, NiV Bangladesh infected Vero cell lysate; lane 4 and 9, HeV infected Vero cell lysate; lane 5 and 10, control Vero cell lysate. After gel separation and transferring, membranes were probed with culture supernatant from 1A11 and 2B10. (B) 293T cells were transfected with NiV Malaysia P, V, W, C or N proteins expressed from plasmids. Ten μL of each cell lysate was separated by SDS-PAGE. Monoclonal antibodies purified from cloned hybridomas were diluted 1 to 2000 and incubated with transferred membranes. Lane M, ladder of MagicMark™ XP Western Protein Standard from Invitrogen.

Figure 2

Epitope mapping of mAb 1A11 C1 using direct ELISA. Synthetic peptides corresponding to the complete NiV N protein sequence (a.a. 1-532) were serial diluted and coated at concentration from 1 μg to 0.1 ng per well (100 μL in volume). A peptide from Alkhurma virus E protein (Alk E, a.a. 143-168) was included as negative control and signal cutoff value (0.17) was calculated based on readings from this peptide. NiV infected Vero lysate diluted 10 fold to 10⁵ fold were served as positive control.

Figure 3

Diagram of antibody epitopes on NiV N protein sequence. In addition to the epitope of mAb 1A11 C1, linear epitope mappings were performed with a panel of polyclonal antibodies on NiV N peptides: NiV and HeV hyperimmune mouse ascites fluid (HMAF), Rabbit anti-HeV serum, NiV infected human convalescent serum and a pool of NiV seropositive swine sera. The boxes with illustrated patterns represent the degree of interaction on direct ELISA, and their a.a. positions in N protein sequence were also indicated above.

Figure 4

Antigen capture ELISAs for the detection of NiV or HeV from infected cell lysate. Serial dilutions of (A) NiV Malaysia prototype, (B) NiV Bangladesh or (C) HeV infected Vero cell lysate was tested with mAbs 1A11 C1 and 2B10 p4 on antigen capture ELISA. Marburg virus (MHF) HMAF was included as negative antibody control. Data points represent the means ± standard deviations from 5 replicates. Signal cutoff values were calculated based on uninfected Vero cell lysate controls.

Figure 5

Sensitivities of antigen capture ELISAs for titrated NiV and HeV stocks. (A) NiV Malaysia prototype stock (4.1 × 10⁶ pfu/mL) and (B) HeV stock (1.9 × 10⁶ pfu/mL) were serial diluted onto wells coated with 1A11 C1 and 2B10 p4. Marburg virus (MHF) HMAF was included as negative antibody control and signals above this antibody control were shown (Adjusted OD). Lassa virus stock (1 × 10⁸ pfu/mL) was used as negative virus control and signal cutoff value was calculated based on its OD readings.