The hybridomas secreting anti-S1 antibodies, F26G18 and P136.8D12, were fused respectively with the YP4 hybridoma to generate quadromas. and virus tropism. Sensitive diagnosis of SARS is essential for the control of the disease in humans. Recombinant SARS-CoV S1 antigen was produced and purified for the development of monoclonal and bi-specific monoclonal antibodies. The hybridomas secreting anti-S1 antibodies, F26G18 and P136.8D12, were fused respectively with the YP4 hybridoma to generate quadromas. The sandwich ELISA was formed by using F26G18 as a coating antibody and biotinylated F26G18 as a detection antibody with a detection limit of 0.037?g/ml (for 7?min. Two ml of PEG was added to the cell pellet, drop-wise over a period of 2?min, with gentle mixing. After adding PEG, the cell suspension was placed at 37?C in a CO2 incubator for 3?min, followed by addition of 20?ml of FBS free RPMI medium to dilute the toxic effects of PEG. Flow cytometer (Epics Elite cell sorter, Coulter, Hialeah, USA) with an argon ion 488?nm air cooled laser (Cross Cancer Institute, University of Alberta) was then used to sort cells with dual fluorescence and were seeded at 1?cell/well in 96 well sterile tissue culture plates with RPMI containing 20% FBS. The plates were incubated at 37?C with 5% CO2. The clones were screened twice using a direct ELISA (see Section 2.7.2). The cloning of quadromas was carried out using the limiting dilution method. The three clones, P135.3F3/YP4 (P144), P136.8D12/YP4 (P145) and F26G18/YP4 (F157), were then selected for recloning (3C4 times) to select positive and highly specific quadromas secreting bsmAb against the SARS-CoV S1 antigen. 2.7. ELISA techniques KI696 isomer 2.7.1. Indirect ELISA Hybridoma culture supernatants were assayed for binding to the SARS-CoV S1 coated 96 KI696 isomer well plates. Plates were coated with 100?l of purified S1 (5?g/ml) in 50?mM carbonate buffer (pH 9.6) and incubated at 4?C overnight. After thorough washing with PBS-T buffer 5 times, the remaining sites on the well surface were blocked with 200?l KI696 isomer of blocking buffer (3% (w/v) BSA in PBS-T) at 37?C for 1?h. After washing, 100?l of hybridoma supernatants (1:2000 dilution in 1% (w/v) BSA in PBS-T) were added to the wells and incubated at 37?C for 1?h, which was followed by additional washing steps. A volume of 100?l of goat anti-mouse IgG HRPO (1:2000 dilution in 1% (w/v) BSA in PBS-T) was added to the wells and incubated at 37?C for 1?h. The plate was washed again with PBS-T. Positive binding was detected with commercial TMB substrate and the plate was read at 650?nm after 5?min using a value <0.05. 3.?Results 3.1. Expression and purification of the S1 fragment of spike protein The purified S1 was analyzed by SDS-PAGE (Fig. 1A). The specificity was determined by Western blotting, which demonstrated that the anti-SARS-CoV S1 mAb (P136.8D12) binds strongly to the S1 antigen (Fig. 1B). A CD282 similar result was also found with anti-SARS-CoV S1 mAb F26G18 (data not shown). There was no cross-reaction with other viral recombinant proteins including nonstructural proteins derived from Dengue virus or Ebola virus (data not shown). Open in a separate window Fig. 1 Reactivity of anti-SARS-CoV S-protein mAb (P136.812) to SARS-CoV S1. (A) SDS-PAGE analysis. Lane M: standard protein molecular weight markers. Lane 1: SARS-CoV S1. (B) Western blot analysis of S-protein probed with anti-SARS-Cov S1 mAb (P136.812) in lane 1. 3.2. Development of anti-SARS-CoV hybridomas and quadromas Hybridomas producing antibodies for SARS-CoV S1 were established successfully from spleenocytes following immunization with S1 antigen. Among six hybridomas against SARS-CoV S1 antigen, three hybridomas of P135.3F3, P1368D12 and F26G18 showed approximately 5-fold higher titer than those of P147.4R4, P147.2R8 and P147.2R16. Table 1, Table 2 show the isotypes of SARS-CoV S1 mAbs and the yield of SARS-CoV S1 purified mAbs, respectively. Table 1 Anti-SARS-CoV spike protein mAbs.
F26G180.813?ml (10?mg)P135.3F30.713?ml (9.1?+?3?=?12?mg)P136.8D120.6810?ml (6.8?mg) Open in a separate KI696 isomer window Table 2 Isotyping of anti-SARS-CoV spike protein mAbs by ELISA.
P135.3F30.0940.0980.0980.717P136.8D120.1660.1770.8090.206F26G180.2170.2200.7570.161 Open in a separate window The hybridomas secreting anti-S1 mAb namely F26G18 was fused respectively with YP4 hybridoma to produce quadromas.