Anti-Ebola MAb 17A3 reacts with bovine and human alpha-2-macroglobulin proteins

Abstract

Monoclonal antibodies (MAbs) were developed against soluble Ebola virus (EBOV) envelope glycoprotein (GP) for the study of the diversity of EBOV envelope and development of diagnostic reagents. Of the three anti-EBOV GP mouse MAbs produced, MAb 15H10 recognized all human EBOV GP species tested (Zaire, Sudan, Ivory Coast), and as well as reacted with the Reston nonhuman primate EBOV GPs. A second MAb, 6D11 recognized EBOV GP species of Sudan and Sudan-Gulu. The third MAb, 17A3, was reported originally in the same article to be EBOV GP specific has now been found to be specific for bovine and human alpha-2-macroglobulin (alpha-2M) proteins which were contaminants in the Ebola envelope protein preparation. Thus, while MAbs 15H10 and 6D11 are indeed EBOV GP specific, MAb 17A3 is an alpha-2-macroglobulin MAb.

Figure 1. Detection of α-2M in the purified recombinant glycoprotein preparations of both Ebola Sudan-Gulu and Zaire strains

The recombinant glycoprotein preparations of both Ebola Sudan-Gulu and Zaire strains that were prepared as described in the original report and the purified bovine and human α-2M proteins were fractionated on 4-20% gradient SDS-PAGE under reducing and non-reducing conditions. Shown are Western blots under reducing (Lanes 1 to 4) and non-reducing conditions (Lanes 5-8); M= Molecular markers; lanes 1 and 5= purified Ebola envelope protein of Sudan-Gulu strain; lanes 2 and 6= purified Ebola envelope protein of Zaire strain; lanes 3 and 7= purified bovine α–2M proteins; and lanes 4 and 8= human α–2M proteins. Anti-α-2M MAb reactivity with the EBOV GP preparations and bovine and human α-2M proteins is shown in panel A, while MAb 17A3 lack of reactivity is shown in panel B. While the anti-alpha-2-macroglobulin antibody reacted with alpha-2-macroglobulins in both Ebola GP preparations as well as with purified bovine and human alpha-2-macroglobulin, MAb 17A3 only reacted weakly with human alpha-2-macroglobulin.

Figure 2. Reactivity of MAb17A3 with bovine and human α-2M proteins

MAb 17A3 reacted the purified bovine (Figure 2A) and human (Figure 2B) α-2M proteins in ELISA. Anti-human α-2M MAb was used as the positive control. An irrelevant MAb, 7E5, was used as the negative control. Open circle indicates the negative MAb 7E5, closed square indicates MAb 17A3, and closed circle is anti-human alpha-2M MAb.

Figure 3. Native gel analysis of glycoproteins of Ebola virus Sudan-Gulu and Zaire

Ebola glycoproteins were fractionated on 3-8% gradient Tris-acetate gel under non-reducing conditions. Ebola virus Sudan-Gulu and Zaire glycoproteins are shown in both Coomassie staining gels and western blot analysis. Similar to Western blot analysis in SDS-PAGE, anti-α 2M MAb detected Ebola glycoproteins bands that MAb 17A3 also detected while MAbs 6D11 and 15H10 did not detect any protein bands. The arrow indicates those bands cut for finding the identity by mass spectroscopy analysis. Lane S indicates the purified Ebola envelope protein of Sudan-Gulu, and Z denotes purified Ebola envelope protein of Zaire strain.