| Summary: | Crotoxin complex CA/CB and crotamine are the main toxins associated with <i>Crotalus</i> envenomation besides the enzymatic activities of phospholipases (PLA<sub>2</sub>) and proteases. The neutralization at least of the crotoxin complex by neutralizing the subunit B could be a key in the production process of antivenoms against crotalids. Therefore, in this work, a Crotoxin B was recombinantly expressed to evaluate its capacity as an immunogen and its ability to produce neutralizing antibodies against crotalid venoms. A Crotoxin B transcript from <i>Crotalus tzabcan</i> was cloned into a pCR<sup>®</sup>2.1-TOPO vector (Invitrogen, Waltham, MA, USA) and subsequently expressed heterologously in bacteria. HisrCrotoxin B was extracted from inclusion bodies and refolded in vitro. The secondary structure of HisrCrotoxin B was comparable to the secondary structure of the native Crotoxin B, and it has PLA<sub>2</sub> activity as the native Crotoxin B. HisrCrotoxin B was used to immunize rabbits, and the obtained antibodies partially inhibited the activity of PLA<sub>2</sub> from <i>C. tzabcan</i>. The anti-HisrCrotoxin B antibodies neutralized the native Crotoxin B and the whole venoms from <i>C. tzabcan, C. s. salvini</i>, and <i>C. mictlantecuhtli.</i> Additionally, anti-HisrCrotoxin B antibodies recognized native Crotoxin B from different <i>Crotalus</i> species, and they could discriminate venom in species with high or low levels of or absence of Crotoxin B.
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