One-step fast gradient method for nanoantibody generation

Abstract

One-step rapid gradient method for rapid generation of nanoantibodies (HHV) against any antigen. Where the method comprises the separation of nanoantibodies against a specific antigen from a nanoantibody expression library through the following steps: (a) binding the antigen of interest to beads made of protein-binding polymers (b) incubating the microorganisms of the expression library with the pearls of stage (a), (c) placing the beads of step (b) in a tube with an inert medium with a density greater than or equal to 1 g/mL and centrifuge for 45 s to 2 minutes at a rate between 150 and 250 g; d) discarding the upper fraction and supernatant with the free microorganisms, and obtain the beads with the linked microorganisms, which correspond to those that express HHV that recognizes the antigen.

1 . Method of separation of nanoantibodies (HHV) against a specific antigen from a nanoantibody expression library CHARACTERIZED in that it comprises the following steps:
 (a) binding the antigen of interest to beads made of protein-binding polymers   (b) incubating the microorganisms of the expression library with the beads of step (a), where the expression library corresponds to microorganisms transformed with the cDNA of fragments corresponding to the HHV domains of HHV-producing animal previously immunized with that antigen of interest, or synthetic libraries;   (c) placing the beads of step (b) in a tube with an inert medium with a density greater than or equal to 1 g/mL and centrifuge for 45 s to 2 minutes at a rate between 150 and 250 g;   d) discarding the upper fraction and supernatant with the free microorganisms, and obtaining the beads with the linked microorganisms, which correspond to those that express HHV that recognizes the antigen.   
     
     
         2 . Method according to  claim 1  CHARACTERIZED in that in step (a) the beads are sepharose, cellulose, latex, agarose or nickel and are modified to bind proteins. 
     
     
         3 . Method according to  claim 2  CHARACTERIZED in that in the beads have a density greater than the medium used in (c). 
     
     
         3 . Method according to  claim 1  CHARACTERIZED in that in step (c) the inert medium is reactive Ficol, percol or sucrose. 
     
     
         4 . Method according to  claim 3  CHARACTERIZED in that in step (a) the beads or equivalents bind stably to the antigen of interest in a time of between 2 to 12 hours. 
     
     
         5 . Method according to  claim 4  CHARACTERIZED in that sites which have not reacted with the antigen are blocked. 
     
     
         6 . Method according to  claim 1  CHARACTERIZED in that the microorganisms of the expression library are treated with a protein expression inducer for 2 to 4 hours prior to step (b). 
     
     
         7 . Method according to  claim 6  CHARACTERIZED in that the protein expression inducer is Isopropyl-β-D-1-tiogalactopyranoside (IPTG) at a concentration between 20 to 100 μM. 
     
     
         8 . Method according to  claim 1  CHARACTERIZED in that in step (b) is incubated for between 20 to 60 minutes at room temperature. 
     
     
         9 . Method according to  claim 1  CHARACTERIZED in that in step (d) the beads are washed with PBS and seeded on agar plates with culture medium in order to obtain isolated colonies. 
     
     
         10 . Method according to  claim 9  CHARACTERIZED in that each colony corresponds to a microorganism expressing an HHV capable of binding to the antigen of interest.