Obtention, purification and characterization of IgY polyclonal antibodies, developed in hens, directed against Colombian isolates of Giardia duodenalis.

Abstract

Introduction. The development of polyclonal antibodies requires laboratory animals, as the usually rabbits, which are bled for obtaining the immunoglobulins. Hens are an alternative to developing IgY polyclonal antibodies.
Objective. In the current study, hens were used to develop IgY anti-Giardia duodenalis antibodies and to evaluate five protocols for its purification.
Materials And Methods. Three hens were immunized intramuscularly immunized with Colombian Giardia duodenalis isolates trophozoites on day 0, 15, 30, 45, 60, 90 and 120. The hen eggs were collected before each immunization, and IgY purified from yolk by delipidation (D) and precipitation (P) using five different protocols: M1: (P: ammonium sulfate/D:dextran sulfate-calcium chloride), M2: (D: dextran sulfate-calcium chloride/P: ammonium sulfate), M3: (D: chloroform/P: ammonium sulfate 50%), M4: (D: solution A/P: solution B) and M5: (D: chloroform/P: ammonium sulfate 30%). The immunochemistry evaluation of IgY anti-Giardia duodenalis was assayed by immunodiffusion, counterimmunoelectrophoresis (CIE) and Western blot. The purity of IgY was assayed by SDS-PAGE under reducing and nonreducing conditions. Immunoglobulin concentration (mg/mL) was estimated by spectrophotometry and densitometry.
Results. IgY anti-Giardia duodenalis by CIE had titres up to 1:32. SDS-PAGE, without 2-mercaptoethanol showed a 180 kd band characteristic of the whole to IgY and, with 2-mercaptoethanol, two bands of 68 and 30 kd, characteristic of its light and heavy chains, respectively. The greatest concentrations of immunoglobulin were recovered by method two (M2), producing 4.6 mg of IgY per mL of initial egg yolk.
Conclusions. The easy and inexpensive production of IgY anti-Giardia duodenalis in hens is an advantage for using it to develop immunoassays that detect the parasite in fecal eluates.