Lyophilization as an alternative for conservation of equine plasma as a source of IgG for neonatal foals

The administration of plasma to foals with failed transfer of passive immunity is of utmost importance. Transporting refrigerated or frozen plasma to farms can be costly and complicated. Lyophilized plasma can facilitate transport and storage by improving and spreading the technology. Lyophilization is the most commonly used method for preparing dehydrated proteins, which must have adequate stability for an extended storage period at room temperature. This work aimed to evaluate the IgG concentration of fresh equine plasma (EP) and post-lyophilization. Plasma was collected from 6 healthy male horses, age 4±2 years, 356±62kg. An aliquot of EP was lyophilized. The lyophilization process was carried out at the Research Support Center of the Federal Rural University of Pernambuco. Before being lyophilized, samples were frozen at -80°C for 24 h and then processed, in an Alpha 1-4 LD Plus Christ apparatus, a drying chamber temperature of -27°C and vacuum of 0.52 mbar, for 24 hours. The lyophilized EP obtained was stored in well-sealed microtubes and frozen at -4°C for further analysis. The lyophilized EP was resuspended in deionized water to maintain the original volume. The concentration of IgG in fresh EP and lyophilized and resuspended EP was measured. Samples were evaluated using Simple Radial Immunodiffusion kits (IDRS) commercially available forequine serum and colostrum: IDRING® Horse IgG Test, by IDBiotech (Issoire/France). Horse IgG test plates contain an agar gel with a specific antiserum against horse IgG. Plate wells are filled with standards of 200, 100, 50, and 25 μg/mL of horse IgG and the EP and lyophylised EP samples. During diffusion on the agar gel, the antibodies specifically react with the antigen to form a precipitation ring. A linear calibration curve is established to determine the samples’ IgG concentrations from the measured diameters to known concentrations in the standard calibration set. The homogeneity of the variance between treatments was verified by the Shapiro-Wilk test. The results were subjected to analysis of variance (ANOVA) to determine the effects of lyophilization on the IgG concentrations. Significance was declared at P<0.05. When significant differences were tested using the Tukey, significance was set at P<0.05. The IgG concentration in fresh plasma was 8.9±3.2 g/L; after lyophilization, the plasma showed an IgG concentration of 7.1±2.2 g/L. No significant differences were found in the plasma IgG concentrations (P>0.05). The results suggest that lyophilization can be an alternative for conserving EP, since it is a very efficient process to maintain IgG concentration. Further studies should be carried out regarding the administration of plasma in the foal and the transfer of immunity.