Objective. In early life, colostrum Immunoglobulins (Igs) absorp&on is cri&cal for the protec&on of the newborn ruminants from infec&ous diseases (Sawyer et al., ...
Comparison of a routine Capillary Electrophoresis method and Single Radial Immunodiffusion assay to measure immunoglobulins in sheep whey colostrum Carlotta Ceniti1, Vincenzo Lopreiato* 1, Valeria Maria Morittu1, Elisabetta Fratto1, Francesca Trimboli1, Genesio Massimini1, Domenico Britti1 1Università
Magna Græcia of Catanzaro, Catanzaro, Italy
Objective In early life, colostrum Immunoglobulins (Igs) absorp7on is cri7cal for the protec7on of the newborn ruminants from infec7ous diseases (Sawyer et al., 1977). Single Radial Immunodiffusion Assay (SRID) is currently considered the gold standard method for the direct measurements of Igs both in colostrum and serum. Capillary Electrophoresis (CE) is an alterna7ve method capable to directly measure Igs in biological liquids. We demonstrated that a rou7ne CE instrument can measure Igs in sheep colostrum (Ceni7 et al., 2016). The aim of this work was to evaluate the rela7onship between SRID and CE for the measurement of Igs concentra7on in sheep colostrum.
Materials and Methods During the summer of 2014, the colostrum from 12 Sarda sheeps, at 6th–7th lacta7ons, was sampled immediately aPer lambing (T0) and at 24 hours post partum (T24). The blood samples were collected from the corresponding 12 newborn lambs at 24 hours aPer birth. Colostrum Igs concentra7on was determined by a rou7ne CE instrument (Minicap, Sebia France), aPer fat and caseins removal by centrifuga7on and enzyme cloTng (rennet), respec7vely. Serum Igs concentra7on was also determined by CE, as rou7ne. Igs peak in the colostrum electropherogram was iden7fied comparing two electropherograms of a same sample obtained before and aPer Ig subtrac7on using Protein G Sepharose coniugate (Gammabind G Sepharose, GE Healthcare). Colostrum and serum IgG concentra7ons were measured by a commercial SRID kit (IDRing® Box – Sheep & Goat IgG TEST; IDBiotech, France) following the manufacturer’s instruc7ons. Before sta7s7cal analysis, all colostrum CE data (T0 and T24) and all colostrum SRID data (T0 and T24) were respec7vely pooled on a same distribu7on. Data were tested for normality of distribu7on using the Shapiro–Wilk test (SAS Ins7tute Inc., Cary, NC, USA). For each sample type (colostrum and serum), linear regression analysis between CE and SRID values was calculated by REG procedure of SAS 9.3 (SAS Ins7tute, Inc., Cary, NC, USA). In addi7on, for each sample type, Pearson correla7on test between the used methods was performed. Sta7s7cal significance was declared at P < 0.05.
Whey colostrum prepara0on
Centrifuga7on
Enzyme cloTng
2500 g x ’ x 4 °C
Rennet
Whey protein electrophoresis paJern
Other whey proteins
Immunoglobulins
Results Table 1. Linear regression rela0onships between IgG concentra0on measured by SRID (y) and Igs concentra0on measured by CE technique (x) observed in sheep colostrum (n=24) and lamb serum (n=12) samples.
Figure 1. ScaJer plot of correla0on between colostrum samples analyzed by CE and SRID (R2=0.98).
Figure 2. ScaJer plot of correla0on between lamb serum samples analyzed by CE and SRID (R2=0.98).
Samples
Parameter Estimate
Colostrum Lamb serum
Intercept
0.402
-0.110
Slope
0.961
0.975
R2
0.98
0.98
