Estimate of serum immunoglobulin G concentration using refractometry with or without caprylic acid fractionation

Abstract

Objectives of this study were to develop a rapid calf-side test to determine serum IgG concentrations using caprylic acid (CA) fractionation, followed by refractometry of the IgG-rich supernatant and compare the accuracy of this method with results obtained using refractometry using raw serum. Serum samples (n=200) were obtained from 1-d-old calves, frozen (-20°C), and shipped to the laboratory. Samples were allowed to thaw for 1h at room temperature. Fractionation with CA was conducted by adding 1mL of serum to a tube containing 45, 60, or 75µL of CA and 0.5, 1.0, or 1.5mL of 0.06 M acetic acid. The tube contents were mixed well, allowed to react for 1 min, and then centrifuged at 3,300 × g for 0, 10, or 20 min at 25°C. The %Brix and refractive index of the fractionated supernatant were determined using a digital refractometer. Nonfractionated serum was analyzed for %Brix (BRn), refractive index (nDn), and IgG concentration by radial immunodiffusion. The mean serum IgG concentration was 19.0 mg/mL [standard deviation (SD)=9.7], with a range of 3.5 to 47.0 mg/mL. The mean serum BRn was 8.6 (SD=0.91), with a range of 6.8 to 11.0. The mean serum nDn was 1.34566 (SD=0.00140), with a range of 1.34300 to 1.34930. Serum nDn was positively correlated with IgG concentration (correlation coefficient=0.86; n=185). Fractionated samples treated with 1mL 0.6 M acetic acid and 60µL of CA and not centrifuged before analysis resulted in a strong relationship between the refractive index of the fractionated supernatant and IgG (correlation coefficient=0.80; n=45). Regression was used to determine cut points indicative of 10, 12, and 14 mg of IgG/mL to determine the sensitivity and specificity of refractometry to identify failure of passive transfer (serum IgG <10 mg/mL at 24 h old). The nDn were 1.34414, 1.34448, and 1.34480 to predict 10, 12, and 14 mg of IgG/mL of serum, respectively. The BRn cut points were 7.6, 7.8, and 8.0, respectively. The nDn cut points of 1.34448 and 1.34480 resulted in similar specificities (82.9%), whereas the 1.34414 cut point had a specificity of 60.0%. The BRn cut point of 7.6 and 7.8%Brix resulted in a similar percentage of correctly classified samples (89.7 and 90.8%, respectively); however, the 7.8% Brix cut point resulted in fewer false positives. These results suggest that Brix refractometry of nonfractionated calf serum provides a strong estimate of IgG concentration and 7.8% Brix may be used as the cut point to identify failure of passive transfer in 1-d-old calves.

Systematic Review and Meta‐Analysis of Diagnostic Accuracy of Serum Refractometry and Brix Refractometry for the Diagnosis of Inadequate Transfer of Passive Immunity in Calves

“…Of these, 115 full-text papers were assessed for eligibility. A total of 13 test accuracy studies (3,788 calves), comprising 11 studies (1,814 calves) of REF 9,12,13,[28][29][30][31][32][33][34] and 5 studies (2,881 calves) of BRIX, 11,12,29,30,35 were included in this systematic review. The characteristics of the studies are presented in Table 1.…”

“…1,8,10 Both RID and TIA are reliable for measuring IgG concentrations but are not practical on farms, are expensive, and the results are typically available after 24-36 hours. 11 For these reasons, other practical methods to estimate serum IgG concentration have been investigated. 12,13 Among these methods, optical or digital refractometry (REF) and Brix refractometry (BRIX) have been suggested as methods to assess serum IgG concentration.…”

Evaluation of 5 methods for diagnosing failure of passive transfer in 160 Holstein calves

“…11,22 The Brix refractometer provides an attractive alternative method to evaluate FTPI in dairy calves as its percentage value approximates the percentage of total solids in plasma or serum. A digital Brix refractometer has been used as an on-farm tool to evaluate colostrum quality, 23,24 and considerable value exists in using the same instrument to also evaluate FTPI in calves. 22 Accordingly, studies were conducted to evaluate the clinical utility of using a digital Brix refractometer to evaluate FTPI using serum samples, 14,22,25,26 with a digital Brix value of <7.8%, <8.3%, <8.4%, <8.6%, or <8.8% used as cut points.…”

Evaluation of an on-farm method to assess colostrum IgG content in sows

“…These results indicate that the Brix refractometer can be a useful on-farm tool and can cheaply generate large scale data, with important implications for improved selection of sows with better maternal characteristics. Future research will investigate whether it could be also possible to assess failed transfer of passive immunity by measuring the piglet serum IgG with the refractometer, as has been done in calves (Chigerwe and Hagey, 2014) and cows (Morrill et al, 2013).…”