Measurement of Bovine IgG by Indirect Competitive ELISA as a Means of Detecting Milk Adulteration

Abstract

The aim of this work was to develop an assay capable of detecting adulteration of high premium milk with milk from cheaper sources. An indirect, competitive ELISA was developed for the rapid detection of cows' milk in the milk of goat, sheep, and buffalo. The assay uses a monoclonal antibody produced against bovine IgG. This antibody recognizes a species-specific epitope on the heavy chain of both bovine IgG1 and IgG2. A peroxidase-conjugated anti-mouse IgG antibody was used to detect bound monoclonal antibody and subsequent enzymatic conversion of substrate resulted in clear differences in absorbance when assaying different mixtures of milks adulterated with cows' milk. Once optimized, the ELISA was found to be highly specific. Detection limits of the assay are 1.0 microg/mL of bovine IgG, or 0.1% (vol/vol) adulteration with cows' milk. The assay was highly reproducible (CV < 10%) and performed equally well when used to detect bovine IgG in mixtures with the 3 types of milk tested. The ELISA performance makes it suitable for development as a kit, for use in the field as a high throughput screening ELISA.

Possibilities of different animal milk detection in milk and dairy products - a review

“…The immunoanalysis employs specific mono-or polyclonal antibodies that react with whey proteins, the individual casein fractions, and peptide fragments of caseins. Reactions were described of the antibodies with, e.g., bovine β-Lg (LE-VIEUX & VENIEN 1994), bovine IgG (HURLEY et al 2004), bovine κ-casein (BITRI et al 1993), bovine β-casein (ANGUITA et al 1997), bovine α S1 -casein (ROLLAND et al 1995), and goat whey proteins (GARCIA et al 1993). By means of cross-reactions with individual milk proteins, it was proved that the antibodies are highly specific and manifest minimum cross-interactions.…”

“…For the detection of basically similar types of milk, such as bovine and buffalo, it seems to be prospective to use monoclonal antibody against bovine immunoglobuline G. The reason is that immunoglobulines are not easily liable to proteolysis and are stronger immunogenes than caseins and other whey proteins. This way of the detection of cow milk in goat, ewe and buffalo milk using indirect ELISA was used by HURLEY et al (2004) who were able to detect 0.1% of bovine IgG in all types of milk. This test is suitable both for raw milk and pasteurised or frozen.…”

“…For this purpose, both indirect (competitive) and sandwich (uncompetitive) ELISA was used. ELISA method was used for the detection of the cow milk presence in goat, ewe, and buffalo milk (HURLEY et al 2004), further in goat and ewe cheese (PIZZANO et al 2000), and of the presence of goat milk in ewe milk (HAZA et al 1996).…”

Inhibition effect of pcDNA-tum-5 on the growth of S180 tumor

“…Assay of protein concentration was performed using indirect competitive ELISA method as described previously (Hurley et al 2004). Briefly, ELISA plates were coated overnight at 4°C with 100 lL/well tum-5 protein (Meng et al 2006) at optimized concentration of 0.2 lg/mL in PBS.…”

12S rRNA mitochondrial gene as marker to trace Sicilian mono-species dairy products

“…Several analytical methods have been applied for species identification in milk and dairy products including immunological (Hurley et al, 2004;López-Calleja et al, 2007a;Xue et al, 2010;Zeleňáková et al, 2008), electrophoretic (Chianese et al, 1990;Mayer, 2005;Molina et al, 1999), chromatographic (Branciari et al, 2000;De Noni et al, 1996;Enne et al, 2005), and spectrometric (Nicolaou et al, 2011). For example, the European Commission Regulation (EC) No 273/2008 laid down detailed rules for the application of EC No 1255/ 1999 as regards methods for the analysis and quality evaluation of milk and milk products.…”