J . Sci. FoodAgric. 1984,35,793-796

Detection and Partial Characterisation of Soluble Proteins of Beef Muscle by Immunoelectrophoresis in Agarose Gels

Carmen Casas, JosC Tormo, Pablo E. Hernandez and BernabC Sanz

Departmento de Higiene y Microbiologia de 10s Alimentos, Facultad de Veterinaria, Universidad Cornplutense, Madrid-3, Spain

(Manuscript received25 July 1983)

Immunoelectrophoresis in agarose gels has been used to detect and partially characterise specific protein precipitin bands of beef proteins (CSP), free of cross-reactions with proteins of pig (PSP) and horse (HSP). Out of the six precipitin bands obtained by reacting CSPs against an anti-CSP antiserum produced by a rabbit, only one band was produced by interaction of the anti-CSP antiserum with PSP and HSP. The other five bands were specific beef muscle soluble proteins. This technique may be useful in detecting the presence of beef in unheated ground meat products.

1. Introduction

A typical adulteration of unheated ground meat products is the addition of vegetable’ or animal proteins,’ not declared as such on the ingredients list. A simple, rapid test would make it possible to determine the addition of undeclared meat in a product, and give the consumers greater protection.

Immunologically, it is possible to differentiate proteins from different animal species using antisera to muscle proteins or to blood serum.3 In an attempt to detect specific protein bands of beef proteins, free of cross-reactions with proteins of pig and horse, the technique of immunoelec- trophoresis in agarose gels has been used. These specific protein bands may indicate the presence of beef in fresh meat products.

As far as the authors are aware, this is the first time that immunoelectrophoresis with anti-(muscle soluble proteins) has been employed in trying to pursue the objective previously stated. The sensitivity of this technique in detecting the presence of species-specific protein bands may be exploited in future studies to isolate these proteins and use them as diagnosis agents for testing unprocessed meat for contamination by extraneous species.

2. Materials and methods

2.1. Preparation of the antigenic extracts Skeletal muscle tissue from beef (Ms . rectusfemoris, Ms. vastus medialis and vastus lateralis), pork (Ms. intercostalis externi and Ms. trapezius) and horse (Ms . glutaeus superficialis and Ms. biceps fernoris), total weight 250 g, was finely triturated, minced and homogenised in 500 ml of a 0.85% saline solution. The soluble proteins were extracted by constant agitation of these homogenates for 1 h at 1°C. The protein extracts were filtrated through a Whatman no. 1 filter paper, lyophilised, and the dried extracts placed in an airtight container and stored at -20°C until use.

2.2. Preparation of the antiserum

Serum containing suitable CSP antibodies was obtained by injecting New Zealand male rabbits subcutaneously with single doses of lyophilised beef protein extracts (48 mg) in 2 ml of deionised and

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distilled water emulsified with 0.5 ml of Freund complete adjuvant (Difco). Fifteen booster doses were applied subcutaneously every 4 days for 62 days. The rabbits were periodically bled from the marginal ear vein, the blood was allowed to clot at room temperature and centrifuged at 1000 X g for 10 min to remove any remaining blood cells present.

2.3. Agarose gel immunoelectrophoresis The basic technique was that of Grabar and Williams4 modified by Scheidegger,’ consequently, only the details relevant to this application are given here. Agarose gel (1%) in veronal buffer, pH 8.6, was used. The gels were punched with a well cutter giving holes 1 mm in diameter and bands of 0 . 2 ~ 5 cm. Samples (1 pl, 24 pg) of the antigenic extracts were deposited in the holes. The plate was subjected to electrophoresis for 4 h at 130 V. When the electrophoresis was concluded, the plate band was filled with 0.3 ml of the corresponding antiserum. Immunodiffusion was performed for 18-24h at 37°C. The protein precipitin bands were visualised with Amido black.6 The immunochemical partial characterisation of these bands comprised the detection of glycoprotein fractions according to NADI’s method’ and lipoprotein fractions with Sudan black.’ The degree of displacement of each band relative to bovine serum albumin (Fraction V Cohen, Sigma Chemical Co) used as standard, was defined as follows.

Band displacement (mm)

V mm-’ s Absolute mobility=

- Band displacement (mm) Bovine serum albumin displacement (mm)

Relative mobility=

Mobility %= Relative mobility x 100

3. Results and discussion

To detect specific protein bands of beef muscle soluble proteins (CSP), free of cross-reactions with soluble muscle proteins of pig (PSP) and horse (HSP), lyophilised soluble protein extracts of each animal species were analysed by immunoelectrophoresis in agarose gels against an anti-CSP antiserum produced by a rabbit.

The lyophilised extracts of soluble protein from beef muscle (CSP), when tested against an homologous antiserum (anti-CSP), allowed the identification of six protein precipitin bands. These had per cent mobilities between 921.1 and 89k0.9 (Table 1 and Figure 1). Two of the six observed bands were arbitrarily defined as major bands (bands 3 and 4) by their strong staining with Amido black. The remaining bands (bands 1 , 2 , 5 and 6) were defined as minor bands by their weak staining

Table 1. Absolute, relative and % mobilities of protein precipitin bands of cow’s muscle soluble proteins (CSP) against a rabbit homologous antiserum (anti-CSP)”

Precipitin Band displacement Absolute Relative % band no. (mm) mobility mobility mobility

1 2 3 4 5 6

B. Albumin D e x t r a n e

3.5 7.5

11.5 15.0 16.5 33.0 37.0 0.0

0.40f0.08 0.86f0.04 1.33f0.11 1.73L0.20 1.90k 0.20 3.81k0.35 4.28f0.34

0.0

0.09 0.20 0.31 0.40 0.44 0.89 1.00 0.0

9+1.15 20f0.50 31f0.17 40 f0.74 442 1.26 89-1-0.89

100 0.0

‘The degree of displacement of each band relative l o bovine serum albumin used as standard, was defined as stated in section 2. The numbers refer to mean values+standard deviations, from 14 independent assays.

Detection of beef muscle proteins 195

- anti-CSP +

5 6

I 0 n i i - c ~ ~ I

onti - CSP

Figure 1. Patterns of protein precipitin bands of soluble proteins from different animals, against antiserum for soluble proteins of beef muscle (anti-CSP). CSP, beef proteins; PSP. pig proteins; HSP, horse proteins.

with the same dye. The partial immunochemical characterisation (Table 2) of these bands to detect the presence of glycoprotein and lipoprotein fractions in their structure gave negative results.

When the lyophilised soluble protein extracts of pig (PSP) and horse (HSP) muscles, were analysed against an anti-CSP antiserum, in both cases the presence of only one protein precipitin band was observed (Figure l), of 20+0.5% mobility. This band has a mobility identical to that of band 2 from the beef soluble protein extracts. Therefore, this band could be responsible for false positive reactions in attempts to quantitatively identify soluble protein extracts from the muscles of different animals by immunodiffusion. It is also important to realise that major (3 and 4) and minor bands (1, 5 and 6) of beef muscle, when assayed against an anti-CSP antiserum, are specific beef protein precipitin bands which do not appear when extracts of soluble proteins of pig and horse muscles are analysed against the same anti-CSP antiserum. These specific beef protein bands could thus serve as an indication for the presence of beef in meat products, when extracts of soluble muscle proteins are analysed against an anti-CSP antiserum.

Immunoelectrophoresis in agarose gels to differentiate proteins from different animal species has been used with plasma and serum proteins antiserum as reference patterns.” The authors believe

Table 2. Detection of protein precipitin bands of soluble proteins of beef muscle (CSP) against a rabbit homologous antiserum (anti-CSP)

Immunisation period Precipitin bands (band no.)

Bleeding (Days) Major Minor

B1 12 B2 28 B3 48 B4 62

- -

3 , 4 - 3, 4 2, 6 3 , 4 1. 2. 5 , 6

796 C. Casas et al.

that the use of antisera for the soluble proteins of muscle instead of those for blood serum proteins, may improve this assay, owing to the elimination of cross-reacting proteins present in the plasma and serum'' which could also be present in the soluble extracts of muscle proteins.

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