J Sci Food Agric 1992. 58, 447-449

Muscle Proteins by Chromatography

Partial Purification of Horse-specific Soluble Immunoadsorption

Rosario Martin, Teresa Garcia, Bernabi Sanz and Pablo E Hernandez Departamento de Nutricion y Bromatologia I11 (Higiene y Tecnologia de 10s Alimentos), Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain

(Received 20 May 1991 ; revised version received 16 September 1991 ; accepted 16 October 1991)

Abstract : Immunoadsorption chromatography has been used to isolate horse- specific soluble muscle proteins from crude horse protein extracts. Horse-specific polyclonal antibodies against soluble horse muscle proteins immobilised on a Protein A-Sepharose CL-4B matrix were used to adsorb the corresponding antigens. Analysis of the crude soluble horse muscle proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed the existence of 20 protein subunits in the gel. SDS-PAGE of the proteins recovered by affinity chromatography showed nine protein subunits, three of which were enriched after the immunopurification step. These affinity-recovered horse-specific soluble muscle proteins may be used as antigens in the development of monoclonal antibodies to detect and quantify horse meat in raw, unheated meat mixtures.

Key words : Horse-specific soluble muscle proteins, horse-specific polyclonal antibodies, immunoadsorption chromatography.

The possibility that unheated ground meat products might contain the flesh of species not indicated by the product description, whether by accident or intention, is certainly not a new problem. Whilst improvements in technology and processing have led to more economical transportation and utilisation of deboned carcass meat, they have also facilitated the use of undeclared meats because unequivocal visual identification of species becomes difficult once meat is off the carcass and cut up.

Most of the immunological methods currently avail- able for meat species analysis use polyclonal antibodies raised against blood proteins (Kang’ethe et a1 1982; Griffiths and Billington 1984; Jones and Patterson 1985, 1986; Patterson and Spencer 1985; Allsup 1987; Cutru- felli et a1 1987) or soluble muscle proteins (Martin et a1 1986, 1988a, b. c). Although immunological methods are sufficiently accurate and sensitive to identify meat from closely related species, they require the production of specific antisera with high titres. The production of species-specific antibodies from the crude antiserum is time consuming and expensive and requires technical expertise. This obviously represents an important prob- lem in the long term. The development of hybridoma

technology (Koller and Milstein 1975) has provided the means for continuous production of monospecific antibodies of known biological activity and consistent specificity from single cell lines, once selected by suitable stringent screening procedures of in-vitro tissue cultures. For meat speciation, monoclonal antibodies of known specificity would permit the universal use of a consistent reagent.

The crude soluble horse muscle proteins (CSHP) were prepared from 1 kg of trimmed, well mixed, hand- defatted horse meat. Representative 100-g samples were thoroughly homogenised in 300 ml of a saline (0.85 % NaCI) solution and the soluble proteins were extracted by gentle agitation for 24 h at 4°C. Insoluble material was removed by centrifugation at 1500 x g for 5 min at 4°C and the supernates were filtered through a Whatman No 1 filter paper and lyophilised.

Soluble horse muscle proteins with horse-specific epitopes were isolated by immunoadsorption chroma- tography. The horse-specific fraction was obtained by passing 30 mg of the lyophilised CSHP diluted in 10 ml of phosphate-buffered saline (PBS), pH 7.2, through a Protein A-Sepharose CL-4B column (Pharmacia Fine

447 J Sci Food Agric 0022-5142/92/$05.00 0 1992 SCI. Printed in Great Britain

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Chemicals, Uppsala, Sweden) containing 571 mg of freeze dried powder coupled to 20 mg of horse-specific antibodies previously produced against soluble horse muscle proteins and rendered species specific by affinity chromatography (Martin et a1 1988a). The adsorbed horse-specific proteins (HSP) were released from the column by elution with 0.05 M diethylamine buffer, pH 11.5, and the eluted fractions showing an absorbance at 280 nm higher than 0.1 were pooled, adjusted to pH 7.2 with 0.5 M sodium phosphate buffer, dialysed overnight against PBS and lyophilised.

The CSHP and HSP were separated electrophoreti- cally in sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE), essentially according to the method described by Laemmli (1970). Separation of 50-pg quantities of each was performed in tubes containing a 30 g litre-' stacking gel and a 125 g litre-' separating gel, at 1.5 mA per tube for 4 h. Gels were stained with 2.5 g litre-' Coomassie Blue G-250 in 450 ml litre-' methanol plus 90 g litre-' acetic acid at 37°C for 2 h and destained in 70 g litre-' acetic acid in 50 ml litre-' methanol. The electropherograms were obtained following a spectro- photometric scanning (580 nm) of the resulting Coo- massie Blue stained gels. The protein markers were from a low molecular weight protein standard (Bio-Rad Laboratories, Richmond, CA, USA).

Hybridoma technology does not require highly puri- fied antigens as a prerequisite to a successful fusion. However, as soluble muscle proteins are highly conserved between species, it is considered convenient to use horse- specific antigens to develop horse-specific monoclonal antibodies. SDS-PAGE of the CSHP fraction revealed the existence in the gel of 20 protein subunits (Fig IA), of an apparent MW between 18 kDa and 100 kDa. Similar results of the affinity-recovered proteins (HSP) showed the presence in the gel of nine protein subunits (Fig IB); three of the subunits of an apparent MW of 37, 70 and 96 kDa were enriched after the immunopurifi- cation step. All adsorbed protein subunits, especially the enriched ones, should have the highest potential to

? i

A B

Fig 1. SDS-PAGE resolution of the crude soluble horse muscle proteins (A) and horse-specific proteins (B) after the immuno-

purification step.

induce the formation of horse-specific monoclonal antibodies. Previous work, using affinity-recovered chicken-specific proteins obtained as described for the isolation of the horse-specific proteins, has resulted in the fast and relatively easy production of monoclonal antibodies specific to soluble chicken muscle proteins (Martin et a1 1989a, b, 1991).

Monoclonal antibodies against soluble horse muscle proteins would then be suitable for various types of immunoassay. ELISA tests using monoclonal antibodies would be readily available for commercialisation into stable kits that contain all the necessary components and supplies for species verification of horse meat in large or small scale inspection programmes.

ACKNOWLEDGEMENTS

This work is supported by Grant No ALI89-0192 from the Comision Interministerial de Ciencia y Tecnologia (CICYT).

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Partial purlJicntion of horse-spec$c soluble muscle proteins by immunoadsorption chromatography 449

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