Production and characterization of monoclonal antibodies specific to chicken muscle soluble proteins

Meat Science 25 (1989) 199 207

Production and Characterization of Monocionai Antibodies Specific to Chicken Muscle Soluble Proteins

Rosario Martin, a R. John Wardale, b Sheila J. Jones, b Pablo E. Hernandez a & Ronald L. S. Patterson b* ° Departamento de Higiene y Tecnologia de los Alimentos, Facultad de Veterinaria. Universidad Complutense, 28040 Madrid, Spain b AFRC Institute of Food Research, Bristol Laboratory, Langford, Bristol BS18 7DY, UK (Received 18 August 1988; revised version received 6 December 1988; accepted 7 December 1988)

A BSTRA C T Three stable hybridoma cell lines ( AH4, BC9 and CF2 ) have been produced which secrete monoclonal antibodies specific Jor chicken and turkey muscle proteins. Partial characterization bv EL1SA and S D S - P A G E immunoblotting indicated that the antibodies failed to cross-react with similar extracts of pork, beef lamb, horse or rabbit. One of the cell lines ( A H4 ) secreted a monoclonal antibody that was also capable of distinguishing between chicken and turkey by indirect ELISA.

INTRODUCTION Poultry (chicken and turkey) tissue represents a major source of protein, generally less expensive than red meat, which is consumed and imported throughout the world. These factors, together with the regulated increasing use of mechanically separated poultry meat, produce a significant potential for the adulteration or substitution of red meat by poultry products. * To whom correspondence should be addressed. 199 Meat Science 0309-1740/89/$03.50 © 1989 Elsevier SciencePublishers Ltd, England. Printed in Great Britain

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Several methods are currently used for identification of meat species and for detection of adulteration with cheaper meats. Non-immunological methods include gel electrophoresis (Babiker et al., 1982; Kim & Shelef, 1986), isoelectrofocussing (King & Kurth, 1982; King, 1984) and chromatographic methods (Carnegie et al., 1983; Ashoor & Osman, 1988; Ashoor et al., 1988). Immunological methods, which are the most commonly applied for meat species analysis in practice, include techniques such as agargel immunodiffusion (AGID) recently modified to the 'dry-disc' type tests tMageau et al., 1984; Jones et al., 1986), immunoelectrophoresis (Casas et al., 1985; Allsup, 1987) and enzyme-linked immunosorbent assay (ELISA) (Kang'ethe et al., 1982; Griffiths & Billington, 1984; Patterson & Jones, 1985; Jones & Patterson, 1985, 1986; Patterson & Spencer, 1985; Martin et al., 1986, 1988a,b,c). Many of the ELISA methods currently available for meat speciation in kit form have used polyclonal antisera raised against various blood proteins present in 'meat' such as albumin. Such antisera require special immunosorbent affinity purification to eliminate significant cross-reactions. Once established, ELISAs also require a continuous consistent supply of suitable antibody reagents to enable large scale production and commercialization. This obviously presents an important problem in the long-term. The development ofhybridoma technology (K/Shler & Milstein, 1975) has provided the means for continuous production of monospecific antibodies of known biological activity and consistent specificity from single immortal cell lines, once selected by suitable stringent screening procedures and invitro tissue culture. For meat speciation, monoclonal antibodies of required specificity would then be suitable for various types of ELISA; for example, "capture' (Jones & Patterson, 1985; Berger et al., 1988; Martin et al., 1988c) or competitive (Dincer et al., 1987) as well as being available for the basic screening procedure called 'indirect' in this study. This report describes the development of monoclonal cell lines capable of identifying and distinguishing between chicken and other meats on the basis of antigenic variants between particular soluble muscle proteins.

MATERIALS AND METHODS

Preparation of chicken-specific proteins Antigenic extracts from chicken meat were prepared from 1 kg of trimmed, well-mixed, hand defatted lean meat. Representative 100g samples were thoroughly homogenized in 300 ml of a saline (0"85% NaCI) solution and the

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soluble proteins were extracted by gentle agitation for 24 h at 4°C. Insoluble material was removed by centrifugation at 1500 × g for 5 min at 4°C and the supernatants filtered through a Whatman No. 1 filter paper and lyophilized. The dried sarcoplasmic proteins were stored in an airtight container at - 2 0 ° C until required for use. Lyophilized solids of other species meats (prepared as above), similar saline extracts, in liquid form, of the chicken organs (heart, kidney, liver and gizzard) and of three heated chicken products were also prepared as test materials for later analysis. Chicken sarcoplasmic proteins with species-specific epitopes were isolated by immuno-adsorption chromatography. The chicken-specific fraction was obtained by passing 30mg of the freeze-dried sarcoplasmic proteins diluted in 10 ml phosphate buffered saline (PBS), pH 7.2, through a Protein A-Sepharose CL4B column (Pharmacia Fine Chemicals, Uppsala, Sweden) containing 571mg of Sepharose coupled to 40mg of chickenspecific polyclonal antibodies previously produced against chicken muscle soluble proteins and rendered species-specific by affinity chromatography (Martin et al., 1988c). The purified chicken-specific proteins (CHSP) were released from the column by elution with 0"05Mdiethylamine buffer, pH 11"5, the eluted fractions showing an absorbance higher than 0"1 at 280 nm were pooled, adjusted to pH 7'2 with 0"5M sodium phosphate buffer, dialyzed overnight against PBS and lyophilized.

Immunization An emulsion was prepared containing equal volumes of CHSP (120/~g of protein/200 ~tliters) and Freund's Complete Adjuvant (Sigma). Five 4-week old female Balb/c mice were immunized intraperitoneally with 0.2 ml of emulsion at 2-week intervals for 6 months. Test sera were obtained by tail vein bleeding and tested for specific antibody production by indirect ELISA. Four days prior to fusion, one mouse showing a high titre by the indirect ELISA was boosted by intraperitoneal injection of ! 20/~g of CHSP in 0.2 ml of distilled water.

Monoclonai antibody production Four days after the booster injection, the mouse producing antibodies reactive in the indirect ELISA was killed by chloroform and the spleen removed and mechanically dissociated by passage through a sterile nylon mesh. Spleen cells were mixed with myeloma cells (P3X63-Ag 8.653) in a 1:5 ratio and fused in polyethylene glycol (mol. wt 1500, Boehringer Mannheim GmbH, W. Germany) according to the procedure of K6hler &

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Milstein (1975). The cells were then collected and distributed at a density of about 105 per well in 96-well sterile cell culture plates (Falcon 3075). Hybridoma colonies were visible in 2 weeks, at which time the medium was tested by indirect ELISA against chicken muscle soluble proteins. Hybridoma supernatants from positive wells were tested against soluble muscle proteins of beef, pork, horse, lamb, rabbit and turkey. Cell lines producing antibodies that were specific for chicken were cloned by limiting dilution in 96-well plates with mouse thymocyte feeder cells (10 7 cells per ml) and expanded before freezing in liquid nitrogen. Hybridomas were grown as ascites tumour cells in Balb/c mice and pretreated by intraperitoneal injection with 0.2ml of pristane (Sigma, T7640) 10 days prior to injection of I × I06 hybridoma cells.

Enzyme-linked immunoassay (ELISA) procedures Hybridoma culture supernatant fluids were screened using the indirect ELISA technique in microtiter plates (Nunc, Denmark). Wells were coated with 5-10 #g of meat sample extracts in 0"15M sodium carbonate-bicarbonate buffer (pH 9"6) and the plates incubated overnight at 4°C. The wells were subsequently washed three times in phosphate buffer saline (PBS) containing 0-5%. Tween 20 (PBST) and then filled with 50/diters of hybridoma supernatants and 50/Aiters of PBST. After incubation for 1 h at ambient temperature, the wells were washed with PBST and 100/Lliters of diluted rabbit anti-mouse immunoglobulins conjugated to peroxidase (Dakopatts, Denmark, P161) (1:1000 in PBST) added and the plates incubated for 1 h at ambient temperature. The wells were washed three times with distilled water before addition of 150 pliters of substrate consisting of 0"03 % 2,2'-azinobis(3-ethyl-benz-thiazoline sulfonic acid) (Sigma, A1888) and 0"2% H202 (6% w/v) in citrate-phosphate buffer pH 4. After 30 rain, the plates were read at 405 nm.

Eiectrophoretic separation and immunobiotting Soluble muscle proteins of chicken, beef, pork, horse and turkey were separated electrophoretically in 10% SDS-polyacrylamide gels (Laemlli, 1970) and transferred to nitrocellulose paper of pore size 0-45 m (Schleicher & Schuel; Towbin et al., 1979). The transfers were incubated with Trisbuffered saline, pH 8.0 (TBS) containing 0"2% Tween 20 (TBST) and then probed with hybridoma supernatants diluted 1:1 in TBST for 3 h at 20°C. After washing (3 x TBST) the transfers were incubated with goat antimouse IgG alkaline phosphatase (1:1000 in TBST, Sigma, A5153) for 2 h at 20°C. After washing, bound monoclonal antibody was detected by adding

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the substrate (1 mg/ml of 4-aminophenylamine diazonium sulphate + 1 mg/ml naphthyl phosphate in 1M tris buffer, pH 9-69).

Monoclonal antibody characterization The testing ofmonoclonal antibody subclass was determined by an agar-gel immunodiffusion technique (Ouchterlony, 1949) against rabbit antisera to mouse IgG1, IgG2a, IgG3 and lgM (Sigma).

RESULTS All immunized mice produced high titres of antibodies against chicken muscle soluble proteins. The mouse showing the least cross-reaction with other animal species by indirect ELISA was chosen for fusion. Screening of hybridoma cell lines by an indirect ELISA indicated the secretion of several monoclonal antibodies against chicken-specific proteins and the cell lines AH4, BC9 and CF2, secreting monoclonal antibodies IgG subclass IgG1, were selected for further characterization. Indirect ELISA demonstrated that these monoclonal antibodies failed to show any cross-reactivity when tested against soluble muscle proteins from the meat species of beef, pork, lamb, horse, rabbit and other proteins also, casein, gelatin and soya protein (Table 1). One of the monoclonal TABLE 1 Comparison of Cross-reactivities Among Several Proteins Toward Chicken Monoclonal Antibodies by the Indirect ELISA Technique

Proteins

Pork Beef Lamb Horse Rabbit Casein Gelatin Soya Turkey Chicken

Monochmal antibodies A H4

C~2

CB9

0'119" 0.118 0.074 0.053 0-079 0.059 0-091 0-059 0"110 1.421

0"118 0-130 0.074 0.063 0.085 0.063 0-096 0"060 1'398 1.933

0"114 0.135 0.082 0'047 0.077 0.080 0"096 0-068 1"570 1.568

" Optical densities at 405 nm.

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3

4

5

1

2

3

4

5

1

2

3

4

5 m

•

-116k -84k

~1~

11-58k -48"5k

-36-5k -26.6k

A H4

BC9

C F2

Fig. 1. SDS-PAGE immunoblots of soluble muscle extracts. Approximately 501Lg of turkey (lane 1), horse (lane 2), pig (lane 3), beef (lane 4) and chicken (lane 5); extracts were probed with monoclonal antibodies AH4, BC9 and CF2. Prestained molecular weight numbers (lane m) are E. coli-galactosidase (116000), rabbit muscle fructose-6-phosphate kinase (84000), chicken muscle pyruvate kinase (58000), porcine heart fumarase (48 500), rabbit muscle lactic dehydrogenase (36 500) and rabbit muscle triosephosphate isomerase {26 600).

antibodies--AH4--also did not cross-react with an extract derived from turkey. These results were confirmed by SDS-PAGE immunotransfer, when proteins from chicken, beef, pork, horse and turkey were probed with the monoclonal antibodies (Fig. 1). A single main band in the chicken and turkey extracts of approximate mol. wt 58 kDa was detected by CF2, whilst BC9 detected a band of approximate mol. wt 47 kDa in both. AH4 appeared to detect the same 47 kDa band but only for chicken. None of the monoclonal antibodies cross-reacted with beef, horse and pig. It was noted that CF2 reacted with the molecular weight marker (mol. wt 58 kDa)--chicken muscle pyruvate kinase--and this reaction was confirmed against pure chicken pyruvate kinase (Sigma) by ELISA and SDS-PAGE immunotransfer (data not shown). The extracts of unheated chicken organs and heated chicken products all gave much lower responses (OD c. 0.5) when compared with the full chicken response of each monoclonal antibody by the indirect ELISA described. However, some increase in response, up to OD 1-0 for kidney and heart, 0.6

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for liver and 0"4 for gizzard, was noted by coating the microtitre plate wells with 10-tbld more protein (50-100pg) from each extract.

DISCUSSION We have produced three monoclonal antibodies which are capable of distinguishing between soluble muscle extracts of the most frequently marketed avian (chicken and turkey) and mammalian (beef, pork, horse and lamb) species of meat animals. One of these antibodies has the added advantage of distinguishing between chicken and turkey extracts by ELISA. Hybridoma technology does not necessarily require highly purified antigens as a prerequisite to a successful fusion. However, as muscle proteins are highly conserved between species, we considered it to be extremely important to use a very specific antigen to prepare our monoclonal antibodies. Previous work using a crude extract of muscle proteins as an immunogen resulted in monoclonal antibodies which cross-reacted with all species tested (unpublished data). For this work, chicken-specific polyclonal antibodies were prepared (Martin et al., 1988c), immobilized on a protein Asepharose CL4B column and used to isolate material enriched in chickenspecific sequences, sufficient for use as an antigen in monoclonal antibody production. Relatively high concentrations (120/tg/injection) of antigen were found to be necessary to stimulate plasma cell secretion of chickenspecific antibodies by the immunized mice. Previous fusions using animals injected with low concentrations (10 pg/injection) but producing high titres of antibody, resulted in few positive hybridoma cell lines, all of which secreted non-specific antibodies. The fusion reported here was highly successful, producing a large number of positive cell lines, three of which secreted the specific monoclonal antibodies that we have described. The protein detected by antibody CF2 was positively identified as pyruvate kinase by virtue of its molecular weight and use of a commercial preparation of the enzyme in ELISA and immunoblotting experiments. Antibodies AH4 and BC9 apparently recognize the same protein of approximate mol. wt 47 kDA. From this molecular weight and previous analysis of the chicken-specific antigen preparation, the protein is probably 3-phosphoglycerate kinase but further characterization will be necessary. It is possible that the preparation from different species of either of these proteins as antigens will provide a means of raising monoclonal antibodies for a range of species-specific immunoassays covering a broad spectrum of meat animals. It should be stressed that one of the monoclonal lines (AH4) does not recognize turkey sarcoplasmic proteins. Chicken and turkey meats are

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c o m m o n l y used, separately or together, in poultry meat products. Determination of the identity of poultry species is important since turkey meat is more expensive than chicken and there may be a tendency to adulterate turkey products with chicken. Furthermore, results obtained from the analysis of chicken muscle and organs indicate that all three monoclonal antibodies are apparently musclespecific when used in this ('indirect') ELISA format. This is due, in part, to the presence of greater amounts of the chicken-specific antigens in the muscle extracts, compared with those from the offals, thus providing more antigen to adhere to the microtitre plate surface during the initial ELISA "coating' step. However, it should be noted that since the antibodies were raised against native proteins they were not ideally suited for detection of heat-denatured chicken meat materials. In this work, we have proved that the monoclonal antibodies AH4, CF2 and BC9 can qualitatively differentiate chicken from other species. Further work will be carried out to apply these monoclonal antibodies to the quantification of chicken proteins in meat products.

ACKNOWLEDGEMENTS This work was supported in part by a British Council award under the British/Spanish Joint Research Programme Acciones Integradas 1987/88, reference number SPN/991/3/24(87/88), and by a grant from the Comunidad A u t o n o m a de Madrid.

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