A fluorescence polarization assay (FPA) was developed to detect Clostridium perfringens type D epsilon antitoxin in serum of goats. Purified epsilon toxin was labelled with fluorescein isothiocyanate and used as a tracer in the test. Seven goat sera with different known concentrations of epsilon antitoxin (as measured by a mouse neutralization test-MNT) were used as positive controls. Sera from eleven colostrum-deprived kids were used as negative controls. The inter-assay variation of ten measurements of the seven positive sera varied between 1.56% and 4.85%. The correlation between the FPA values and concentration of epsilon antitoxin (as measured by the MNT) was 0.90. This test appears to be a good alternative to the MNT without most of the inconveniences of the later.

Palabras clave: ensayo de fluorescencia polarizada, cabras, Clostridium perfringens, anticuerpos contra toxina epsilon

Key words: fluorescence polarization assay, goats, Clostridium perfringens, epsilon toxin antibodies

INTRODUCTION

Enterotoxaemia (pulpy kidney disease) is produced in several animal species by epsilon toxin, a major exotoxin produced by Clostridium perfringens type D (Niilo, 1980). The commercial vaccines against pulpy kidney disease consist, therefore of a formalin-inactivated epsilon toxin (British Pharmacopeia, 1993).

To evaluate the response of animals to enterotoxaemia vaccines, antibody titres against C. perfringens epsilon toxin are usually measured in sera using the toxin neutralisation test in mice (MNT) (British Pharmacopeia, 1993) or, more recently ELISA techniques (Sojka et al, 1989, Wood, 1991, Uzal et al, 1997). The MNT is cumbersome, time consuming, slow, expensive, requires the use of living animals and can be imprecise (British Pharmacopeia, 1993). The ELISA tests are quicker to perform than the MNT, taking a few hours to complete, but they still require multiple manipulations and reagents, and expensive equipment (Nielsen et al, 1992).

The homogeneous fluorescence polarisation assays (FPA) are single-step assays that have been used to detect a variety of substances in biological samples (Jenkins, 1992, Nielsen et al, 1996). They are based on the principle that a fluorescent dye can be excited by plane-polarised light, and a stationary molecule, excited with polarised light, will emit light in the same plane. However, molecules are constantly rotating around an axis, and this rotation rate can be assessed by measuring the light intensity in the horizontal and vertical planes. Small molecules (such as antigen alone) rotate at a faster rate emitting more depolarised light. If a small fluorescent antigen with rapid movement becomes bound to antibody, the rotation time will increase as a result of formation of the antigen-antibody complex, thus confining emitted light more to a single plane (more polarised). Thus, the time a molecule takes to rotate through a given angle is related to its size and is used in the FPA as an indicator of the presence of antibodies in the sample (Watanabe and Miyai, 1988). A FPA for the measurement of goat antibodies to C. perfringens type D epsilon toxin is reported in this communication.

MATERIAL AND METHODS

Freeze-dried, activated C. perfringens epsilon toxin was obtained from a commercial batch for vaccine production at Commonwealth Serum Laboratories (Melbourne, Australia). The toxin was reconstituted in sterile distilled water and dialyzed against 10mM Tris, pH 7.5, at 4ºC, overnight. Two columns (1.0 x 10cm) were prepared with DEAE Sepharose CL6B (Pharmacia, Sweden) equilibrated in 10mM Tris, pH 7.5. The toxin was applied to one column and the effluent was monitored at 220nm. The initially eluted peak was saved, dialyzed against distilled water and freeze-dried. This freeze-dried toxin was dissolved in 10mM Tris, pH 7.5, and applied to the second DEAE Sepharose column. The first peak was dialyzed and freeze dried. The toxin was dissolved in distilled water and applied to a Sephadex G25 fine column (25 x 1cm), equilibrated in 10mM Tris, pH 7.5, and the first peak was retained and freeze-dried. This last product was labelled with fluorescein isothiocyanate (FITC) using the method described by Lin et al ( 1996). Briefly, lyophilized epsilon toxin (1 mg, in 0.5 ml of 0.85% saline) was mixed with 0.15% ml of FITC (1 mg/ml) in 0.15 M Na₂HPO₄-NaOH (pH 9.5) and incubated at 37ºC for 1.5 hr. FITC-labelled epsilon toxin was purified by chromatography on a column of Sephadex G-25 (1 x 23 cm) pre-equilibrated with 0.1 M Na₂HPO₄-NaH₂PO₄ buffer (pH 7.0) containing 0.04% NaN₃. The degree of incorporation of fluorescein groups into epsilon toxin was determined by independent measurements of the concentrations of FITC and protein in a given sample. The FITC concentration was estimated spectrophotometrically, taking the molar absorption coefficient to be 7.45 x 10⁴ M^-1 cm^-1 (Bernardt et al, 1983). The concentration of epsilon toxin in this sample was determined by the colorimetric method of Smith et al (1985), using a bicinchoninic acid protein assay kit (Pierce Life Science and Analytical Research Products, Canada) and bovine serum albumin as standard. Under the condition employed, reaction of purified epsilon toxin with FITC resulted in incorporation of 2.37 fluorescein groups per epsilon toxin molecule.

A hyperimmune goat serum with a concentration of anti-epsilon toxin antibody of 723 IU/ml (measured by MNT) was used. The MNT was performed as previously described (British Pharmacopoeia, 1993). The goat hyperimmune serum was diluted in negative serum obtained from a colostrum-deprived goat kid with no antibodies against epsilon toxin, in order to obtain final dilutions equivalent to the following concentrations of epsilon antitoxin per ml: 13.44, 6.72, 3.60, 1.80, 0.40, 0.10 and 0.02 IU. These diluted sera were used as positive controls. Sera from eleven colostrum-deprived kids, obtained between 1 to 4 weeks after birth, were used as negative controls.

All the sera were diluted 1:100 in 2 ml of 0.01 M Na₂HPO₄-NaH₂PO4 buffer, pH 7.4 containing 0.15 M NaCl, 0.1 % sodium azide and 0.05% lithium dodecyl sulphate (diluent buffer) and placed in 12 x 75 mm glass tubes. A serum blank measurement was obtained with the Fluorescence Polarisation Analyser (FPM-1, Jolley Consulting and Research, Round Lake, Il 60073, USA). Twenty microliters of epsilon toxin-FITC conjugate (tracer), diluted 1:30 in diluent buffer, were added and after mixing and incubating at room temperature for at least two minutes, the fluorescence polarization (FP) of the tracer was measured in millipolarisation units (mP), with the blank subtracted. The latter determination was used as an indicator of the amount of antibody in the serum sample.

All the sera were tested ten times. The correlation coefficient between the IU values of the control positive sera and the results of the FPA readings was calculated using the means of the readings of 10 different runs, and the inter-assay variation was calculated for every control sera and expressed as coefficient of variation (CV).

RESULTS AND DISCUSSION

The inter-assay variation of the ten measurements of the seven positive control sera are presented in table 1. It varied between 1.56 % and 4.85 % .

The correlation coefficient between FP values and concentration of epsilon anti-toxin (as measured by MNT) was 0.90.

The mP obtained after testing the negative control sera (from colostrum-deprived kids), were always lower than the mP given by the weakest positive control serum (0.02 IU/ml).

Each test was performed in approximately 2 minutes.

A quick and simple in vitro test to detect C. perfringens epsilon antitoxin in serum of animals is necessary to test potency of enterotoxaemia vaccines and also as a tool in enterotoxaemia research.

Conventionally, epsilon toxin antibodies are measured in serum of animals by MNT (British Pharmaopeia, 1993). This test uses mice to determine neutralisation of epsilon toxin by serum antibodies, is laborious, requires complex standardisation processes and the results can not be read before 72 hours. The FPA technique offers several major advantages over the MNT, including its simplicity (one step), rapidity (2 minutes per sample) and the fact that it does not require the use of living animals. However, a disadvantage of this test is the requirement of the fluorescent polarization analyzer, which is a relatively expensive piece of equipment.

Indirect and competitive ELISAs have been used to measure epsilon antitoxin in serum of rabbits (Sojka et al, 1989, Wood, 1991) and of goats (Uzal et al, 1997). The correlation between these ELISA techniques and MNT varied between 0.93 and 0.99 (Sojka et al, 1989, Wood, 1991, Uzal et al, 1997). In the FPA here reported the correlation between this technique and the MNT was slightly lower (0.90), which could be a disadvantage in comparison with the ELISAs. However, the FPA is a very simple test that requires minimal manipulations and it can be performed in only a few minutes. The inter-assay variation was always very low, indicating that the test is highly reproducible.

The relative high correlation found between MNT and FPA indicates that both techniques were measuring similar entities. If this is the case, the FPA would be a good alternative to the MNT. We presented here preliminary results using a reduced number of sera; the FPA should be further validated using a larger number of sera.

RESUMEN

Se desarrolló un test de fluorescencia polarizada (TFP) para detectar antitoxina epsilon de Clostridium perfringens tipo D en suero de cabras. Se marcó toxina epsilon purificada con isothiocianato de fluoresceína y se la usó como tracer en el test. Siete sueros caprinos con diferentes concentraciones conocidas de antitoxina epsilon (titulados por una prueba de seroneutralización en ratones-PSR) se utilizaron como controles positivos. Sueros de once chivitos deprivados de calostro se utilizaron como controles negativos. La variación entre ensayos de diez mediciones de los siete sueros positivos control varió entre 1.56% y 4.85%. La correlación entre los valores de TFP y la concentración de antitoxina epsilon (medida por PSR) fue de 0.90. Este test parece ser una buena alternativa para la PSR sin los inconvenientes de esta última.

ACKNOWLEDGMENTS

Thanks are due to the following organisations for help in different ways: Fundación Antorchas, the National Institute of Agricultural Technology and the National Council of Scientific and Technical Research (the three form Argentina), the Dairy Goat Association and Commonwealth Serum Laboratories (both from Australia), and the Animal Disease Research Institute (Canada). F.A. Uzal was supported by and by an Australian Overseas Postgraduate Research Scholarship.
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Aceptado: 13.4.99.

* Dirección actual: Unidad de Salud Animal, Instituto
Nacional de Tecnología Agropecuaria, CC 277, (8400)
Bariloche, Argentina.

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