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An investigation into the proteins of horse sweat (Equus caballus)
Comp. Biochem. Physiol. Vol. 73B, No. 2, pp. 375 to 378, 1982 Printed in Great Britain.
0305-0491/82/100375-04503.00/0 © 1982 Pergamon Press Ltd
AN INVESTIGATION INTO THE PROTEINS OF HORSE SWEAT (EQUUS CABALLUS) P. D. ECKERSALL*,M. G. KERR*fand D. H. SNowt *Department of Veterinary Biochemistry, -;-Department of Veterinary Pharmacology, University of Glasgow Veterinary School, Bearsden Road, Bearsden, Glasgow G61 IQH, U.K. (Received 20 January 1982) Abstract 1. Equine sweat has a high protein content which was previously thought to consist mostly of serum albumin. 2. Investigation of adrenaline and heat-induced equine sweat has now revealed that serum albumin contributes less than l°J0 to the total secreted protein. 3. The major equine sweat protein consists of two groups of differing molecular weight, the greater of which was glycosylated. 4. The major sweat proteins had low isoelectric points.
INTRODUCTION Equine sweat is unusual in that a relatively high concentration of protein is present, particularly at the onset of secretion, in comparison to other species (Jenkinson et al., 1974). Human sweat has, for instance, ten times less protein than the equine secretion. The rate of protein lost in heat-induced equine sweat decreases with the length of time of secretion and an exponential decrease has been reported (Kerr et al., 1980). Early investigators of equine sweat (Smith, 1890; Jirka & Kotas, 1959) assumed that serum albumin contributed the major portion of the sweat protein although the presence of mucoprotein was recognised. More recently equine sweat protein were separated into three anodal bands on cellulose acetate electrophoresis (Jenkinson et al., 1974) but these were neither characterised or identified. A number of other serum protein, as well as albumin, have been identified immunologically in human, ovine and bovine sweat (Page & Remington, 1967; Jenkinson et al., 1979; Lloyd et al., 1977). The protein of equine sweat has now been investigated, revealing that the constituents responsible for the high protein content were hitherto unknown proteins and not serum albumin. MATERIALS AND METHODS
different times, were pooled and concentrated by dialysis against polyethylene glycol. Heat-induced sweat (Kerr et al., 1980I was also taken from eight of the horses and ponies. Electrophoresis in the presence of sodium dodecyl sulphate (Laemmli, 1970) was performed in 15~ (w/v) polyacrylamide gels (SDS-PAGE)+. Isoelectric focusing was performed (Radola, 1980) on ultrathin (100 #rot polyacrylamide in a pH gradient ofpH 2.5-8.0 (1.2°,,, w/v pH 2.5 5.0 and 1.21'; w/v pH 5.(~8.0, pharmalyte: Pharmacia Uppsala, Swedenl. Gel filtration on sephadex G-100 (Pharmacia, Uppsala, Sweden) was carried out in a 85 × 1.5cm column with a 0.05 M phosphate buffer pH 7.0 (0.02°; w/'v sodium azide) at 40C. Eluates were monitored for protein by absorbance at E280 and for carbohydrate by the phenol-sulphuric acid reaction (Williams & Chase, 1968). Gel filtration and SDSPAGE were calibrated with standard proteins. Protein was assayed by the method of Bradford (1976) using bovine serum albumin as standard. Equine serum albumin was estimated by radial immunodiffusion (Ouchterlony & Nilsson, 1973) using monospecific antiserum to ESA raised in rabbits (Nordic Immunologicals). Immunoelectrophoresis (Arquembourg, 1975) was performed at 5v/cm for 1.25hr in a barbitone buffer pH 8.6 (20 mmol/l sodium barbitone plus 4.3 mmol/l dietthylbarbituric acid). Antiserum to equine serum was obtained from Nordic Immunologicals.
RESULTS
Equine sweat samples were collected following adrenaline infusion (0.18 gg/kg/min) from an area of the neck which had been previously clipped but was thoroughly washed just prior to collection. Sweat was scraped off with a plastic spatula, centrifuged at 700 0 and stored at -20'C. Samples were taken from nine thoroughbred horses and ponies (Equus caballus). Sequential samples were collected from one horse at intervals of 30-60rain over a 3-hr period. A further sweat sample was collected 24 hr later. For gel filtration sweat samples from the same horse, at
Abbreviations: SDS-PAGE--sodium dodecyl sulphate polyacrylamide gel electrophoresis. 375
The major components of equine sweat protein, which are responsible for the unusual high total protein concentration, were found to consist of five proteins ranging from 11,000 to 28,000 in molecular weight by SDS-PAGE (Fig. la). Minor proteins were also apparent, one of which corresponded to serum albumin. On isoelectric focusing, the major sweat proteins had an acidic pI exhibiting heterogeneity in the region pH 3.0-3.5 (Fig. lb). Sequential sweat samples (Fig. 2) showed that the exponential drop in protein secreted was mirrored by similar drops in the major proteins. The total protein dropped from 16.6g/1, which
376
P.D. ECKERSALL¢-'tal,
MW
Sw •
......
C
H
L
.....
4, 3,.
Se
U
.....
21P ii
ta~ 3"0 •
(~) 3"5 •
4"0 •
4"5 •
5"0
~
U
pn
V
Sw L
@
H °
C Se
Fig. 1. Equine sweat protein separated by (a) SDS-PAGE (5/~g) (b) isoelectric focusing (50/lg). Sw: equine sweat, C: peak C from G-100, H: peak H fl'om G-100, L: peak L from G-100. Se: equine serum protein at 1:30 dilution and MW: mol. wt × 10 4.
exceeded the capacity of the gel causing some distortion, to 0.61g/1 when protein was hardly stainable. The 24-hr sweat sample showed a substantial recovery of protein with concentration rising to 3.2 g/l. Gel filtration of equine sweat (Fig. 3) revealed two protein peaks, called L and H, which were calculated to elute with molecular weights of 33,000 and 49,000 respectively. A minor protein peak eluted with the void volume (peak C). The carbohydrate content of the eluates revealed that peak H contained over 90!~,
of the carbohydrate content of the sweat. Separation of peaks H and L was not complete but by SDSP A G E (Fig. la) H was predominantly composed ot proteins of 28~000 and 22,000 in molecular weight while L had proteins of 18,000 and 12,000 in molecular weight. Peaks H and L had similar low isoelectric points (Fig. lb). The content of serum albumin in equine sweat was low but variable whether the sweat was adrenaline or heat induced (Table 1) and in only one out of fifteen
Proteins of horse sweat
3.0
3.5
4"0
377
4"5
pH
5-0
®
5 6 7 8
l
9
Se Fig. 2. Equine sweat protein on isoelectric focusing. Sweat taken at (1) 0rain (2) lOmin (3) 30rain (4) 60 min (5) 90 min (6) 120 rain (7) 150 rain (8) 180 rain after start of adrenaline infusion and (9) 24 hr later. Se : equine serum protein at 1 : 30 dilution.
,2t
H
i )'0 t
~0 t/
I
J I I
.15
E E
C
)o~
/'
o! 0-2
10
20 FRACTION
30 NUMBER
40
50
60
Fig. 3. G-100 sephadex separation of 1.5 ml of equine sweat protein (14 g/l) in 0.05 M pH 7.0 phosphate buffer flow rate = 10ml/hr, fraction volume = 2.2 ml.
378
P.D. ECKERSALL et al. Table 1. Albumin content of equine sweat estimated by radial immunodiffusion (121
Adrenaline Induced (n - 9) Heat Induced (n = 8)
Albumin g/]
Total Protein g/1
i!,, Albumin
0.037 -t- 0,030"
13.5 + 7.6
0.41 _+ 0.51
0.017 4- 0.015
8.55 _+ 2.2
0.19 + 0.15
* Mean 4 SD. samples did the albumin rise above l°i, of the total protein. O n immunoelectrophoresis against antiserum to equine serum, equine sweat only showed a very faint precipitin line which had the mobility of equine serum albumin.
tions to further characterise these proteins may elucidate these problems. Acknowledqement This study was carried out under a grant from the Horserace Betting Levy Board. M. G. Kerr is a Horserace Betting Levy Board Research Scholar. REFERENCES
DISCUSSION C o n t r a r y to early reports (Smith, 1890; Jirka & Kotas, 1959) the major proteins of equine sweat were neither equine serum albumin, nor were they derived from other serum proteins. Albumin was found in equine sweat but only as a small percentage ( < 1°0) of the total protein. Similarly other serum proteins may be present at low levels. The major sweat proteins had lower molecular weights than albumin and were also more acidic (Fig. 1). Perhaps they are in some way related to the h u m a n sweat antigens (Page & Remington, 1967; Wilkinson et al., 1971) which were more anodic on electrophoresis than a l b u m i n ; however, these antigens were not precipitable by 15'~; (w/v) trichloroacetic acid. The elution volumes found for the two peaks on gel filtration which do not correspond to the molecular weights of the proteins determined by S D S - P A G E suggest that in the non-denaturing conditions of the column some form of aggregation is taking place. Both peaks H and L eluted with molecular weights in excess of those determined on S D S - P A G E indicating their similarity. The proteins of both peaks also have similar pl. It is possible that the proteins of the two peaks have a c o m m o n primary structure but in H a conjugated glycosyl residue leads to a higher molecular weight. Whether the presence of the glycosyl residues are artifacts of production or secretion, or whether they affect the function, will await further investigation. Glycosylated and non-glycosylated proteins which are otherwise identical have been reported in other secretions, for instance h u m a n salivary ~-amy[ase consists of two such falnilies of enzyme (Keller et al., 1971). Perhaps they are a feature of secretory proteins. The probable source of the sweat protein is in the secretory vesicles recently observed by M o n t g o m e r y et al. (1981) in the fundus sweat cells of the equine sweat gland where b o t h protein and glycoprotein have been identified. These storage vesicles became depleted during sweating which would explain the progressive drop in protein following stimulation. The function of these proteins is unknown. Speculation may include a role in secretion or a post secretory function such as antibacterial action, lnvestiga-
ARQUEMBOURG P. O. (1975) lmmunoeh, ctrophoresi.s, 2nd edn. pp. 14 37. Karger, Basle. BRAI)FORD M. M. (1976) A rapid and sensitive method for the quantitative of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248 254. JENKINSON D. M., MABON R. M. & MANSON W. (1974) Sweat proteins. Br. J. Derm. 90, 175 181. JENKINSON D. M., LLOYD D. H. & MABON R. M. (1979) The antigenic composition and source of soluble proteins on the surface of the skin of sheep. ,I. comp. Path. Ther. 89, 43 50. JIRKAS M. & KOTAS J. 119591 Some observations on the chemical composition of horse sweat..I. Physiol.. Lomt. 147, 74 77. KELLER P. J., KAUFFMAND. L., AI.LAN B. J. & WILIAAMSB. L. (1971) Further studies on the structural differences between the isoenzymes of human parotid >amylase. Biochemistry 10, 4867 4874. KERR M. G., MUXRO C. D. & Sxow D. H. (1980) Equine sweat composition during prolonged heat exposure. J Physiol.. Lond. 307, 52 53. LAEMMLI U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature. Lond. 227, 68(~685. LLOYI) D. H., MAUON R. M. & JENKINSIIN D. M. (19771 The antigenic constituents of cattle skin washings..1. Comp. Path. Ther. 87, 75 82. MONTGOMERY I., JENKINSON D. M. & ELDfR H. Y. (1981) The effects of thermal stimulation on the ultrastructure of the fundus and duct of the equine sweat gland. J. Anat. [In pressl. OUCHTERLONY 0. ~,d Nn.ssoN L. A. (1973) In thmdhook O] Experimental Immunology, (Edited by WEre D. M.) 2nd edn, Vol. 1, pp. 19.1 19.39. Blackwell Scientific Publications, Oxford. PAGE C. O. & REMINGION J. S. (1967) hnmunological studies in normal human sweat. J. Lah. elm. Med. 69, 634 650. RAI)OI.A B. J. (1980) Ultrathin-layer isoelectric focusing ila 50 100/tin polyacrylamide gels on silanizcd gklss plates or polyester films. Electrol~hore.sis i, 43 56. SMrrH F. (18901 Note on composition of the sweat of the horse. J. Physiol.. Lond. I I, 497 501. WILKINSON R. D., ELI/! J. D., MIOT M-J. & WAKKARY J. A. (1971) Autoimmunity to human sweat. J. incest. Derm. 57, 401 406. WILLIAMS C. A. & CHASe M. W. (19681 Methods in lmmunochemistrv, Vol. 1I, pp. 287 288. Academic Press, London.
0305-0491/82/100375-04503.00/0 © 1982 Pergamon Press Ltd
AN INVESTIGATION INTO THE PROTEINS OF HORSE SWEAT (EQUUS CABALLUS) P. D. ECKERSALL*,M. G. KERR*fand D. H. SNowt *Department of Veterinary Biochemistry, -;-Department of Veterinary Pharmacology, University of Glasgow Veterinary School, Bearsden Road, Bearsden, Glasgow G61 IQH, U.K. (Received 20 January 1982) Abstract 1. Equine sweat has a high protein content which was previously thought to consist mostly of serum albumin. 2. Investigation of adrenaline and heat-induced equine sweat has now revealed that serum albumin contributes less than l°J0 to the total secreted protein. 3. The major equine sweat protein consists of two groups of differing molecular weight, the greater of which was glycosylated. 4. The major sweat proteins had low isoelectric points.
INTRODUCTION Equine sweat is unusual in that a relatively high concentration of protein is present, particularly at the onset of secretion, in comparison to other species (Jenkinson et al., 1974). Human sweat has, for instance, ten times less protein than the equine secretion. The rate of protein lost in heat-induced equine sweat decreases with the length of time of secretion and an exponential decrease has been reported (Kerr et al., 1980). Early investigators of equine sweat (Smith, 1890; Jirka & Kotas, 1959) assumed that serum albumin contributed the major portion of the sweat protein although the presence of mucoprotein was recognised. More recently equine sweat protein were separated into three anodal bands on cellulose acetate electrophoresis (Jenkinson et al., 1974) but these were neither characterised or identified. A number of other serum protein, as well as albumin, have been identified immunologically in human, ovine and bovine sweat (Page & Remington, 1967; Jenkinson et al., 1979; Lloyd et al., 1977). The protein of equine sweat has now been investigated, revealing that the constituents responsible for the high protein content were hitherto unknown proteins and not serum albumin. MATERIALS AND METHODS
different times, were pooled and concentrated by dialysis against polyethylene glycol. Heat-induced sweat (Kerr et al., 1980I was also taken from eight of the horses and ponies. Electrophoresis in the presence of sodium dodecyl sulphate (Laemmli, 1970) was performed in 15~ (w/v) polyacrylamide gels (SDS-PAGE)+. Isoelectric focusing was performed (Radola, 1980) on ultrathin (100 #rot polyacrylamide in a pH gradient ofpH 2.5-8.0 (1.2°,,, w/v pH 2.5 5.0 and 1.21'; w/v pH 5.(~8.0, pharmalyte: Pharmacia Uppsala, Swedenl. Gel filtration on sephadex G-100 (Pharmacia, Uppsala, Sweden) was carried out in a 85 × 1.5cm column with a 0.05 M phosphate buffer pH 7.0 (0.02°; w/'v sodium azide) at 40C. Eluates were monitored for protein by absorbance at E280 and for carbohydrate by the phenol-sulphuric acid reaction (Williams & Chase, 1968). Gel filtration and SDSPAGE were calibrated with standard proteins. Protein was assayed by the method of Bradford (1976) using bovine serum albumin as standard. Equine serum albumin was estimated by radial immunodiffusion (Ouchterlony & Nilsson, 1973) using monospecific antiserum to ESA raised in rabbits (Nordic Immunologicals). Immunoelectrophoresis (Arquembourg, 1975) was performed at 5v/cm for 1.25hr in a barbitone buffer pH 8.6 (20 mmol/l sodium barbitone plus 4.3 mmol/l dietthylbarbituric acid). Antiserum to equine serum was obtained from Nordic Immunologicals.
RESULTS
Equine sweat samples were collected following adrenaline infusion (0.18 gg/kg/min) from an area of the neck which had been previously clipped but was thoroughly washed just prior to collection. Sweat was scraped off with a plastic spatula, centrifuged at 700 0 and stored at -20'C. Samples were taken from nine thoroughbred horses and ponies (Equus caballus). Sequential samples were collected from one horse at intervals of 30-60rain over a 3-hr period. A further sweat sample was collected 24 hr later. For gel filtration sweat samples from the same horse, at
Abbreviations: SDS-PAGE--sodium dodecyl sulphate polyacrylamide gel electrophoresis. 375
The major components of equine sweat protein, which are responsible for the unusual high total protein concentration, were found to consist of five proteins ranging from 11,000 to 28,000 in molecular weight by SDS-PAGE (Fig. la). Minor proteins were also apparent, one of which corresponded to serum albumin. On isoelectric focusing, the major sweat proteins had an acidic pI exhibiting heterogeneity in the region pH 3.0-3.5 (Fig. lb). Sequential sweat samples (Fig. 2) showed that the exponential drop in protein secreted was mirrored by similar drops in the major proteins. The total protein dropped from 16.6g/1, which
376
P.D. ECKERSALL¢-'tal,
MW
Sw •
......
C
H
L
.....
4, 3,.
Se
U
.....
21P ii
ta~ 3"0 •
(~) 3"5 •
4"0 •
4"5 •
5"0
~
U
pn
V
Sw L
@
H °
C Se
Fig. 1. Equine sweat protein separated by (a) SDS-PAGE (5/~g) (b) isoelectric focusing (50/lg). Sw: equine sweat, C: peak C from G-100, H: peak H fl'om G-100, L: peak L from G-100. Se: equine serum protein at 1:30 dilution and MW: mol. wt × 10 4.
exceeded the capacity of the gel causing some distortion, to 0.61g/1 when protein was hardly stainable. The 24-hr sweat sample showed a substantial recovery of protein with concentration rising to 3.2 g/l. Gel filtration of equine sweat (Fig. 3) revealed two protein peaks, called L and H, which were calculated to elute with molecular weights of 33,000 and 49,000 respectively. A minor protein peak eluted with the void volume (peak C). The carbohydrate content of the eluates revealed that peak H contained over 90!~,
of the carbohydrate content of the sweat. Separation of peaks H and L was not complete but by SDSP A G E (Fig. la) H was predominantly composed ot proteins of 28~000 and 22,000 in molecular weight while L had proteins of 18,000 and 12,000 in molecular weight. Peaks H and L had similar low isoelectric points (Fig. lb). The content of serum albumin in equine sweat was low but variable whether the sweat was adrenaline or heat induced (Table 1) and in only one out of fifteen
Proteins of horse sweat
3.0
3.5
4"0
377
4"5
pH
5-0
®
5 6 7 8
l
9
Se Fig. 2. Equine sweat protein on isoelectric focusing. Sweat taken at (1) 0rain (2) lOmin (3) 30rain (4) 60 min (5) 90 min (6) 120 rain (7) 150 rain (8) 180 rain after start of adrenaline infusion and (9) 24 hr later. Se : equine serum protein at 1 : 30 dilution.
,2t
H
i )'0 t
~0 t/
I
J I I
.15
E E
C
)o~
/'
o! 0-2
10
20 FRACTION
30 NUMBER
40
50
60
Fig. 3. G-100 sephadex separation of 1.5 ml of equine sweat protein (14 g/l) in 0.05 M pH 7.0 phosphate buffer flow rate = 10ml/hr, fraction volume = 2.2 ml.
378
P.D. ECKERSALL et al. Table 1. Albumin content of equine sweat estimated by radial immunodiffusion (121
Adrenaline Induced (n - 9) Heat Induced (n = 8)
Albumin g/]
Total Protein g/1
i!,, Albumin
0.037 -t- 0,030"
13.5 + 7.6
0.41 _+ 0.51
0.017 4- 0.015
8.55 _+ 2.2
0.19 + 0.15
* Mean 4 SD. samples did the albumin rise above l°i, of the total protein. O n immunoelectrophoresis against antiserum to equine serum, equine sweat only showed a very faint precipitin line which had the mobility of equine serum albumin.
tions to further characterise these proteins may elucidate these problems. Acknowledqement This study was carried out under a grant from the Horserace Betting Levy Board. M. G. Kerr is a Horserace Betting Levy Board Research Scholar. REFERENCES
DISCUSSION C o n t r a r y to early reports (Smith, 1890; Jirka & Kotas, 1959) the major proteins of equine sweat were neither equine serum albumin, nor were they derived from other serum proteins. Albumin was found in equine sweat but only as a small percentage ( < 1°0) of the total protein. Similarly other serum proteins may be present at low levels. The major sweat proteins had lower molecular weights than albumin and were also more acidic (Fig. 1). Perhaps they are in some way related to the h u m a n sweat antigens (Page & Remington, 1967; Wilkinson et al., 1971) which were more anodic on electrophoresis than a l b u m i n ; however, these antigens were not precipitable by 15'~; (w/v) trichloroacetic acid. The elution volumes found for the two peaks on gel filtration which do not correspond to the molecular weights of the proteins determined by S D S - P A G E suggest that in the non-denaturing conditions of the column some form of aggregation is taking place. Both peaks H and L eluted with molecular weights in excess of those determined on S D S - P A G E indicating their similarity. The proteins of both peaks also have similar pl. It is possible that the proteins of the two peaks have a c o m m o n primary structure but in H a conjugated glycosyl residue leads to a higher molecular weight. Whether the presence of the glycosyl residues are artifacts of production or secretion, or whether they affect the function, will await further investigation. Glycosylated and non-glycosylated proteins which are otherwise identical have been reported in other secretions, for instance h u m a n salivary ~-amy[ase consists of two such falnilies of enzyme (Keller et al., 1971). Perhaps they are a feature of secretory proteins. The probable source of the sweat protein is in the secretory vesicles recently observed by M o n t g o m e r y et al. (1981) in the fundus sweat cells of the equine sweat gland where b o t h protein and glycoprotein have been identified. These storage vesicles became depleted during sweating which would explain the progressive drop in protein following stimulation. The function of these proteins is unknown. Speculation may include a role in secretion or a post secretory function such as antibacterial action, lnvestiga-
ARQUEMBOURG P. O. (1975) lmmunoeh, ctrophoresi.s, 2nd edn. pp. 14 37. Karger, Basle. BRAI)FORD M. M. (1976) A rapid and sensitive method for the quantitative of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248 254. JENKINSON D. M., MABON R. M. & MANSON W. (1974) Sweat proteins. Br. J. Derm. 90, 175 181. JENKINSON D. M., LLOYD D. H. & MABON R. M. (1979) The antigenic composition and source of soluble proteins on the surface of the skin of sheep. ,I. comp. Path. Ther. 89, 43 50. JIRKAS M. & KOTAS J. 119591 Some observations on the chemical composition of horse sweat..I. Physiol.. Lomt. 147, 74 77. KELLER P. J., KAUFFMAND. L., AI.LAN B. J. & WILIAAMSB. L. (1971) Further studies on the structural differences between the isoenzymes of human parotid >amylase. Biochemistry 10, 4867 4874. KERR M. G., MUXRO C. D. & Sxow D. H. (1980) Equine sweat composition during prolonged heat exposure. J Physiol.. Lond. 307, 52 53. LAEMMLI U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature. Lond. 227, 68(~685. LLOYI) D. H., MAUON R. M. & JENKINSIIN D. M. (19771 The antigenic constituents of cattle skin washings..1. Comp. Path. Ther. 87, 75 82. MONTGOMERY I., JENKINSON D. M. & ELDfR H. Y. (1981) The effects of thermal stimulation on the ultrastructure of the fundus and duct of the equine sweat gland. J. Anat. [In pressl. OUCHTERLONY 0. ~,d Nn.ssoN L. A. (1973) In thmdhook O] Experimental Immunology, (Edited by WEre D. M.) 2nd edn, Vol. 1, pp. 19.1 19.39. Blackwell Scientific Publications, Oxford. PAGE C. O. & REMINGION J. S. (1967) hnmunological studies in normal human sweat. J. Lah. elm. Med. 69, 634 650. RAI)OI.A B. J. (1980) Ultrathin-layer isoelectric focusing ila 50 100/tin polyacrylamide gels on silanizcd gklss plates or polyester films. Electrol~hore.sis i, 43 56. SMrrH F. (18901 Note on composition of the sweat of the horse. J. Physiol.. Lond. I I, 497 501. WILKINSON R. D., ELI/! J. D., MIOT M-J. & WAKKARY J. A. (1971) Autoimmunity to human sweat. J. incest. Derm. 57, 401 406. WILLIAMS C. A. & CHASe M. W. (19681 Methods in lmmunochemistrv, Vol. 1I, pp. 287 288. Academic Press, London.