Archs oral Bid. Vol.3.5,No. 9, pp. 741-746,1990 Printedin Great Britain.,411rightsreserved
0003-9969/!30 $3.00+ 0.00 Copyright0 1990Pergamon Press plc
AN ENZYME-LINKED IMMUNOASSAY SALIVARY ALBUMIN
FOR HUMAN
D. SWEENEY and J. A. BEELEY* Oral Biochemistry Unit (Oral Biology Group), University of Glasgow Dental Hospital and School, 378 Sauchiehall St, Glasgow G2 352, Scotland (Received 17 October 1989; accepted 28 March 1990) Abstract-An ELISA method has been developed for the determination of albumin in human parotid saliva. The ra.nge of sensitivity is 2-80 pg/l and it is suitable for use with volumes of 5 ~1 or less of freshly collected parotid fluid, without prior sample preparation. Calibration data were fitted by microcomputer to an exponential function. The albumin concentration of parotid saliva was 1.9 k 1.53 mg/l, but levels fell on sampb: storage. Isoelectric focusing, followed by immunoblotting with antiserum to human serum albumin, showed identical double bands of pI 4.9-5.0 in albumin from both serum and saliva, thereby confirming that the antigens from the two sources are identical. Key words: parotid saliva, serum albumin, immunoassay.
tedious, requires larger volumes than may be available from a diseased gland and can lead to losses and alterations in protein structure, resulting in differing reactivity to antisera immunoglobulins. For adequate sensitivity with parotid saliva, prior concentration of parotid saliva samples for rocket immunoelectrophoresis is also desirable. Immunodiffusion methods rely on measuring very small differences in the zones of migration of antigens and are therefore very imprecise. This has produced highly variable values for salivary albumin (Hattingh, 1979). A far more sensitive, rapid and more reliable method is therefore required. Salivary albumin has not been proved to be structurally identical to plasma albumin, an important consideration in immunoassays, where it is assumed that the cross-reactivities of the standard and antigen in the sample are identical. We now describe an ELISA method suitable for measuring albumin in small samples of parotid saliva and other oral secretions.
:[NTRODUCTION Human saliva has a protein content of approx. OS-3 mg/ml, with albumin constituting about 1% of this in whole saliva and about 0.1% in parotid saliva (Mason and Chisholm, 1975). It probably results from passive diffusion from the plasma into the salivary ducts (Oppenheim, 1970) and, in the case of whole saliva, from the gingival crevice as well. Elevated levels of parotid salivary albumin have been associated with parotitis (Tabak et al., 1978), Sjagrens’s syndrome (Stuchell et al., 1978) and sarcoidosis (Beeley and Chisholm, 1976). Increased levels in whole saliva are also an early indicator of mucositis (Izutsu et al., 1981), gingivitis (Oppenheim, 1970; Meechan, 1983), inflammation and damage to oral tissues in general. The role of albumin, if any, in duct saliva is unknown. It could conceivably assist transport of as yet unspecified plasma constituents between the salivary glands and plasma. Because of the low levels of albumin in saliva, especially parotid Isaliva, where the normal level is of the order of l-2 mg/l, and the consequent difficulties in accurately assaying this protein, the potential diagnostic value of analysing salivary albumin has not been fully exploited. Parotid salivary albumin has been measured largely by single radial immunodiffusion (Stuchell et al., 1978; Beeleq and Chisholm, 1976; Izutsu et al., 1981) and electro-immunodiffusion (rocket electrophoresis; Seiner, Merrill and Claman, 1968; Lindsay et al., 1979). It is generally accepted that the earlier gel and paper electrophoresis and bromo-cresol green staining methods substantially overestimate albumin due to contaminating proteins (Ferguson, 1975). Single radial hnmunodiffusion requires prior concentration of parotid and minor gland salivas. This is
MATERIALSAND
METHODS
Saliva
Parotid saliva was collected between 1000 and 1600 h, after at least 1 h of fasting, from 32 healthy individuals, aged between 17 and 62 yr. A modified Carlsson-Crittenden cup (Stephen and Speirs, 1976) was used and salivary flow was stimulated by 10% (w/v) citric acid. Saliva was collected into ice-cooled containers and either assayed immediately or stored frozen at -25°C until used. Flow rates were in the range 0.3-2.0 ml/min. Antisera
*To whom correspondence should be addressed. Abbreviation: ELISA, enzyme-linked immunosorbent assay.
and conjugates
Sheep antiserum to human serum albumin, donkey anti-rabbit IgG conjugated to horseradish peroxidase, donkey anti-sheep/goat IgG and sheep peroxidase-anti-peroxidase complex were obtained from
741
142
D. SWEENEY and J. A. BEELEY
the Scottish Antibody Production Unit, Law Hospital, Carluke, Lanarkshire, U.K. Rabbit antiserum to human serum albumin (ORCB 05) was obtained from Behring, Salisbury Road, Hounslow, Middlesex, England. Donkey anti-sheep IgG conjugated to alkaline phosphatase was obtained from Sigma Chemical Company Ltd, Fancy Rd, Poole, Dorset, U.K. Preparation of immunoglobulin fractions IgG was fractionated from the sheep and rabbit anti-albumin antisera by n-octanoic acid precipitation (Steinbuch and Audran, 1969), followed by ion-exchange adsorption. The pH of 1 ml of antiserum was adjusted to 5.0 with 0.1 M acetic acid and 88 ~1 of n-octanoic acid were added slowly with stirring. Stirring was continued for 30 min at room temperature, after which the suspension was centrifuged at 15OOg for 20 min at 16°C and the supernatant retained. The pellet was washed with 1 ml of 0.1 M sodium bicarbonate and centrifuged again. The pooled supematants were dialysed overnight at 4°C against 0.1 M sodium bicarbonate and then concentrated about 2-fold by covering the dialysis bag with solid polyethylene glycol 10,000 (BDH Chemicals Ltd, Poole, Dorset, U.K.). Dialysis was then continued for 72 h against phosphate-buffered saline (buffer A; NaCl 8.0 g/l, KC1 0.2 g/l, Na,HPO, 1.15 g/l, KH,PO, 0.2 g/l, pH 7.4) containing 0.015% (w/v) thimerosal (Sigma). Sheep and rabbit anti-albumin fractions contained about 40 and 5 mg/ml protein respectively at this stage. About 0.5 ml of partially purified IgG was dialysed overnight against 0.1 M tris-HCl buffer, pH 7.0, and QAE-Sephadex A-50 (Pharmacia-LKB plc, Phannacia House, Midsummer Boulevard, Milton Keynes, Bucks, U.K.) equilibrated against the same buffer, was added to give a final volume of l-2ml. After brief mixing, the suspension was centrifuged at 1500 g for 20 min at 4°C and the supernatant dialyzed against 0.05 M sodium phosphate buffer, pH 7.2, overnight at 4°C. The final protein concentrations of the sheep and rabbit IgG solutions were about 10 and 1 mg/ml respectively. Protein assay Protein was estimated by the BCA method [Pierce and Warriner (U.K.) Ltd, 44 Upper Northgate St, Chester, U.K.; Smith et al., 19851 using bovine albumin (Pentex Bovine Albumin 81-001; Miles Scientific, Stoke Poges, Slough, Bucks, U.K.) standardized by its specific extinction (A, 1% w/v, 1 cm, 280 nm, was determined as 6.55). Albumin standards Standard human serum (ORDT 07) and purified human albumin (ORHA 21) were obtained from Behring. Purified human serum albumin solution (118 g/l) was obtained from the Scottish Blood Transfusion Service, Protein Fractionation Centre, Ellen’s Glen Rd, Edinburgh, U.K. For ELISA calibration curves, a stock standard solution of serum albumin was prepared from standard human serum (Behring) by dilution with 0.1 M NaCl to give a solution containing 250 mg/l albumin.
This standard was compared with the solution of purified human serum albumin (Scottish Blood Transfusion Service) and with the purified serum albumin (Behring), standardized by its specific extinction (A, 1% w/v, 1 cm, 280 nm was determined as 5.5 using the pure protein) ELISA method Purified sheep anti-human albumin IgG was diluted (typically l/1000) with 0.05 M carbonate-bicarbonate buffer (Na,CO, 1.59 g/l, NaHCO, 2.93 g/l, pH 9.6) and samples (100 pl) were added to the wells of microtitre plates (Alpha Laboratories Ltd, Eastleigh, Hampshire, U.K.). Plates were left overnight at 4°C. The plates were then washed repeatedly with buffer A containing 0.5 g/l Tween 20 (buffer B). The stock standard serum albumin solution was initially diluted l/1000 (250 pg/l) with buffer B and serial 2-fold dilutions of this were prepared to give 8 standard solutions in the concentration range 1.9-250 pg/l albumin. Saliva was diluted with buffer B to give approximate albumin concentrations in the same range (l/5&1/100 for normal parotid saliva). Serum albumin standards and saliva samples (100 p 1)were added to the wells in quadruplicate and the plates allowed to stand for at least 90min at room temperature. After washing with buffer B, 100 ~1 of purified rabbit anti-albumin IgG, appropriately diluted (typically l/5000) with the same buffer, were added to the wells and the plates were left at room temperature for a further 90 min. After washing, 100 ~1 of donkey anti-rabbit IgG-horse radish peroxidase conjugate, diluted l/1000 with buffer B, were added to the wells and the plates again left at room temperature for 90min, or at 4°C overnight, and then washed. All incubations were carried out in a humid chamber. Freshly prepared substrate solution at room temperature (100 ~1: 3.7 mM o -phenylenediamine (Sigma) and 3.5 mM hydrogen peroxide in McIlvaine’s buffer, pH 5.0-prepared by mixing 48.5ml 0.1 M citric acid with 51.5ml 0.2M Na,HPO,) was added to the wells, and the plates were left in the dark at room temperature. The reaction was stopped after a suitable interval (usually about 10 min) by addition of 1.0 M sulphuric acid (50 ~1). The incubation period was chosen to give an absorbance reading of about l-l.5 for the highest standard. Plates were read on a Minireader II plate reader (Dynatech Laboratories Ltd, Billingshurst, Sussex, U.K.) with a 490 mn filter fitted. Calculation of results The absorbance values of the standards were fitted to the function (Beynon, 1985): A,=A,,+(A,-A,,)*exp(-kc) using an iterative curve-fitting computer program (Enzfltter; Elsevier-BIOSOFT, 68 Hill’s Rd, Cambridge, U.K.). A,,, and A, are the absorbance maximum and minimum respectively, A, is the absorbance at concentration c, and k is a constant. AO, A, and k were estimated by the program using absorbance values from the albumin standards, and albumin
143
Immunoassay of salivary albumin
chloride, in 50 mM sodium carbonate/bicarbonate buffer, pH 9.61 until clearly defined bands were obtained. The membranes were then washed with water. Due to the low sensitivity of this method at the low loads of albumin attainable with some unconcentrated parotid salivas (approx. 0.3 pg maximum), attempts were made to amplify the detection sensitivity using a peroxidase-anti-peroxidase immunocomplex procedure (Mason et al., 1982). After blotting, nitrocellulose membranes were blocked in buffer C containing 5% (w/v) skimmed milk powder (Marvela, Premier Brands U.K. Ltd, Birmingham, U.K.) for 1 h at room temperature and placed in the sheep anti-human albumin IgG fraction diluted l/400 with the same solution. After standing overnight at 4°C the membranes were washed in 3 changes of buffer C and placed in donkey antisheep/goat IgG antiserum diluted l/400 with buffer C. After standing for 2 h at room temperature the membranes were washed as before and placed in sheep peroxidaseanti-peroxidase complex, diluted l/400 with buffer B, and left at room temperature for 2 h. Sequential addition of donkey anti-sheep/goat IgG and sheep peroxidase-anti-peroxidase complex was repeated twice more to further amplify the sensitivity, and the washed membranes were then placed in substrate solution [O.O3%(w/v) H202, O.O15%(w/v) Cchloro-1-naphthol in phosphate/citrate buffer, pH 6.0, prepared by mixing 0.2M Na,HPO, and 0.1 M citric acid] until bands developed. The reaction was stopped by washing the membranes with water.
values of saliva samples were calculated by substituting the absorbance values obtained, in the expression: \. c =
In
(Al - &ax)~1
7-d
,
>,.
(“1, - ‘%EkX~ K
Isoelectric focusing Isoelectric focusing was carried out in thin layers (0.8 mm) of polyacrylamide (T = 4.85%; C = 0.15%) in a LKB 2117 Multiphor apparatus (PharmaciaLKB plc) accordinl; to the manufacturer’s instructions (LKB Application note No. 75). Parotid saliva (dialysed against distilled water), purified human albumin (Behring) and serum from human volunteers were focused in gradients of pH 4-6 for 3 h at 10 W (1200 V maximum) at 10°C. The pH gradient of the gels was determined by means of Isoelectric Point Markers, pI range 2.4-5.65 (BDH). Western blotting Isoelectric focusing gels were blotted onto nitrocellulose membranes (Hybond-C, Amersham International, Aylesbury, Bucks, U.K.) on an LKB Multiphor II Nova Blot according to the manufacturer’s instructions l(LKB manual 90-02-3379), using a discontinuous buffer system. Blots were probed for human albumin by a modification of the method described by Blake et al. (1984). In brief, blots were washed in 3 changes of buffer A containing 5 g/l Tween 20 (buffer C) and placed in the sheep antihuman albumin IgG fraction, diluted (typically l/1000) with the same buffer. After standing 2 h at room temperature, the membranes were washed as before and then placed in donkey anti-sheep IgG-alkaline phosphatase conjugate, diluted l/l000 with buffer C, and left overnight at 4°C. After washing, the membranes were blotted on tissue and placed in substrate solution [O.Ol% (w/v) nitro blue tetrazolium containing 0.005% (w/v) 5-bromo-4chloro-indoxyl phosphate and 4 mM magnesium
RESULTS
Figure l(A) shows a calibration curve for the ELISA of albumin in the range 1.95-25Opg/l. The data fitted closely to the monoexponential function, A, = A,,, + (A, - A,,,).exp(-kc).
(A)
.-
2.0
./’ / , (B)
o.oL___ i
-2 1 0
0.4
I 1
I 2
I 3
CAlbuminl
0
40
60
120
[Albumid
160
I 4
(pg
I1
I 5
10’
I
200
24
j_
(pg/l)
Fig. I. (A) Fit of calibration data for the determination of albumin by an ELISA method to a mono-exponential function. (B) Linear transformation of the data shown in (A). The points shown are the means of quadruplicate measurements.
D. SWEENEY amd J. A.
144 0.6 t
01' 0
10
20
30
[Albumin]
40
50
60
70
00
(pg / 1)
Fig. 2. Calibration curve routinely used for the determination of salivary albumin by the ELISA method. 0: Standard human serum (Behring); values obtained using purified human serum albumin (Scottish Antibody Production Unit or Behnng) could be superimposed on this curve. 0: Normal parotid saliva (see text). All analyses were performed in quadruplicate. Trueness of fit was confirmed by the observations that residuals were small and random and that plots of ln(Amax- A3 vs albumin concentration, a linear transformation of the function, were satisfactory in the range 1.95-62.5 pg/l [Fig. l(B)]. (Distortion of the fit above this concentration was obviously due to the transformation as the error distribution was satisfactory in the exponential plot.) The shape of the curve, as used routinely, is shown in Fig. 2. Curves obtained using the 3 different standard albumins could be superimposed. The lower limit of albumin, differing from 0 at the 95% confidence limit, was 0.8 pg/l; the
BEELEY
working lower limit was 1.95pg/l, the value of the lowest standard, which had a typical coefficient of variation of 6.3%. The upper limit chosen was 80 pg/l, as above this value experimental error became significant due to flattening of the curve. The albumin content of parotid saliva was determined by this procedure and a range of dilutions was then assayed; Fig. 2 shows that salivary albumin responds in exactly the same way as serum albumin and the results are not affected by the extent of dilution of the sample. Purified human serum albumin added to diluted parotid saliva was recovered at 103.5% (average of quadruplicate measurements). The mean albumin level of stimulated parotid saliva from the 32 healthy individuals, calculated using the log normal distribution, was 1.9 mg/l (SD f 1.53). The measured albumin levels in samples stored at -25°C dropped over time. For example, the mean albumin concentration of 9 parotid saliva samples dropped from 1.94 to 1.40 mg/l during 2 months of storage. In order to ascertain the identity of serum and salivary albumin, samples were subjected to isoelectric focusing followed by immunoblotting using antiserum to human serum albumin as a probe. Amplification by the sheep peroxide-anti-peroxide complex resulted in a 3-fold increase in sensitivity of detection as compared with conventional immunoblotting. The results are shown diagrammatically in Fig. 3; identical band patterns were formed with both procedures. Purified human serum albumin appeared heterogeneous, focusing as a 2-component zone of p1 approx. 4.9-5.0, with evidence of microheterogeneity within each zone. The patterns obtained with salivary and serum albumin were identical (Fig. 3, tracks b PI
(+I
b
c
d
-
3.65
-
3.95
-
5.65
t-1
Fig. 3. Isoelectric focusing of serum and salivary albumins followed by immunoblotting with antiserum to human serum albumin. a-Purified human serum albumin (Behring); b-human parotid saliva (dialysed); c-human serum; d-human parotid saliva after concentration with polyethylene glycol. (Samples b and c were from the same subject.) The samples in each track contained -0.3 yg albumin.
hnmunoassay
and c). Albumin in saliva that had been concentrated by means of polyethylene glycol, however, showed increased heterogeneity and changes in p1 (Fig. 3, track d), and focused in the pH range 4.9-5.1 with some minor components of p1 as high as 5.3. IMSCUSSION
Published values for albumin levels in stimulated normal parotid saliva, measured by single radial immunodiffusion, have ranged from 0.26 to 25 mg/l (Tabak et al., 1978; Hattingh, 1979). Immunodiffusion is an inherently inaccurate method at low concentrations of antigen, and the parotid saliva was concentrated, freeze-dried or stored for long periods before analysis, giving rise to further sources of error. Such sample preparation may also cause loss of or changes in the albumin, as we have now shown. In addition, with single radial immunodiffusion there are difficulties in accurately measuring the diameters of the precipitin rings and a need for samples of sufficient size (several ml). Solid-phase immunofluorescent radial im!munodiffusion, a more sensitive modification of single radial immunodiffusion, still has a lower limit of sensitivity for albumin of 16.3 mg/l (Izutsu et (zZ.,1980). Electra-immunodilfusion offers considerable advantages over radial immunodiffusion both in terms of accuracy and sample size, and analyses can be performed on 2 ~1 samples of freshly collected saliva. With this ta:hnique, Lindsay et al. (1979) found albumin levels in stimulated parotid saliva of 6 f 3 mg/l (range 2--15). However, whilst the technique was used successfully for submandibular, sublingual and minor gland secretions where the albumin levels were 11 + 7, 35 + 16 and 63 &-30 mg/l, respectively, in the case of parotid secretion the levels were so low as to make precise measurement of rocket heights impossible. The introduction of enzyme immunoassays has brought large increases in sensitivity and has, for example, allowed the monitoring of urinary albumin levels in diabetic patients with microalbuminuria (Fielding, Price and Houlton, 1983; Coppo et al., 1985; Feldt-Rasmussen, Dinesen and Deckert, 1985). However, the lower limit of sensitivity of these procedures was inadequate for assay of small volumes of parotid saliva and further refinement of the technique was necessary. Our ELBA meth.od has the level of sensitivity necessary and is well suited for determining albumin in saliva from all glands, and other fluids where the concentrations are as low as 5 pg/l, or where only very small volumes of sample are available. The lower limit of sensitivity is 2 pg/l and the practical range is 2-80 pg/l. The method is precise and easy, and with computer-aided curve fitting, variations due to operator judgement are (eliminated. Curves can be fitted by eye, however. Degradation of salivary proteins during sample concentration resulted in alterations in the pattern of the albumin bands in isoelectric focusing (Fig. 3, track d). Similar observations were made after freezing and thawing. Measured levels of salivary albumin dropped by approx. 28% over 2 months of storage, a finding which reinforces
145
of salivary albumin
the need to carry out assays
on freshly collected samples. The albumin level of normal parotid saliva, determined using freshly collected material, was 1.9 + 1.53 mg/l. Antibody purification procedures are simple and need only be carried out infrequently; the requirement is for only a small amount of commonly available antisera. Saliva samples can be assayed immediately after collection. The method appears to be specific, as calibration curves prepared simultaneously with standard human albumin and purified human albumin standardized by specific absorption measurements could be superimposed. Parotid saliva is normally diluted 1 in 100 and the response is directly proportional to dilution over a wide range of concentrations. It has been suggested that salivary albumin may not be identical to that of plasma (Ferguson, 1975), and that differences in antigenic determinants may result in low estimates of salivary albumin with immunological methods that use antisera raised against the serum antigen. This proposal, however, is not borne out by our findings. Comparison of freshly collected saliva with serum from the same subject by isoelectric focusing, followed by immunoblotting, consistently indicated that the albumins in these 2 fluids are identical in p1. Small differences in molecular structure, if present, should have been easily detected by this high resolution method. Our procedure adequately overcomes the problems encountered with earlier procedures for the analysis of salivary albumin and the sensitivity is adequate for the accurate monitoring of changes in albumin levels associated with various pathological disorders. Acknowledgements-The financial assistance of the Wood Boyd fund from the University of Glasgow Faculty of Medicine for part of this work is gratefully acknowledged.
REFERENCES
Beeley J. A. and Chisholm D. M. (1976) Sarcoidosis with salivary gland involvement: biochemical studies on parotid saliva. .I. Lab. clin. Med. 88, 276-281. Beynon R. J. (1985) A general purpose non-linear curve fitting program for the British Broadcasting Corporation Microcomputer. Comput. Appl. Biosci. 1, 11l-l 15. Blake M. S., Johnston K. H., Russell-Jones G. J. and Gotschlich E. C. (1984) A rapid, sensitive method for detection of alkaline phosphatase-conjugated antiantibodv on Western blots. Analvt. Biochem. 136. 175-179: Coppo R., Cerutti F., Amore A., Guidoni C., Saitta M., Piccoli G. and Nicola P. (1985) An enzyme immunoassay to detect microalbuminuha in diabetic patients. Diabe;. Care 8, 525-526. Feldt-Rasmussen B., Dinesen B. and Deckert M. (1985) Enzyme immunoassay: an improved determination of urinary albumin in diabetics with incipient nephropathy. Stand. J. Clin. Lab. Invest. 45. 539-544. Ferauson D. B. (1975) Saliva&_ glands and saliva. In: Applied Physiology of’the Mouth (Edited by Lavelle C. L. B.) DD. 145-179. Wright. Bristol. Fielding B. A., Price b. .A. and Houlton C. A. (1983) Enzyme immunoassay for urinary albumin. Clin. Chem. 29, 355-351. Hattingh J. (1979) Albumins in saliva: what concentration? S. Afr. J. Sci. 75, 184186. Izutsu K., Truelove E., Felton S., Siegel I., Madden P. and Schubert M. (19801 \ , A solid ohase immunofluorescent I
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D. SWEENEY and J. A. BEELEY
assay for the measurement of salivary immunoglobulin and albumin levels. J. dent. Res. 59, 1192-1196. Izutsu K. T., Truelove E. L., Bleyer W. A., Anderson W. M., Schubert M. M. and Rice J. C. (1981) Whole saliva albumin as an indicator of stomatitis in cancer therapy patients. Cancer 4& 1450-1454. Lindsay J. C. B., Meechan J. G., Stephen K. W. and Beeley J. A. (1979) Albumin concentrations in human saliva from individual glands. J. dent. Res. 58, 1244. Mason D. K. and Chisholm D. M. (1975) Saliuary Glands in Health and Disease, pp. 42-50. Saunders, London. Mason D. Y., Cordell J. L., Abdulaziz Z., Naiem M. and Bordenave G. (1982) Preparation of peroxidase antineroxidase (PAP) complexes for immunohistological labeling of monoclonal- antibodies. J. Histochem. Cytochem. 30, 1114-1122. Meechan J. G. (1983) Salivary albumin concentration and periodontal disease. J. dent. Res. 62, 439. Oppenheim F. G. (1970) Preliminary observations on the presence and origin of serum albumin in human saliva. Helv. odonr. Acta 14, 10-17.
Seiner A. C., Merrill D. A. and Claman H. N. (1968) Salivary immunoglobulin and albumin: development during the newborn period. J. Pediat. 72, 685-689.
Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenxano M. D., Fujimonto E. K., Goeke N. M., Olson B. J. and Klenk D. C. (1985) Measurement of protein using bicinchoninic acid. Analyt. Biochem. 150, 7685. Steinbuch M. and Audran R. (1969) The isolation of IgG from mammalian sera with the aid of caprylic acid. Archs Biochem. Biophys. 134, 279-284.
Stephen K. W. and Speirs C. F. (1976) Methods for collecting individual components of mixed saliva. Br. J. clin. Pharmac. 3, 3 16-3 19. Stuchell R. N., Mandel I. D., Herrera M. and Baurmash H. (1978) Clinical utilisation of sialochemistry in Sjogren’s syndrome. J. oral Path. 13, 303-309. Tabak K., Mandel I. D., Herrera M. and Baurmash H. (1978) Changes in lactoferrin and other proteins in a case of chronic recurrent parotitis. J. oral Puth. 7, 91-99.
0003-9969/!30 $3.00+ 0.00 Copyright0 1990Pergamon Press plc
AN ENZYME-LINKED IMMUNOASSAY SALIVARY ALBUMIN
FOR HUMAN
D. SWEENEY and J. A. BEELEY* Oral Biochemistry Unit (Oral Biology Group), University of Glasgow Dental Hospital and School, 378 Sauchiehall St, Glasgow G2 352, Scotland (Received 17 October 1989; accepted 28 March 1990) Abstract-An ELISA method has been developed for the determination of albumin in human parotid saliva. The ra.nge of sensitivity is 2-80 pg/l and it is suitable for use with volumes of 5 ~1 or less of freshly collected parotid fluid, without prior sample preparation. Calibration data were fitted by microcomputer to an exponential function. The albumin concentration of parotid saliva was 1.9 k 1.53 mg/l, but levels fell on sampb: storage. Isoelectric focusing, followed by immunoblotting with antiserum to human serum albumin, showed identical double bands of pI 4.9-5.0 in albumin from both serum and saliva, thereby confirming that the antigens from the two sources are identical. Key words: parotid saliva, serum albumin, immunoassay.
tedious, requires larger volumes than may be available from a diseased gland and can lead to losses and alterations in protein structure, resulting in differing reactivity to antisera immunoglobulins. For adequate sensitivity with parotid saliva, prior concentration of parotid saliva samples for rocket immunoelectrophoresis is also desirable. Immunodiffusion methods rely on measuring very small differences in the zones of migration of antigens and are therefore very imprecise. This has produced highly variable values for salivary albumin (Hattingh, 1979). A far more sensitive, rapid and more reliable method is therefore required. Salivary albumin has not been proved to be structurally identical to plasma albumin, an important consideration in immunoassays, where it is assumed that the cross-reactivities of the standard and antigen in the sample are identical. We now describe an ELISA method suitable for measuring albumin in small samples of parotid saliva and other oral secretions.
:[NTRODUCTION Human saliva has a protein content of approx. OS-3 mg/ml, with albumin constituting about 1% of this in whole saliva and about 0.1% in parotid saliva (Mason and Chisholm, 1975). It probably results from passive diffusion from the plasma into the salivary ducts (Oppenheim, 1970) and, in the case of whole saliva, from the gingival crevice as well. Elevated levels of parotid salivary albumin have been associated with parotitis (Tabak et al., 1978), Sjagrens’s syndrome (Stuchell et al., 1978) and sarcoidosis (Beeley and Chisholm, 1976). Increased levels in whole saliva are also an early indicator of mucositis (Izutsu et al., 1981), gingivitis (Oppenheim, 1970; Meechan, 1983), inflammation and damage to oral tissues in general. The role of albumin, if any, in duct saliva is unknown. It could conceivably assist transport of as yet unspecified plasma constituents between the salivary glands and plasma. Because of the low levels of albumin in saliva, especially parotid Isaliva, where the normal level is of the order of l-2 mg/l, and the consequent difficulties in accurately assaying this protein, the potential diagnostic value of analysing salivary albumin has not been fully exploited. Parotid salivary albumin has been measured largely by single radial immunodiffusion (Stuchell et al., 1978; Beeleq and Chisholm, 1976; Izutsu et al., 1981) and electro-immunodiffusion (rocket electrophoresis; Seiner, Merrill and Claman, 1968; Lindsay et al., 1979). It is generally accepted that the earlier gel and paper electrophoresis and bromo-cresol green staining methods substantially overestimate albumin due to contaminating proteins (Ferguson, 1975). Single radial hnmunodiffusion requires prior concentration of parotid and minor gland salivas. This is
MATERIALSAND
METHODS
Saliva
Parotid saliva was collected between 1000 and 1600 h, after at least 1 h of fasting, from 32 healthy individuals, aged between 17 and 62 yr. A modified Carlsson-Crittenden cup (Stephen and Speirs, 1976) was used and salivary flow was stimulated by 10% (w/v) citric acid. Saliva was collected into ice-cooled containers and either assayed immediately or stored frozen at -25°C until used. Flow rates were in the range 0.3-2.0 ml/min. Antisera
*To whom correspondence should be addressed. Abbreviation: ELISA, enzyme-linked immunosorbent assay.
and conjugates
Sheep antiserum to human serum albumin, donkey anti-rabbit IgG conjugated to horseradish peroxidase, donkey anti-sheep/goat IgG and sheep peroxidase-anti-peroxidase complex were obtained from
741
142
D. SWEENEY and J. A. BEELEY
the Scottish Antibody Production Unit, Law Hospital, Carluke, Lanarkshire, U.K. Rabbit antiserum to human serum albumin (ORCB 05) was obtained from Behring, Salisbury Road, Hounslow, Middlesex, England. Donkey anti-sheep IgG conjugated to alkaline phosphatase was obtained from Sigma Chemical Company Ltd, Fancy Rd, Poole, Dorset, U.K. Preparation of immunoglobulin fractions IgG was fractionated from the sheep and rabbit anti-albumin antisera by n-octanoic acid precipitation (Steinbuch and Audran, 1969), followed by ion-exchange adsorption. The pH of 1 ml of antiserum was adjusted to 5.0 with 0.1 M acetic acid and 88 ~1 of n-octanoic acid were added slowly with stirring. Stirring was continued for 30 min at room temperature, after which the suspension was centrifuged at 15OOg for 20 min at 16°C and the supernatant retained. The pellet was washed with 1 ml of 0.1 M sodium bicarbonate and centrifuged again. The pooled supematants were dialysed overnight at 4°C against 0.1 M sodium bicarbonate and then concentrated about 2-fold by covering the dialysis bag with solid polyethylene glycol 10,000 (BDH Chemicals Ltd, Poole, Dorset, U.K.). Dialysis was then continued for 72 h against phosphate-buffered saline (buffer A; NaCl 8.0 g/l, KC1 0.2 g/l, Na,HPO, 1.15 g/l, KH,PO, 0.2 g/l, pH 7.4) containing 0.015% (w/v) thimerosal (Sigma). Sheep and rabbit anti-albumin fractions contained about 40 and 5 mg/ml protein respectively at this stage. About 0.5 ml of partially purified IgG was dialysed overnight against 0.1 M tris-HCl buffer, pH 7.0, and QAE-Sephadex A-50 (Pharmacia-LKB plc, Phannacia House, Midsummer Boulevard, Milton Keynes, Bucks, U.K.) equilibrated against the same buffer, was added to give a final volume of l-2ml. After brief mixing, the suspension was centrifuged at 1500 g for 20 min at 4°C and the supernatant dialyzed against 0.05 M sodium phosphate buffer, pH 7.2, overnight at 4°C. The final protein concentrations of the sheep and rabbit IgG solutions were about 10 and 1 mg/ml respectively. Protein assay Protein was estimated by the BCA method [Pierce and Warriner (U.K.) Ltd, 44 Upper Northgate St, Chester, U.K.; Smith et al., 19851 using bovine albumin (Pentex Bovine Albumin 81-001; Miles Scientific, Stoke Poges, Slough, Bucks, U.K.) standardized by its specific extinction (A, 1% w/v, 1 cm, 280 nm, was determined as 6.55). Albumin standards Standard human serum (ORDT 07) and purified human albumin (ORHA 21) were obtained from Behring. Purified human serum albumin solution (118 g/l) was obtained from the Scottish Blood Transfusion Service, Protein Fractionation Centre, Ellen’s Glen Rd, Edinburgh, U.K. For ELISA calibration curves, a stock standard solution of serum albumin was prepared from standard human serum (Behring) by dilution with 0.1 M NaCl to give a solution containing 250 mg/l albumin.
This standard was compared with the solution of purified human serum albumin (Scottish Blood Transfusion Service) and with the purified serum albumin (Behring), standardized by its specific extinction (A, 1% w/v, 1 cm, 280 nm was determined as 5.5 using the pure protein) ELISA method Purified sheep anti-human albumin IgG was diluted (typically l/1000) with 0.05 M carbonate-bicarbonate buffer (Na,CO, 1.59 g/l, NaHCO, 2.93 g/l, pH 9.6) and samples (100 pl) were added to the wells of microtitre plates (Alpha Laboratories Ltd, Eastleigh, Hampshire, U.K.). Plates were left overnight at 4°C. The plates were then washed repeatedly with buffer A containing 0.5 g/l Tween 20 (buffer B). The stock standard serum albumin solution was initially diluted l/1000 (250 pg/l) with buffer B and serial 2-fold dilutions of this were prepared to give 8 standard solutions in the concentration range 1.9-250 pg/l albumin. Saliva was diluted with buffer B to give approximate albumin concentrations in the same range (l/5&1/100 for normal parotid saliva). Serum albumin standards and saliva samples (100 p 1)were added to the wells in quadruplicate and the plates allowed to stand for at least 90min at room temperature. After washing with buffer B, 100 ~1 of purified rabbit anti-albumin IgG, appropriately diluted (typically l/5000) with the same buffer, were added to the wells and the plates were left at room temperature for a further 90 min. After washing, 100 ~1 of donkey anti-rabbit IgG-horse radish peroxidase conjugate, diluted l/1000 with buffer B, were added to the wells and the plates again left at room temperature for 90min, or at 4°C overnight, and then washed. All incubations were carried out in a humid chamber. Freshly prepared substrate solution at room temperature (100 ~1: 3.7 mM o -phenylenediamine (Sigma) and 3.5 mM hydrogen peroxide in McIlvaine’s buffer, pH 5.0-prepared by mixing 48.5ml 0.1 M citric acid with 51.5ml 0.2M Na,HPO,) was added to the wells, and the plates were left in the dark at room temperature. The reaction was stopped after a suitable interval (usually about 10 min) by addition of 1.0 M sulphuric acid (50 ~1). The incubation period was chosen to give an absorbance reading of about l-l.5 for the highest standard. Plates were read on a Minireader II plate reader (Dynatech Laboratories Ltd, Billingshurst, Sussex, U.K.) with a 490 mn filter fitted. Calculation of results The absorbance values of the standards were fitted to the function (Beynon, 1985): A,=A,,+(A,-A,,)*exp(-kc) using an iterative curve-fitting computer program (Enzfltter; Elsevier-BIOSOFT, 68 Hill’s Rd, Cambridge, U.K.). A,,, and A, are the absorbance maximum and minimum respectively, A, is the absorbance at concentration c, and k is a constant. AO, A, and k were estimated by the program using absorbance values from the albumin standards, and albumin
143
Immunoassay of salivary albumin
chloride, in 50 mM sodium carbonate/bicarbonate buffer, pH 9.61 until clearly defined bands were obtained. The membranes were then washed with water. Due to the low sensitivity of this method at the low loads of albumin attainable with some unconcentrated parotid salivas (approx. 0.3 pg maximum), attempts were made to amplify the detection sensitivity using a peroxidase-anti-peroxidase immunocomplex procedure (Mason et al., 1982). After blotting, nitrocellulose membranes were blocked in buffer C containing 5% (w/v) skimmed milk powder (Marvela, Premier Brands U.K. Ltd, Birmingham, U.K.) for 1 h at room temperature and placed in the sheep anti-human albumin IgG fraction diluted l/400 with the same solution. After standing overnight at 4°C the membranes were washed in 3 changes of buffer C and placed in donkey antisheep/goat IgG antiserum diluted l/400 with buffer C. After standing for 2 h at room temperature the membranes were washed as before and placed in sheep peroxidaseanti-peroxidase complex, diluted l/400 with buffer B, and left at room temperature for 2 h. Sequential addition of donkey anti-sheep/goat IgG and sheep peroxidase-anti-peroxidase complex was repeated twice more to further amplify the sensitivity, and the washed membranes were then placed in substrate solution [O.O3%(w/v) H202, O.O15%(w/v) Cchloro-1-naphthol in phosphate/citrate buffer, pH 6.0, prepared by mixing 0.2M Na,HPO, and 0.1 M citric acid] until bands developed. The reaction was stopped by washing the membranes with water.
values of saliva samples were calculated by substituting the absorbance values obtained, in the expression: \. c =
In
(Al - &ax)~1
7-d
,
>,.
(“1, - ‘%EkX~ K
Isoelectric focusing Isoelectric focusing was carried out in thin layers (0.8 mm) of polyacrylamide (T = 4.85%; C = 0.15%) in a LKB 2117 Multiphor apparatus (PharmaciaLKB plc) accordinl; to the manufacturer’s instructions (LKB Application note No. 75). Parotid saliva (dialysed against distilled water), purified human albumin (Behring) and serum from human volunteers were focused in gradients of pH 4-6 for 3 h at 10 W (1200 V maximum) at 10°C. The pH gradient of the gels was determined by means of Isoelectric Point Markers, pI range 2.4-5.65 (BDH). Western blotting Isoelectric focusing gels were blotted onto nitrocellulose membranes (Hybond-C, Amersham International, Aylesbury, Bucks, U.K.) on an LKB Multiphor II Nova Blot according to the manufacturer’s instructions l(LKB manual 90-02-3379), using a discontinuous buffer system. Blots were probed for human albumin by a modification of the method described by Blake et al. (1984). In brief, blots were washed in 3 changes of buffer A containing 5 g/l Tween 20 (buffer C) and placed in the sheep antihuman albumin IgG fraction, diluted (typically l/1000) with the same buffer. After standing 2 h at room temperature, the membranes were washed as before and then placed in donkey anti-sheep IgG-alkaline phosphatase conjugate, diluted l/l000 with buffer C, and left overnight at 4°C. After washing, the membranes were blotted on tissue and placed in substrate solution [O.Ol% (w/v) nitro blue tetrazolium containing 0.005% (w/v) 5-bromo-4chloro-indoxyl phosphate and 4 mM magnesium
RESULTS
Figure l(A) shows a calibration curve for the ELISA of albumin in the range 1.95-25Opg/l. The data fitted closely to the monoexponential function, A, = A,,, + (A, - A,,,).exp(-kc).
(A)
.-
2.0
./’ / , (B)
o.oL___ i
-2 1 0
0.4
I 1
I 2
I 3
CAlbuminl
0
40
60
120
[Albumid
160
I 4
(pg
I1
I 5
10’
I
200
24
j_
(pg/l)
Fig. I. (A) Fit of calibration data for the determination of albumin by an ELISA method to a mono-exponential function. (B) Linear transformation of the data shown in (A). The points shown are the means of quadruplicate measurements.
D. SWEENEY amd J. A.
144 0.6 t
01' 0
10
20
30
[Albumin]
40
50
60
70
00
(pg / 1)
Fig. 2. Calibration curve routinely used for the determination of salivary albumin by the ELISA method. 0: Standard human serum (Behring); values obtained using purified human serum albumin (Scottish Antibody Production Unit or Behnng) could be superimposed on this curve. 0: Normal parotid saliva (see text). All analyses were performed in quadruplicate. Trueness of fit was confirmed by the observations that residuals were small and random and that plots of ln(Amax- A3 vs albumin concentration, a linear transformation of the function, were satisfactory in the range 1.95-62.5 pg/l [Fig. l(B)]. (Distortion of the fit above this concentration was obviously due to the transformation as the error distribution was satisfactory in the exponential plot.) The shape of the curve, as used routinely, is shown in Fig. 2. Curves obtained using the 3 different standard albumins could be superimposed. The lower limit of albumin, differing from 0 at the 95% confidence limit, was 0.8 pg/l; the
BEELEY
working lower limit was 1.95pg/l, the value of the lowest standard, which had a typical coefficient of variation of 6.3%. The upper limit chosen was 80 pg/l, as above this value experimental error became significant due to flattening of the curve. The albumin content of parotid saliva was determined by this procedure and a range of dilutions was then assayed; Fig. 2 shows that salivary albumin responds in exactly the same way as serum albumin and the results are not affected by the extent of dilution of the sample. Purified human serum albumin added to diluted parotid saliva was recovered at 103.5% (average of quadruplicate measurements). The mean albumin level of stimulated parotid saliva from the 32 healthy individuals, calculated using the log normal distribution, was 1.9 mg/l (SD f 1.53). The measured albumin levels in samples stored at -25°C dropped over time. For example, the mean albumin concentration of 9 parotid saliva samples dropped from 1.94 to 1.40 mg/l during 2 months of storage. In order to ascertain the identity of serum and salivary albumin, samples were subjected to isoelectric focusing followed by immunoblotting using antiserum to human serum albumin as a probe. Amplification by the sheep peroxide-anti-peroxide complex resulted in a 3-fold increase in sensitivity of detection as compared with conventional immunoblotting. The results are shown diagrammatically in Fig. 3; identical band patterns were formed with both procedures. Purified human serum albumin appeared heterogeneous, focusing as a 2-component zone of p1 approx. 4.9-5.0, with evidence of microheterogeneity within each zone. The patterns obtained with salivary and serum albumin were identical (Fig. 3, tracks b PI
(+I
b
c
d
-
3.65
-
3.95
-
5.65
t-1
Fig. 3. Isoelectric focusing of serum and salivary albumins followed by immunoblotting with antiserum to human serum albumin. a-Purified human serum albumin (Behring); b-human parotid saliva (dialysed); c-human serum; d-human parotid saliva after concentration with polyethylene glycol. (Samples b and c were from the same subject.) The samples in each track contained -0.3 yg albumin.
hnmunoassay
and c). Albumin in saliva that had been concentrated by means of polyethylene glycol, however, showed increased heterogeneity and changes in p1 (Fig. 3, track d), and focused in the pH range 4.9-5.1 with some minor components of p1 as high as 5.3. IMSCUSSION
Published values for albumin levels in stimulated normal parotid saliva, measured by single radial immunodiffusion, have ranged from 0.26 to 25 mg/l (Tabak et al., 1978; Hattingh, 1979). Immunodiffusion is an inherently inaccurate method at low concentrations of antigen, and the parotid saliva was concentrated, freeze-dried or stored for long periods before analysis, giving rise to further sources of error. Such sample preparation may also cause loss of or changes in the albumin, as we have now shown. In addition, with single radial immunodiffusion there are difficulties in accurately measuring the diameters of the precipitin rings and a need for samples of sufficient size (several ml). Solid-phase immunofluorescent radial im!munodiffusion, a more sensitive modification of single radial immunodiffusion, still has a lower limit of sensitivity for albumin of 16.3 mg/l (Izutsu et (zZ.,1980). Electra-immunodilfusion offers considerable advantages over radial immunodiffusion both in terms of accuracy and sample size, and analyses can be performed on 2 ~1 samples of freshly collected saliva. With this ta:hnique, Lindsay et al. (1979) found albumin levels in stimulated parotid saliva of 6 f 3 mg/l (range 2--15). However, whilst the technique was used successfully for submandibular, sublingual and minor gland secretions where the albumin levels were 11 + 7, 35 + 16 and 63 &-30 mg/l, respectively, in the case of parotid secretion the levels were so low as to make precise measurement of rocket heights impossible. The introduction of enzyme immunoassays has brought large increases in sensitivity and has, for example, allowed the monitoring of urinary albumin levels in diabetic patients with microalbuminuria (Fielding, Price and Houlton, 1983; Coppo et al., 1985; Feldt-Rasmussen, Dinesen and Deckert, 1985). However, the lower limit of sensitivity of these procedures was inadequate for assay of small volumes of parotid saliva and further refinement of the technique was necessary. Our ELBA meth.od has the level of sensitivity necessary and is well suited for determining albumin in saliva from all glands, and other fluids where the concentrations are as low as 5 pg/l, or where only very small volumes of sample are available. The lower limit of sensitivity is 2 pg/l and the practical range is 2-80 pg/l. The method is precise and easy, and with computer-aided curve fitting, variations due to operator judgement are (eliminated. Curves can be fitted by eye, however. Degradation of salivary proteins during sample concentration resulted in alterations in the pattern of the albumin bands in isoelectric focusing (Fig. 3, track d). Similar observations were made after freezing and thawing. Measured levels of salivary albumin dropped by approx. 28% over 2 months of storage, a finding which reinforces
145
of salivary albumin
the need to carry out assays
on freshly collected samples. The albumin level of normal parotid saliva, determined using freshly collected material, was 1.9 + 1.53 mg/l. Antibody purification procedures are simple and need only be carried out infrequently; the requirement is for only a small amount of commonly available antisera. Saliva samples can be assayed immediately after collection. The method appears to be specific, as calibration curves prepared simultaneously with standard human albumin and purified human albumin standardized by specific absorption measurements could be superimposed. Parotid saliva is normally diluted 1 in 100 and the response is directly proportional to dilution over a wide range of concentrations. It has been suggested that salivary albumin may not be identical to that of plasma (Ferguson, 1975), and that differences in antigenic determinants may result in low estimates of salivary albumin with immunological methods that use antisera raised against the serum antigen. This proposal, however, is not borne out by our findings. Comparison of freshly collected saliva with serum from the same subject by isoelectric focusing, followed by immunoblotting, consistently indicated that the albumins in these 2 fluids are identical in p1. Small differences in molecular structure, if present, should have been easily detected by this high resolution method. Our procedure adequately overcomes the problems encountered with earlier procedures for the analysis of salivary albumin and the sensitivity is adequate for the accurate monitoring of changes in albumin levels associated with various pathological disorders. Acknowledgements-The financial assistance of the Wood Boyd fund from the University of Glasgow Faculty of Medicine for part of this work is gratefully acknowledged.
REFERENCES
Beeley J. A. and Chisholm D. M. (1976) Sarcoidosis with salivary gland involvement: biochemical studies on parotid saliva. .I. Lab. clin. Med. 88, 276-281. Beynon R. J. (1985) A general purpose non-linear curve fitting program for the British Broadcasting Corporation Microcomputer. Comput. Appl. Biosci. 1, 11l-l 15. Blake M. S., Johnston K. H., Russell-Jones G. J. and Gotschlich E. C. (1984) A rapid, sensitive method for detection of alkaline phosphatase-conjugated antiantibodv on Western blots. Analvt. Biochem. 136. 175-179: Coppo R., Cerutti F., Amore A., Guidoni C., Saitta M., Piccoli G. and Nicola P. (1985) An enzyme immunoassay to detect microalbuminuha in diabetic patients. Diabe;. Care 8, 525-526. Feldt-Rasmussen B., Dinesen B. and Deckert M. (1985) Enzyme immunoassay: an improved determination of urinary albumin in diabetics with incipient nephropathy. Stand. J. Clin. Lab. Invest. 45. 539-544. Ferauson D. B. (1975) Saliva&_ glands and saliva. In: Applied Physiology of’the Mouth (Edited by Lavelle C. L. B.) DD. 145-179. Wright. Bristol. Fielding B. A., Price b. .A. and Houlton C. A. (1983) Enzyme immunoassay for urinary albumin. Clin. Chem. 29, 355-351. Hattingh J. (1979) Albumins in saliva: what concentration? S. Afr. J. Sci. 75, 184186. Izutsu K., Truelove E., Felton S., Siegel I., Madden P. and Schubert M. (19801 \ , A solid ohase immunofluorescent I
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D. SWEENEY and J. A. BEELEY
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