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Quantitative radial immunodiffusion assay for serum amyloid A protein
Journal of Immunological Methods, 59 (1983) 95-103
95
Elsevier Biomedical Press
Quantitative Radial Immunodiffusion Assay for Serum Amyloid A Protein Robin E. Chambers and John T. Whicher Department of Chemical Pathology, Bristol Royal Infirmary, Brz~tol BS2 8HW, U.K.
(Received 18 May 1982, accepted 17 November 1982)
A radial irnmunodiffusion assay for serum amyloid A protein (SAA) using a commercially available antiserum is described. Serum is applied untreated to 1Z agarose gels prepared in 0.02 M barbitone buffer, pH 8.6, containing 40 g/l polyethylene glycol 6000. Incubation is carried out overnight at 37°C. The assay combines the advantages of simplicity, rapidity, specificity and stability, and avoids the hazards associated with the previously described radioimmunoassays. The method has sufficient sensitivity to measure SAA in the majority (99%) of normal subjects, and confirms the behaviour of SAA as a very sensitive acute phase reactant in inflammatory disease. The method is ideally suited to the rapid processing of a large number of samples. Key words: serum amyloid A protein - - radial immunodiffusion assay - - acute-phase protein - amyloidosis
Introduction There is currently m u c h interest in the measurement of serum amyloid A protein (SAA), a normal al globulin which is thought to be the circulating precursor of tissue amyloid AA. S A A behaves as an acute-phase protein ( M c A d a m et al., 1978) and significantly increased levels have been reported in a n u m b e r of inflammatory diseases (Rosenthal and Franklin, 1975; Gorevic et al., 1976; Benson and Cohen, 1979; Rosenthal and Sullivan, 1979). The original estimations of SAA concentration were qualitative, m a d e by double i m m u n o d i f f u s i o n techniques (Levin et al., 1973; H u s b y and Natvig, 1974; Kronvall et al., 1975; Meretoja et al., 1976), although H u s b y and Natvig (1974) also used radial immunodiffusion to correlate S A A concentration with precipitation titre in strongly reacting sera. These techniques were not sufficiently sensitive to detect SAA in the majority of normal individuals. More recently, sensitive radioimmunoassays capable of quantitating S A A in normal subjects have been described. In some assays serum is analysed untreated (Rosenthal and Franklin, 1975; Benson and Cohen, 1979), but in others S A A is first denatured either with acid (Sipe et al., 1976) or alkali (Van Rijswijk, 1981). All radioimmunoassays suffer from certain inherent disadvantages in that they tend to be technically complex, use hazardous and 0022-1759/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
96 unstable reagents and require isotope counting equipment. There is therefore a need for a simple, sensitive and quantitative assay for SAA. The availability of a commercial antiserum that reacted strongly by double immunodiffusion with sera from patients with active inflammatory disease prompted us to re-assess radial immunodiffusion as a suitable assay for SAA, thereby overcoming the problems associated with the use of radioactive materials. We report here the details of an agarose gel radial immunodiffusion assay for SAA which possesses the advantages of speed, stability and simplicity, which has the desired sensitivity and which is suited to the processing of large numbers of samples.
Materials and Methods
Commercial antiserum Anti-tissue A component was purchased from Atlantic Antibodies, American Hospital Supply (U.K.) (Didcot, Oxfordshire). Reference material and samples Purified amyloid AA protein was kindly supplied by Dr K.P.W.J. McAdam, Tufts University School of Medicine, (Boston, MA). Alkali-degraded amyloid (DAM) was prepared by overnight incubation in 0.1 M N a O H (Pras et al., 1969) of amyloid AA extracted by the technique of Pras et al. (1968) from the spleen of a patient with secondary amyloidosis. A reference serum pool was prepared by combining human sera with raised SAA concentrations. The pool was calibrated against the degraded amyloid and aliquots stored at - 2 0 ° C for future use. SAA was measured in sera collected from normal healthy blood donors (n = 200) and from patients suffering from chronic inflammatory disease (n = 120). Gel diffusion Radial immunodiffusion and Ouchterlony double immunodiffusion were performed in 1% agarose gels prepared in 0.02 M barbitone buffer, p H 8.6, containing 40 g/1 polyethylene glycol 6000 (PEG). For quantitative radial immunodiffusion, 12 /~1 antiserum/ml of agarose was used, and test and reference sera (5 #l) were pipetted into 2.5 mm wells. Dilutions of the reference serum and patients' sera with SAA concentrations greater than 180 m g / l were made in 0.02 M barbitone buffer, p H 8.6. For concentrations less than 10 mg/l, 20/~1 of neat serum (4 mm wells) were used. All incubations were carried out in a moist chamber overnight at 37°C after which the gels were thoroughly washed in hypertonic saline before staining. Ring diameters were measured and a calibration graph constructed from ring diameter squared plotted against SAA concentration. Pre-incubation of serum with acid or alkali Ten sera with normal or raised SAA concentrations were pre-treated with either 10% formic acid at 37°C for 24 h (Hijmans and Sipe, 1979) or 0.1 M N a O H at room
97
temperature for 6 h (Van Rijswijk, 1981). SAA was then measured by radial immunodiffusion in the treated and original sera.
Results
Assay conditions The described conditions were chosen to give optimum immunoprecipitation. Incorporation of PEG into the gel was found to be necessary for precipitation to occur, the best results being obtained with a PEG concentration of 40 g/l. Concentrations greater than 50 g/1 resulted in incomplete dissolution of agarose during gel preparation whereas concentrations less than 40 g/1 resulted in diminished ring intensity. No precipitation tings were visible in gels that did not contain PEG. The pH of the medium was not critical, immunoprecipitation occurring equally within the pH range of 7.0-8.6. However, in gels of pH greater than 9.0 or less than 6.5, satisfactory results could not be obtained. Similarly, incubation at a temperature of 37°C was most satisfactory. At room temperature, faint precipitation rings were visible after 3 days, but at 4°C, no rings could be detected even after incubation for 1 week. Incubation at temperatures above 37°C resulted in the non-specific precipitation of serum proteins and also the antiserum in the gel.
Fig. 1. Ouchterlony double immunodiffusion analysis of alkali-degraded amyloid (DAM), purified protein AA and the reference serum pool (SAA) (outer wells) tested against the Atlantic antiserum (central well).
98 P r e - i n c u b a t i o n of serum with 10% formic acid resulted in a m a r k e d decrease in apparent SAA concentration (mean =-66%, range - 3 8 % to - 7 9 % ) , whereas p r e - i n c u b a t i o n in 0.1 M N a O H resulted in only a small decrease ( m e a n =- - 8 % , r a n g e + 3 % to - 13%). N o significant increase in S A A c o n c e n t r a t i o n was observed with either procedure.
Specificity of antiserum Double immunodiffusion A A p r o t e i n tested against p r e c i p i t i n line o n l y could be Similarly, d o u b l i n g dilutions
of the reference serum p o o l (SAA), D A M a n d purified the A t l a n t i c a n t i s e r u m is shown in Fig. 1. A single seen with each antigen, with fusion between each pair. of serum p r o d u c e d only single lines over the range from
Fig. 2. Ouchterlony double immunodiffusion analysis of doubling dilutions of the reference serum pool (outer wells) tested against the Atlantic antiserum (central wells). On the original plate, a faint precipitation line, which is not apparent in the photograph, was visible at the 1/64 dilution. The top well (N) contained neat serum.
99 n e a t serum to a 1 / 6 4 dilution. N o p r e c i p i t i n lines were visible at dilutions greater t h a n 1 / 6 4 (Fig. 2). A d s o r p t i o n o f the a n t i s e r u m b y i n c u b a t i o n with purified A A p r o t e i n overnight at 3 7 ° C resulted in d i s a p p e a r a n c e of the i m m u n o p r e c i p i t a t i o n rings with 20 sera c o n t a i n i n g n o r m a l or elevated S A A concentrations. A n t i s e r u m that h a d been inc u b a t e d overnight at 37°C with 0.02 M b a r b i t o n e b u f f e r in place of A A p r o t e i n p r o d u c e d the e x p e c t e d rings when similarly tested. N o p r e c i p i t a t i o n rings were o b s e r v e d in gels from which a n t i s e r u m h a d b e e n omitted. Precision W i t h i n - a s s a y p r e c i s i o n was d e t e r m i n e d f r o m replicate (n = 20) analyses m a d e s i m u l t a n e o u s l y on 2 serum samples, one with a n o r m a l a n d one with a raised S A A c o n c e n t r a t i o n . T h e results were 16.89 __+ 1.03 m g / 1 ( m e a n + S.D.), coefficient of v a r i a t i o n (CV) = 6.1% a n d 132.50 + 7.02 mg/1, CV = 5.3%.
0
O
Fig. 3. Radial immunodiffusion plate demonstrating the detection of SAA in normal sera (lower 4 rows of wells). The top 5 wells are dilutions of the reference serum to give standards of 2, 5, 10, 20 and 40 mg/l. The wells are 4 mm in diameter and contained 20 ~1 of sample•
100
Between assay precision was determined from serial analyses (n = 20) made over 3 months on 2 other serum pools. The results were 18.05 + 2.72 mg/1, CV = 15.1%, and 111.30+ 13.67 mg/l, CV = 12.3%. SAA concentrations in normal people and in inflammatory disease SAA was detected in 198 (99%) of 200 normal individuals, the remaining 2 individuals having SAA concentrations below the lower limit of detection of the assay (2 mg/1). The derived normal range was less than 5-30 m g / l (5th-95th percentiles). A typical radial immunodiffusion plate containing normal sera is shown in Fig. 3. Marked increases in SAA concentration were found in chronic inflammatory disease (for example, rheumatoid arthritis), the changes in SAA concentration closely paralleling (r = 0.77) those of C-reactive protein (CRP) (Fig. 4). A typical plate of increased SAA sera is shown in Fig. 5.
10000 5000
.
r=0.77
. .
!1"
~.-.
2500 •
";.s'e..
-~..
1000 •
500
• •
oo
E 250
100
i,
~ 50
%
25
10 5 2.5
I
I
I
I
I
I
I
2.5
5
10
25
50
100
250
I
500 1000
CRPCONCENTRATION(mg/I)
Fig. 4. Relationship of SAA and CRP concentration in patients (n = 120) with inflammatory disease. Both axes are plotted on log 10 scales.
101
Fig. 5. Radial immunodiffusion plate demonstrating increased SAA concentrations in patients with inflammatory disease. The top 2 rows of wells are dilutions of the reference serum to give standards over the range 5-180 rag/l, together with one quality control serum. The wells are 2.5 mm in diameter and contained 5/~l of sample.
Discussion The method described here is a conventional radial immunodiffusion assay, the only unusual features being the incorporation of P E G and incubation at 37°C, b o t h of which enhance immunoprecipitation. It thus combines the advantage of simplicity, i.e., no requirement for complex techniques or equipment with speed and reliability, and also avoids the hazardous and unstable reagents necessary for radioimmunoassay. U p to 60 samples can be analysed simultaneously on a l0 c m x 20 c m plate. The precision figures are typical of a gel diffusion technique. The Atlantic anti-tissue A antiserum appears to be specific for SAA. Single lines of identity were seen for SAA, D A M and purified A A protein in double immunodiffusion assays (Figs. 1 and 2) and adsorption of the antiserum with pure A A protein
102 abolished its SAA-precipitating ability. Non-specific protein precipitation is not a problem, although some diffuse background staining can be seen in gels that have not been kept adequately moist during incubation, or have not been thoroughly washed in hypertonic saline. The assay is sufficiently sensitive to measure SAA in the majority of normal people (Fig. 3), without recourse to the denaturation procedures that enhance immunoreactivity in some radioimmunoassays. Indeed, treatment of serum with 10% formic acid (Hijmans and Sipe, 1979) caused a marked decrease (up to 79%) in the apparent concentration of SAA rather than the expected increase. A similar finding has been reported by Benson and Cohen (1979). Preincubation in 0.1 M N a O H (Van Rijswijk, 1981) had less effect, the maximum decrease being 13%, but again no significant increase was observed. These discrepancies are probably due to differences between antisera used by each group. Each antiserum is individually prepared, usually against AA protein. Consequently, it is unlikely that any 2 antisera will be directed against identical antigenic determinants on SAA. It has been suggested (Van Rijswijk, 1981) that denaturation causes the SAA molecule to unfold, thereby unmasking previously hidden determinants. If an antiserum is directed against one of more of these epitopes, then denaturation will result in an apparent increase in SAA concentration. If, on the other hand, an antiserum recognises epitopes that are readily accessible but destroyed by unfolding, denaturation will result in an apparent decrease in immunoreactivity. The Atlantic antiserum presumably belongs to the latter group. It is of course also possible that diffusion through the agarose-PEG gel may induce some conformational change in the SAA molecule that results in increased reactivity with this particular antiserum. One of the major problems in the estimation of SAA concentrations is the absence of a suitable standard. Results in this and previously described assays (Rosenthal and Franklin, 1975; Benson and Cohen, 1979; Hijmans and Sipe, 1979; Van Rijswijk, 1981) have therefore been expressed relative to arbitrary standards (purified AA protein, DAM or a human serum pool calibrated from them). This fact, combined with the differences in technique and, in particular, the antiserum (as discussed above) probably accounts for the variation in the normal ranges reported. The normal range determined with this assay (less than 5-30 mg/1) is similar to that found by Hijmans and Sipe (1979) with radioimmunoassay after formic acid denaturation. Clinically, SAA measurements will probably be of value in monitoring inflammatory disease. SAA is known to behave as an acute-phase protein with a time course similar to CRP (McAdam et al., 1978) and markedly increased levels have been found in acute infections (Gorevic et al., 1976) and rheumatic and neoplastic diseases (Rosenthal and Franklin, 1975; Benson and Cohen, 1979; Rosenthal and Sullivan, 1979). Using radial immunodiffusion we also have found increased SAA concentrations in inflammatory disease. The changes in SAA concentration correlate well ( r = 0.77) with those of CRP (Fig. 4), although SAA appears to be the more sensitive. Van Rijswijk (1981) has also reported good correlation between SAA and CRP concentrations, and between SAA level and inflammatory activity. The radial immunodiffusion assay described offers a simple, cheap and non-
103 hazardous alternative to radioimmunoassay
allowing the measurement
of S A A as a
practical procedure in clinical laboratories.
References Benson, M.D. and A.S. Cohen, 1979, Arthr. Rheum. 22, 36. Gorevic, P.D., C.J. Rosenthal and E.C. Franklin, 1976, Clin. Immunol. Immunopathol. 6, 83. Hijmans, W. and J.D. Sipe, 1979, Clin. Exp. Immunol. 35, 96. Husby, G. and J.B. Natvig, 1974, J. Clin. Invest. 53, 1054. Kronvall, G., G. Husby, D. Samuel, G. Bjune and H. Wheate, 1975, Infect. Immun. 11,969. Levin, M., M. Pras and E.C. Franklin, 1973, J. Exp. Med. 138, 373. McAdam, K.P.WJ., R.J. Elin, J.D. Sipe and S.M. Wolff, 1978, J. Clin. Invest. 61,390. Meretoja, J., J.B. Natvig and G. Husby, 1976 Scand. J. Immunol. 5, 169. Pras, M., M. Schubert, D. Zucker-Franklin, A. Rimon and E.C. Franklin, 1968, J. Clin. Invest. 47, 924. Pras, M., D. Zucker-Franklin, A. Rimon and E.C. Franklin, 1969, J. Exp. Med. 130, 777. Rosenthal, C.J. and E.C. Franklin, 1975, J. Clin. Invest. 55, 746. Rosenthal, C.J. and L.M. Sullivan, 1979, Ann. Int. Med. 91,383. Sipe, J.D., T.F. lgnaczak, P.S. Pollock and G.G. Glenner, 1976, J. Immunol. 116, 1151. Van Rijswijk, M.H., 1981, Amyloidosis (PhD Thesis, University of Groningen, Netherlands).
95
Elsevier Biomedical Press
Quantitative Radial Immunodiffusion Assay for Serum Amyloid A Protein Robin E. Chambers and John T. Whicher Department of Chemical Pathology, Bristol Royal Infirmary, Brz~tol BS2 8HW, U.K.
(Received 18 May 1982, accepted 17 November 1982)
A radial irnmunodiffusion assay for serum amyloid A protein (SAA) using a commercially available antiserum is described. Serum is applied untreated to 1Z agarose gels prepared in 0.02 M barbitone buffer, pH 8.6, containing 40 g/l polyethylene glycol 6000. Incubation is carried out overnight at 37°C. The assay combines the advantages of simplicity, rapidity, specificity and stability, and avoids the hazards associated with the previously described radioimmunoassays. The method has sufficient sensitivity to measure SAA in the majority (99%) of normal subjects, and confirms the behaviour of SAA as a very sensitive acute phase reactant in inflammatory disease. The method is ideally suited to the rapid processing of a large number of samples. Key words: serum amyloid A protein - - radial immunodiffusion assay - - acute-phase protein - amyloidosis
Introduction There is currently m u c h interest in the measurement of serum amyloid A protein (SAA), a normal al globulin which is thought to be the circulating precursor of tissue amyloid AA. S A A behaves as an acute-phase protein ( M c A d a m et al., 1978) and significantly increased levels have been reported in a n u m b e r of inflammatory diseases (Rosenthal and Franklin, 1975; Gorevic et al., 1976; Benson and Cohen, 1979; Rosenthal and Sullivan, 1979). The original estimations of SAA concentration were qualitative, m a d e by double i m m u n o d i f f u s i o n techniques (Levin et al., 1973; H u s b y and Natvig, 1974; Kronvall et al., 1975; Meretoja et al., 1976), although H u s b y and Natvig (1974) also used radial immunodiffusion to correlate S A A concentration with precipitation titre in strongly reacting sera. These techniques were not sufficiently sensitive to detect SAA in the majority of normal individuals. More recently, sensitive radioimmunoassays capable of quantitating S A A in normal subjects have been described. In some assays serum is analysed untreated (Rosenthal and Franklin, 1975; Benson and Cohen, 1979), but in others S A A is first denatured either with acid (Sipe et al., 1976) or alkali (Van Rijswijk, 1981). All radioimmunoassays suffer from certain inherent disadvantages in that they tend to be technically complex, use hazardous and 0022-1759/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
96 unstable reagents and require isotope counting equipment. There is therefore a need for a simple, sensitive and quantitative assay for SAA. The availability of a commercial antiserum that reacted strongly by double immunodiffusion with sera from patients with active inflammatory disease prompted us to re-assess radial immunodiffusion as a suitable assay for SAA, thereby overcoming the problems associated with the use of radioactive materials. We report here the details of an agarose gel radial immunodiffusion assay for SAA which possesses the advantages of speed, stability and simplicity, which has the desired sensitivity and which is suited to the processing of large numbers of samples.
Materials and Methods
Commercial antiserum Anti-tissue A component was purchased from Atlantic Antibodies, American Hospital Supply (U.K.) (Didcot, Oxfordshire). Reference material and samples Purified amyloid AA protein was kindly supplied by Dr K.P.W.J. McAdam, Tufts University School of Medicine, (Boston, MA). Alkali-degraded amyloid (DAM) was prepared by overnight incubation in 0.1 M N a O H (Pras et al., 1969) of amyloid AA extracted by the technique of Pras et al. (1968) from the spleen of a patient with secondary amyloidosis. A reference serum pool was prepared by combining human sera with raised SAA concentrations. The pool was calibrated against the degraded amyloid and aliquots stored at - 2 0 ° C for future use. SAA was measured in sera collected from normal healthy blood donors (n = 200) and from patients suffering from chronic inflammatory disease (n = 120). Gel diffusion Radial immunodiffusion and Ouchterlony double immunodiffusion were performed in 1% agarose gels prepared in 0.02 M barbitone buffer, p H 8.6, containing 40 g/1 polyethylene glycol 6000 (PEG). For quantitative radial immunodiffusion, 12 /~1 antiserum/ml of agarose was used, and test and reference sera (5 #l) were pipetted into 2.5 mm wells. Dilutions of the reference serum and patients' sera with SAA concentrations greater than 180 m g / l were made in 0.02 M barbitone buffer, p H 8.6. For concentrations less than 10 mg/l, 20/~1 of neat serum (4 mm wells) were used. All incubations were carried out in a moist chamber overnight at 37°C after which the gels were thoroughly washed in hypertonic saline before staining. Ring diameters were measured and a calibration graph constructed from ring diameter squared plotted against SAA concentration. Pre-incubation of serum with acid or alkali Ten sera with normal or raised SAA concentrations were pre-treated with either 10% formic acid at 37°C for 24 h (Hijmans and Sipe, 1979) or 0.1 M N a O H at room
97
temperature for 6 h (Van Rijswijk, 1981). SAA was then measured by radial immunodiffusion in the treated and original sera.
Results
Assay conditions The described conditions were chosen to give optimum immunoprecipitation. Incorporation of PEG into the gel was found to be necessary for precipitation to occur, the best results being obtained with a PEG concentration of 40 g/l. Concentrations greater than 50 g/1 resulted in incomplete dissolution of agarose during gel preparation whereas concentrations less than 40 g/1 resulted in diminished ring intensity. No precipitation tings were visible in gels that did not contain PEG. The pH of the medium was not critical, immunoprecipitation occurring equally within the pH range of 7.0-8.6. However, in gels of pH greater than 9.0 or less than 6.5, satisfactory results could not be obtained. Similarly, incubation at a temperature of 37°C was most satisfactory. At room temperature, faint precipitation rings were visible after 3 days, but at 4°C, no rings could be detected even after incubation for 1 week. Incubation at temperatures above 37°C resulted in the non-specific precipitation of serum proteins and also the antiserum in the gel.
Fig. 1. Ouchterlony double immunodiffusion analysis of alkali-degraded amyloid (DAM), purified protein AA and the reference serum pool (SAA) (outer wells) tested against the Atlantic antiserum (central well).
98 P r e - i n c u b a t i o n of serum with 10% formic acid resulted in a m a r k e d decrease in apparent SAA concentration (mean =-66%, range - 3 8 % to - 7 9 % ) , whereas p r e - i n c u b a t i o n in 0.1 M N a O H resulted in only a small decrease ( m e a n =- - 8 % , r a n g e + 3 % to - 13%). N o significant increase in S A A c o n c e n t r a t i o n was observed with either procedure.
Specificity of antiserum Double immunodiffusion A A p r o t e i n tested against p r e c i p i t i n line o n l y could be Similarly, d o u b l i n g dilutions
of the reference serum p o o l (SAA), D A M a n d purified the A t l a n t i c a n t i s e r u m is shown in Fig. 1. A single seen with each antigen, with fusion between each pair. of serum p r o d u c e d only single lines over the range from
Fig. 2. Ouchterlony double immunodiffusion analysis of doubling dilutions of the reference serum pool (outer wells) tested against the Atlantic antiserum (central wells). On the original plate, a faint precipitation line, which is not apparent in the photograph, was visible at the 1/64 dilution. The top well (N) contained neat serum.
99 n e a t serum to a 1 / 6 4 dilution. N o p r e c i p i t i n lines were visible at dilutions greater t h a n 1 / 6 4 (Fig. 2). A d s o r p t i o n o f the a n t i s e r u m b y i n c u b a t i o n with purified A A p r o t e i n overnight at 3 7 ° C resulted in d i s a p p e a r a n c e of the i m m u n o p r e c i p i t a t i o n rings with 20 sera c o n t a i n i n g n o r m a l or elevated S A A concentrations. A n t i s e r u m that h a d been inc u b a t e d overnight at 37°C with 0.02 M b a r b i t o n e b u f f e r in place of A A p r o t e i n p r o d u c e d the e x p e c t e d rings when similarly tested. N o p r e c i p i t a t i o n rings were o b s e r v e d in gels from which a n t i s e r u m h a d b e e n omitted. Precision W i t h i n - a s s a y p r e c i s i o n was d e t e r m i n e d f r o m replicate (n = 20) analyses m a d e s i m u l t a n e o u s l y on 2 serum samples, one with a n o r m a l a n d one with a raised S A A c o n c e n t r a t i o n . T h e results were 16.89 __+ 1.03 m g / 1 ( m e a n + S.D.), coefficient of v a r i a t i o n (CV) = 6.1% a n d 132.50 + 7.02 mg/1, CV = 5.3%.
0
O
Fig. 3. Radial immunodiffusion plate demonstrating the detection of SAA in normal sera (lower 4 rows of wells). The top 5 wells are dilutions of the reference serum to give standards of 2, 5, 10, 20 and 40 mg/l. The wells are 4 mm in diameter and contained 20 ~1 of sample•
100
Between assay precision was determined from serial analyses (n = 20) made over 3 months on 2 other serum pools. The results were 18.05 + 2.72 mg/1, CV = 15.1%, and 111.30+ 13.67 mg/l, CV = 12.3%. SAA concentrations in normal people and in inflammatory disease SAA was detected in 198 (99%) of 200 normal individuals, the remaining 2 individuals having SAA concentrations below the lower limit of detection of the assay (2 mg/1). The derived normal range was less than 5-30 m g / l (5th-95th percentiles). A typical radial immunodiffusion plate containing normal sera is shown in Fig. 3. Marked increases in SAA concentration were found in chronic inflammatory disease (for example, rheumatoid arthritis), the changes in SAA concentration closely paralleling (r = 0.77) those of C-reactive protein (CRP) (Fig. 4). A typical plate of increased SAA sera is shown in Fig. 5.
10000 5000
.
r=0.77
. .
!1"
~.-.
2500 •
";.s'e..
-~..
1000 •
500
• •
oo
E 250
100
i,
~ 50
%
25
10 5 2.5
I
I
I
I
I
I
I
2.5
5
10
25
50
100
250
I
500 1000
CRPCONCENTRATION(mg/I)
Fig. 4. Relationship of SAA and CRP concentration in patients (n = 120) with inflammatory disease. Both axes are plotted on log 10 scales.
101
Fig. 5. Radial immunodiffusion plate demonstrating increased SAA concentrations in patients with inflammatory disease. The top 2 rows of wells are dilutions of the reference serum to give standards over the range 5-180 rag/l, together with one quality control serum. The wells are 2.5 mm in diameter and contained 5/~l of sample.
Discussion The method described here is a conventional radial immunodiffusion assay, the only unusual features being the incorporation of P E G and incubation at 37°C, b o t h of which enhance immunoprecipitation. It thus combines the advantage of simplicity, i.e., no requirement for complex techniques or equipment with speed and reliability, and also avoids the hazardous and unstable reagents necessary for radioimmunoassay. U p to 60 samples can be analysed simultaneously on a l0 c m x 20 c m plate. The precision figures are typical of a gel diffusion technique. The Atlantic anti-tissue A antiserum appears to be specific for SAA. Single lines of identity were seen for SAA, D A M and purified A A protein in double immunodiffusion assays (Figs. 1 and 2) and adsorption of the antiserum with pure A A protein
102 abolished its SAA-precipitating ability. Non-specific protein precipitation is not a problem, although some diffuse background staining can be seen in gels that have not been kept adequately moist during incubation, or have not been thoroughly washed in hypertonic saline. The assay is sufficiently sensitive to measure SAA in the majority of normal people (Fig. 3), without recourse to the denaturation procedures that enhance immunoreactivity in some radioimmunoassays. Indeed, treatment of serum with 10% formic acid (Hijmans and Sipe, 1979) caused a marked decrease (up to 79%) in the apparent concentration of SAA rather than the expected increase. A similar finding has been reported by Benson and Cohen (1979). Preincubation in 0.1 M N a O H (Van Rijswijk, 1981) had less effect, the maximum decrease being 13%, but again no significant increase was observed. These discrepancies are probably due to differences between antisera used by each group. Each antiserum is individually prepared, usually against AA protein. Consequently, it is unlikely that any 2 antisera will be directed against identical antigenic determinants on SAA. It has been suggested (Van Rijswijk, 1981) that denaturation causes the SAA molecule to unfold, thereby unmasking previously hidden determinants. If an antiserum is directed against one of more of these epitopes, then denaturation will result in an apparent increase in SAA concentration. If, on the other hand, an antiserum recognises epitopes that are readily accessible but destroyed by unfolding, denaturation will result in an apparent decrease in immunoreactivity. The Atlantic antiserum presumably belongs to the latter group. It is of course also possible that diffusion through the agarose-PEG gel may induce some conformational change in the SAA molecule that results in increased reactivity with this particular antiserum. One of the major problems in the estimation of SAA concentrations is the absence of a suitable standard. Results in this and previously described assays (Rosenthal and Franklin, 1975; Benson and Cohen, 1979; Hijmans and Sipe, 1979; Van Rijswijk, 1981) have therefore been expressed relative to arbitrary standards (purified AA protein, DAM or a human serum pool calibrated from them). This fact, combined with the differences in technique and, in particular, the antiserum (as discussed above) probably accounts for the variation in the normal ranges reported. The normal range determined with this assay (less than 5-30 mg/1) is similar to that found by Hijmans and Sipe (1979) with radioimmunoassay after formic acid denaturation. Clinically, SAA measurements will probably be of value in monitoring inflammatory disease. SAA is known to behave as an acute-phase protein with a time course similar to CRP (McAdam et al., 1978) and markedly increased levels have been found in acute infections (Gorevic et al., 1976) and rheumatic and neoplastic diseases (Rosenthal and Franklin, 1975; Benson and Cohen, 1979; Rosenthal and Sullivan, 1979). Using radial immunodiffusion we also have found increased SAA concentrations in inflammatory disease. The changes in SAA concentration correlate well ( r = 0.77) with those of CRP (Fig. 4), although SAA appears to be the more sensitive. Van Rijswijk (1981) has also reported good correlation between SAA and CRP concentrations, and between SAA level and inflammatory activity. The radial immunodiffusion assay described offers a simple, cheap and non-
103 hazardous alternative to radioimmunoassay
allowing the measurement
of S A A as a
practical procedure in clinical laboratories.
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