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Sandwich enzyme immunoassay for serum amyloid A protein (SAA)
169
Clinica Chimica Acta, 179 (1989) 169-176 Elsevier
CCA 04343
Short communication
Sandwich enzyme immunoassay for serum amyloid A protein ( SAA) Toshiyuki Yamada ‘, Kazuaki Uchiyama ‘, Minoru Yakata ’ and Fumitake Gejyo 2 of Laboratory Medicine and 2 Depa~t~eni of ~~ternat Medicine, Niigata ~‘~~v~rsitySchool of Medicine, Asahi~achi Niigata fJapan~
’ ~e~art~nt (Received
23 May 1988, revision received 3 October
Key words: Serum amyloid
A protein
1988; accepted
after revision 24 October
(SAA); Enzyme immunoassay
(EIA); Normal
1988)
value
Serum amyloid A (SAA), a putative serum precursor of the AA protein which constitutes amyloid fibrils in secondary amyloidosis [l], has been clinically evaluated as one of the sensitive acute-phase reactants in serum [2,3]. The SAA concentration has been analyzed by radial i~unodiffusion (RID) f4,5] and radioi~unoassay (RIA) [6-81. The RID method was very simple and suitable for the analysis of SAA in its increased states, but could not determine the normal range of SAA because of low sensitivity. At present there are only a few descriptions outlining the use of enzyme immunoassay (EIA) in determining the concentration of SAA in serum [8,9]. It has also been mentioned that EIA may be unsuitable for this purpose because of poor reproducibility of results [S]. In this study, we attempted to develop a sensitive and highly reproducible EIA method for the measurement of SAA in serum, and to evaluate the normal range of the SAA. Materials and methods
Amyloid fibrils were extracted from kidney sections taken from a 60-yr-old female who died from renal failure due to secondary amyloidosis [lo]. The AA protein was purified from this fibrils by two sequential gel filtrations on Sephadex G-100 (Pharmacia Fine Chemicals, Uppsala, Sweden) under 6 mol/l guanidine HCl [II]. SAA was purified from pooled sera with high concentrations of C-reactive protein (CRP) by sequential gel filtrations on Sephadex G-100, G-75, G-50, under
Correspondence to: Dr. T. Yamada, Department Medicine, Asabimacbi Niigata 951, Japan.
~9-8981/89/$03.50
0 1989 Elsevier Scien,
of Laboratory
Publishers
Medicine,
B.V. (Biomedical
Niigata
Division)
University
School of
170
10% formic acid and a final passage through a Sepharose 4B (pharmacia) coupled with antihuman whole serum (Dako, CA, USA) [12].
column
Preparation of antibodies The antiserum against AA was produced in rabbits by immunization with AA purified as above. One milligram of AA in PBS was mixed with an equal amount of Freund’s complete adjuvant and injected subcutaneously into the footpads and dorsa of the animals. Booster doses were administered biweekly using one-fifth of the first dose, and the animals were bled 3 mth after the first injection. The resulting antiserum was adsorbed to normal human serum. The antiserum was further purified by sedimentation with 40% ammonium sulfate and by affinity chromatography with Sepharose 4B coupled AA. Conjugation of biotin to anti-AA One ml of anti-AA, adjusted to 1 mg/ml in 0.1 mol/l bicarbonate buffer, pH 8.0, was incubated with 60 ~1 of NHS-biotin (Sigma Chemical Co., St. Louis, MO, USA), and solubilized at 1 mg/ml in dimethyl sulfoxide for 3 h at room temperature. After being dialysed to PBS, it was stored at 4°C. Radial immunodiffusion assay for SAA and assay standard The radial immunodiffusion (RID) for. SAA was developed according to the previously described method [5]. Briefly, 1.2% agarose containing 10% anti-AA antiserum, prepared as above, and 3% polyethylene glycol 6000 was incubated with 5 ~1 of the sample for 12 h at 37°C. For assay standardization, purified SAA, 1 mg/ml in 0.1 mol/l bicarbonate buffer, pH 8.0, was reconstituted 1 : 9 with a normal serum diluted 1 : 10 with 0.1 mol/l PBS, pH 7.1 and used as the primary standard with an SAA concentration of 100 pg/ml. The normal serum was selected from among 50 normal sera after it was confirmed to show the lowest absorbance in preliminary EIA tests for SAA. The assay range for SAA in this RID method was from 3.0 to 70 pg/rnl. Pooled sera collected from patients suffered from several inflammatory diseases was calibrated against that primary standard in RID assay and used as the standard serum for every assay of RID and EIA. This standard serum was divided into aliquots and stored at -70°C. Enzyme immunoassay for SAA Polystyrene microtitration plates (Nunc, Roskilde, Denmark) were coated with 200 ~1 of anti-AA adjusted to 2 pg/ml in 0.05 mol/l carbonate-bicarbonate buffer, pH 9.6, for 12 h at 4“ C. After being washed with PBS containing 0.05% Tween-20, which was used as the washing buffer for each procedure, the plates were recoated with 210 ~1 of PBS containing 3% bovine serum albumin (BSA) (Sigma) for 3 h at room temperature. After 3 washings, the plates were filled with PBS containing 0.1% sodium azide, then sealed and stored at 4’C until assayed. The plates were then incubated with 200 ~1 of standard serum and the test samples diluted with 1% BSA-PBS for 1 h in a moist chamber at 37°C. After 7 washings, the plates were incubated with 200 ~1 of biotin-labelled anti-AA diluted 1 : 1000 for 1 h at 37 o C.
171
The washings were repeated, and the plates were incubated with 200 ~1 of peroxidaseconjugated streptavidine (Bethesda Research Laboratories, Gaithersburg, MD, USA) diluted 1: 5 000 for 1 h at room temperature. The plates were again washed and 200 ~1 of a freshly prepared substrate solution (0.04% orthophenyldiamine in 0.1 mol/l citrate-O.2 mol/l phosphate buffer, pH 5.0) containing 0.01% H,O, was added. The colour reaction was stopped after 20 min with 50 ~1 of 2.5 mol/l sulphuric acid. The absorbance was read using a multiscan plate reader (Bio Rad, Richmond, CA, USA) at 492 nm. The SAA concentration of the test samples was determined by the calibration curve obtained from dilutions of standard serum prepared as described above. Serum samples One hundred and sixty-seven serum samples were collected from healthy adults aged between 20 and 69 yr (106 subjects were male, 61 were female). These samples were used for the analysis of the normal value of SAA. Thirty-five other sera were collected from several patients who suffered from inflammatory diseases. These sera were used to investigate the relationship between the EIA and RID assays. All samples were stored at -70°C. Pretreatment of samples Pretreatment studies of samples with 10% formic acid and 0.1 mol/l performed as previously described [8].
NaOH
were
Results Assay conditions of EIA for SAA The application of purified SAA diluted with PBS or 1% BSA-PBS as an assay standard was unsuccessful because of its different dilution patterns compared to
E 1.5 = c
I
0
10
L
20
Fig. 1. Calibration curve for SAA obtained blank absorbance with only 1% BSA-PBS.
30
40 ng/ml
; S *T
by diluting standard serum. The dashed line indicates
the
172 TABLE I Precision assays of EIA for SAA Within assay (n = 10)
Between assay (5 days)
Mean (&ml)
SD
cv (W)
Mean @g/m0
SD
cv (8)
0.198 0.657 2.510
0.032 0.050 0.219
16.0 7.6 8.7
0.197 0.650 2.480
0.021 0.050 0.220
10.5 7.7 8.9
Recovery test Added purified SAA (prot. cont.; pg/ml)
Recovered SAA (calculated; j.~g/ml)
(S)
0.20 0.40 0.80 1.60
O.tP+0.02 * 0.39f0.04 0.73 f 0.07 1.42~0.18
95.0 i 97.5 * 91.3* 88.8 +
a Meanf
Recovery 10.0 10.0 8.7 11.3
SD.
those of serum samples in both the RID and ETA methods. The problem was not improved when the samples were treated with acid or alkali. Thus, pooled sera with a concentration of SAA determined as described above were used as the assay standard. The calibration cmve for the pooled sera is shown in Fig. 1. The assay range was from 1.5 to 30 ng/ml. All of the serum samples could be assayed from a 1: 100 dilution. Fretreatment of 8 samples with acid and alkali caused a IO-X% decrease in the absorbance compared with samples which underwent no treatment_
w
5
10
50
SOQ
100 R
I
D
~000
rsrml
Fig 2. Relationship between the HA and RID assay for SAA on 35 patients’ sera.
173
However, the calculated SAA values of these samples showed no significant changes after the same treatment of standard serum as test samples.
Assay precision
The results for precision both within and between assays and for the recovery test, which was performed by adding purified SAA to a serum sample containing 1.0 ~g/ml of SAA, are summarized in Table I. Good reproducibilities and average recoveries were obtained. The RID results for the 35 samples obtained from the patients corresponded well to those of EIA, as seen in Fig. 2.
Normal values
The histogram of the SAA values on 167 healthy subjects showed a logarithmic normal distribution, thus the statistical analyses described below were performed after logarithmic conversion of the raw data. The SAA results of 5 of the 167 persons showed extremely high values (6.0-12.5 pg/ml). These subjects were excluded from the analysis below after it was confirmed their level of CRP had been raised. In total, of the remaining subjects, no significant difference of the SAA value between sexes was found. The SAA values of each age group (over 10 year intervals) are shown in Fig. 3. The mean values of SAA for each group in the 20- to 59-yr old range were significantly higher than those for all other groups (p -C0.05). The mean for subjects aged 20-59 (n = 132) was 0.81 pg/ml and the range (+ 2 SD) was 0.22-3.0 pg/ml. For those aged 60-69 (n = 30), the mean was 1.1 pg/ml and the range was 0.37-3.3 pg/ml.
w/ml < 5.0;:
'
_ .
.
1.0: i c?5;i : .
I : 0.1
I
. 1
(20)
!T
4
l
l
4 .
1 i
:
I
,
20-29
it. Ii 1 ji .
.
: : :
30-39
40-49
(34)
(47)
50-59
60-69
(31)
(30)
yr (n)
Fig. 3. Age distribution of SAA in 162 healthy subjects. f , mean* old group.
SD. * p c
0.05 vs. each 20- to 59-yr
174
Discussion The method described in this paper achieved a sensitive, reproducible and rapid measurement of SAA in serum sufficient for practical use. Among previous studies which described the use of EIA for SAA, Marhaug [8] claimed that EIA using plastic plates was not suitable for SAA assay in normal serum because of the high background absorbance and the nonspecific binding of SAA to plastic. In our study, we also found the background absorbance to be comparatively high, probably because of the use of three incubation processes. However, the problem of nonspecific binding seemed to be overcome by the use of very dilute sera. This was achieved by applying the avidin-biotin method which is highly sensitive. Denaturation of the sera was studied because SAA exists in high-density lipoprotein (HDL) particles as one of the apolipoproteins [13]. Such treatments seemed unnecessary in terms pooled serum which was used as an assay standard from our results. Purified SAA did not show an identical reaction by sequential dilution to serum samples and it indicated that there were immunoreactive differences between a one-time purified SAA and native SAA in serum, as discussed elsewhere [14]. The reconstituted SAA with normal dilute sera containing a negligible amount of SAA in the RID assay showed a reaction by dilution similar to that of serum samples. Pooled sera, with an SAA value determined by RID assay, was utilized as the assay standard in EIA testing after test results showing good recovery were obtained. Godenir et al. [14] succeeded in standardizing the SAA assay on RIA by the use of HDL, the SAA value of which was determined by the protein staining after electrophoretic separation, as assay standard. Our normal value obtained here was close to theirs. SAA values obtained from healthy subjects varied between individuals and showed age variations in agreement with the previous descriptions [2]. Evaluation of these variation and clinical significance of minute changes of SAA should be investigated further by applying this sensitive method. Acknowledgement We thank Mr. M. Wada, Eiken Chemical antiserum.
Co., Ltd., for his help in producing
the
References 1 Husby G, Natvig JB. A serum component related to non-immunoglobulin amyloid protein AS, a possible precursor of the fibrils. J Chn Invest 1974;53:1054-1061. 2 Rosenthal CJ, Franklin EC. Variation with age and disease of an amyloid A protein-related serum component. J Clin Invest 1975;55:746-753. 3 McAdam KPWJ, Elin R, Sipe JD, Wolff SM. Changes in human serum amyloid A and C-reactive protein after etiocholanolone-induced inflammation. J Clin Invest 1978;61:390-394. 4 Maury CPJ, Teppo AM, Wegelius 0. Relationship between urinary sialylated saccharides, serum amyloid A protein and C-reactive protein in rheumatoid arthritis and systemic lupus erythematosus. Ann Rheum Dis 1982;41:268-271.
175 5 Chambers RE, Whither JT. Quantitative radial immunodiffusion assay for serum amyloid A protein. J Immunol Meth 1983;59:95-103. 6 Benson MD, Cohen AS. Serum amyloid A protein in amyloidosis, rheumatic, and neoplastic diseases. Arthritis Rheum 1979;22:36-42. 7 De Beer FC, Dyck RF, Pepys MB. Solid phase immunoradiometric assay for serum amyloid A protein using magnetisable cellulose particles. J Immunol Meth 1982;54:213-219. 8 Marhaug M. Three assays for the characterization and quantitation of human serum amylod A. Stand J Immunol 1983;18:329-338. 9 Doepel FM, Glorioso JC, Newcomer CE, Skinner M, Abrams CD. Enzyme-linked immunosorbent assay of serum protein SAA in rhesus monkeys with secondary amyloidosis. Lab Invest 1981;45:7-15. 10 Pras M, Schubert M, Zucker-Franklin D, Rimon A, Franklin EC. The characterization of soluble amyloid prepared in water. J Clin Invest 1968;47:924-933. 11 Glenner GG, Harada M, Isersky C. The purification of amyloid fibril proteins. Prep Biochem 1972;2:39-51. 12 Marhaug G, Husby G. Characterization of human amyloid-related protein SAA as a polymorphic protein: association with albumin and prealbumin in serum. Clin Exp Immunol 1981;45:89-106. 13 Benditt EP, Erikson N. Amyloid protein SAA is associated with high density lipoprotein from human serum. Proc Nat1 Acad Sci USA 1977;74:4025-4028. 14 Godenir NL, Jeenah MS, Coetzee GA, Van der Westhuyzen DR, Strachan AF, De Beer FC. Standardization of the quantitation of serum amyloid A protein (SAA) in human serum. J Immunol Meth 1985:83:217-225.
Clinica Chimica Acta, 179 (1989) 169-176 Elsevier
CCA 04343
Short communication
Sandwich enzyme immunoassay for serum amyloid A protein ( SAA) Toshiyuki Yamada ‘, Kazuaki Uchiyama ‘, Minoru Yakata ’ and Fumitake Gejyo 2 of Laboratory Medicine and 2 Depa~t~eni of ~~ternat Medicine, Niigata ~‘~~v~rsitySchool of Medicine, Asahi~achi Niigata fJapan~
’ ~e~art~nt (Received
23 May 1988, revision received 3 October
Key words: Serum amyloid
A protein
1988; accepted
after revision 24 October
(SAA); Enzyme immunoassay
(EIA); Normal
1988)
value
Serum amyloid A (SAA), a putative serum precursor of the AA protein which constitutes amyloid fibrils in secondary amyloidosis [l], has been clinically evaluated as one of the sensitive acute-phase reactants in serum [2,3]. The SAA concentration has been analyzed by radial i~unodiffusion (RID) f4,5] and radioi~unoassay (RIA) [6-81. The RID method was very simple and suitable for the analysis of SAA in its increased states, but could not determine the normal range of SAA because of low sensitivity. At present there are only a few descriptions outlining the use of enzyme immunoassay (EIA) in determining the concentration of SAA in serum [8,9]. It has also been mentioned that EIA may be unsuitable for this purpose because of poor reproducibility of results [S]. In this study, we attempted to develop a sensitive and highly reproducible EIA method for the measurement of SAA in serum, and to evaluate the normal range of the SAA. Materials and methods
Amyloid fibrils were extracted from kidney sections taken from a 60-yr-old female who died from renal failure due to secondary amyloidosis [lo]. The AA protein was purified from this fibrils by two sequential gel filtrations on Sephadex G-100 (Pharmacia Fine Chemicals, Uppsala, Sweden) under 6 mol/l guanidine HCl [II]. SAA was purified from pooled sera with high concentrations of C-reactive protein (CRP) by sequential gel filtrations on Sephadex G-100, G-75, G-50, under
Correspondence to: Dr. T. Yamada, Department Medicine, Asabimacbi Niigata 951, Japan.
~9-8981/89/$03.50
0 1989 Elsevier Scien,
of Laboratory
Publishers
Medicine,
B.V. (Biomedical
Niigata
Division)
University
School of
170
10% formic acid and a final passage through a Sepharose 4B (pharmacia) coupled with antihuman whole serum (Dako, CA, USA) [12].
column
Preparation of antibodies The antiserum against AA was produced in rabbits by immunization with AA purified as above. One milligram of AA in PBS was mixed with an equal amount of Freund’s complete adjuvant and injected subcutaneously into the footpads and dorsa of the animals. Booster doses were administered biweekly using one-fifth of the first dose, and the animals were bled 3 mth after the first injection. The resulting antiserum was adsorbed to normal human serum. The antiserum was further purified by sedimentation with 40% ammonium sulfate and by affinity chromatography with Sepharose 4B coupled AA. Conjugation of biotin to anti-AA One ml of anti-AA, adjusted to 1 mg/ml in 0.1 mol/l bicarbonate buffer, pH 8.0, was incubated with 60 ~1 of NHS-biotin (Sigma Chemical Co., St. Louis, MO, USA), and solubilized at 1 mg/ml in dimethyl sulfoxide for 3 h at room temperature. After being dialysed to PBS, it was stored at 4°C. Radial immunodiffusion assay for SAA and assay standard The radial immunodiffusion (RID) for. SAA was developed according to the previously described method [5]. Briefly, 1.2% agarose containing 10% anti-AA antiserum, prepared as above, and 3% polyethylene glycol 6000 was incubated with 5 ~1 of the sample for 12 h at 37°C. For assay standardization, purified SAA, 1 mg/ml in 0.1 mol/l bicarbonate buffer, pH 8.0, was reconstituted 1 : 9 with a normal serum diluted 1 : 10 with 0.1 mol/l PBS, pH 7.1 and used as the primary standard with an SAA concentration of 100 pg/ml. The normal serum was selected from among 50 normal sera after it was confirmed to show the lowest absorbance in preliminary EIA tests for SAA. The assay range for SAA in this RID method was from 3.0 to 70 pg/rnl. Pooled sera collected from patients suffered from several inflammatory diseases was calibrated against that primary standard in RID assay and used as the standard serum for every assay of RID and EIA. This standard serum was divided into aliquots and stored at -70°C. Enzyme immunoassay for SAA Polystyrene microtitration plates (Nunc, Roskilde, Denmark) were coated with 200 ~1 of anti-AA adjusted to 2 pg/ml in 0.05 mol/l carbonate-bicarbonate buffer, pH 9.6, for 12 h at 4“ C. After being washed with PBS containing 0.05% Tween-20, which was used as the washing buffer for each procedure, the plates were recoated with 210 ~1 of PBS containing 3% bovine serum albumin (BSA) (Sigma) for 3 h at room temperature. After 3 washings, the plates were filled with PBS containing 0.1% sodium azide, then sealed and stored at 4’C until assayed. The plates were then incubated with 200 ~1 of standard serum and the test samples diluted with 1% BSA-PBS for 1 h in a moist chamber at 37°C. After 7 washings, the plates were incubated with 200 ~1 of biotin-labelled anti-AA diluted 1 : 1000 for 1 h at 37 o C.
171
The washings were repeated, and the plates were incubated with 200 ~1 of peroxidaseconjugated streptavidine (Bethesda Research Laboratories, Gaithersburg, MD, USA) diluted 1: 5 000 for 1 h at room temperature. The plates were again washed and 200 ~1 of a freshly prepared substrate solution (0.04% orthophenyldiamine in 0.1 mol/l citrate-O.2 mol/l phosphate buffer, pH 5.0) containing 0.01% H,O, was added. The colour reaction was stopped after 20 min with 50 ~1 of 2.5 mol/l sulphuric acid. The absorbance was read using a multiscan plate reader (Bio Rad, Richmond, CA, USA) at 492 nm. The SAA concentration of the test samples was determined by the calibration curve obtained from dilutions of standard serum prepared as described above. Serum samples One hundred and sixty-seven serum samples were collected from healthy adults aged between 20 and 69 yr (106 subjects were male, 61 were female). These samples were used for the analysis of the normal value of SAA. Thirty-five other sera were collected from several patients who suffered from inflammatory diseases. These sera were used to investigate the relationship between the EIA and RID assays. All samples were stored at -70°C. Pretreatment of samples Pretreatment studies of samples with 10% formic acid and 0.1 mol/l performed as previously described [8].
NaOH
were
Results Assay conditions of EIA for SAA The application of purified SAA diluted with PBS or 1% BSA-PBS as an assay standard was unsuccessful because of its different dilution patterns compared to
E 1.5 = c
I
0
10
L
20
Fig. 1. Calibration curve for SAA obtained blank absorbance with only 1% BSA-PBS.
30
40 ng/ml
; S *T
by diluting standard serum. The dashed line indicates
the
172 TABLE I Precision assays of EIA for SAA Within assay (n = 10)
Between assay (5 days)
Mean (&ml)
SD
cv (W)
Mean @g/m0
SD
cv (8)
0.198 0.657 2.510
0.032 0.050 0.219
16.0 7.6 8.7
0.197 0.650 2.480
0.021 0.050 0.220
10.5 7.7 8.9
Recovery test Added purified SAA (prot. cont.; pg/ml)
Recovered SAA (calculated; j.~g/ml)
(S)
0.20 0.40 0.80 1.60
O.tP+0.02 * 0.39f0.04 0.73 f 0.07 1.42~0.18
95.0 i 97.5 * 91.3* 88.8 +
a Meanf
Recovery 10.0 10.0 8.7 11.3
SD.
those of serum samples in both the RID and ETA methods. The problem was not improved when the samples were treated with acid or alkali. Thus, pooled sera with a concentration of SAA determined as described above were used as the assay standard. The calibration cmve for the pooled sera is shown in Fig. 1. The assay range was from 1.5 to 30 ng/ml. All of the serum samples could be assayed from a 1: 100 dilution. Fretreatment of 8 samples with acid and alkali caused a IO-X% decrease in the absorbance compared with samples which underwent no treatment_
w
5
10
50
SOQ
100 R
I
D
~000
rsrml
Fig 2. Relationship between the HA and RID assay for SAA on 35 patients’ sera.
173
However, the calculated SAA values of these samples showed no significant changes after the same treatment of standard serum as test samples.
Assay precision
The results for precision both within and between assays and for the recovery test, which was performed by adding purified SAA to a serum sample containing 1.0 ~g/ml of SAA, are summarized in Table I. Good reproducibilities and average recoveries were obtained. The RID results for the 35 samples obtained from the patients corresponded well to those of EIA, as seen in Fig. 2.
Normal values
The histogram of the SAA values on 167 healthy subjects showed a logarithmic normal distribution, thus the statistical analyses described below were performed after logarithmic conversion of the raw data. The SAA results of 5 of the 167 persons showed extremely high values (6.0-12.5 pg/ml). These subjects were excluded from the analysis below after it was confirmed their level of CRP had been raised. In total, of the remaining subjects, no significant difference of the SAA value between sexes was found. The SAA values of each age group (over 10 year intervals) are shown in Fig. 3. The mean values of SAA for each group in the 20- to 59-yr old range were significantly higher than those for all other groups (p -C0.05). The mean for subjects aged 20-59 (n = 132) was 0.81 pg/ml and the range (+ 2 SD) was 0.22-3.0 pg/ml. For those aged 60-69 (n = 30), the mean was 1.1 pg/ml and the range was 0.37-3.3 pg/ml.
w/ml < 5.0;:
'
_ .
.
1.0: i c?5;i : .
I : 0.1
I
. 1
(20)
!T
4
l
l
4 .
1 i
:
I
,
20-29
it. Ii 1 ji .
.
: : :
30-39
40-49
(34)
(47)
50-59
60-69
(31)
(30)
yr (n)
Fig. 3. Age distribution of SAA in 162 healthy subjects. f , mean* old group.
SD. * p c
0.05 vs. each 20- to 59-yr
174
Discussion The method described in this paper achieved a sensitive, reproducible and rapid measurement of SAA in serum sufficient for practical use. Among previous studies which described the use of EIA for SAA, Marhaug [8] claimed that EIA using plastic plates was not suitable for SAA assay in normal serum because of the high background absorbance and the nonspecific binding of SAA to plastic. In our study, we also found the background absorbance to be comparatively high, probably because of the use of three incubation processes. However, the problem of nonspecific binding seemed to be overcome by the use of very dilute sera. This was achieved by applying the avidin-biotin method which is highly sensitive. Denaturation of the sera was studied because SAA exists in high-density lipoprotein (HDL) particles as one of the apolipoproteins [13]. Such treatments seemed unnecessary in terms pooled serum which was used as an assay standard from our results. Purified SAA did not show an identical reaction by sequential dilution to serum samples and it indicated that there were immunoreactive differences between a one-time purified SAA and native SAA in serum, as discussed elsewhere [14]. The reconstituted SAA with normal dilute sera containing a negligible amount of SAA in the RID assay showed a reaction by dilution similar to that of serum samples. Pooled sera, with an SAA value determined by RID assay, was utilized as the assay standard in EIA testing after test results showing good recovery were obtained. Godenir et al. [14] succeeded in standardizing the SAA assay on RIA by the use of HDL, the SAA value of which was determined by the protein staining after electrophoretic separation, as assay standard. Our normal value obtained here was close to theirs. SAA values obtained from healthy subjects varied between individuals and showed age variations in agreement with the previous descriptions [2]. Evaluation of these variation and clinical significance of minute changes of SAA should be investigated further by applying this sensitive method. Acknowledgement We thank Mr. M. Wada, Eiken Chemical antiserum.
Co., Ltd., for his help in producing
the
References 1 Husby G, Natvig JB. A serum component related to non-immunoglobulin amyloid protein AS, a possible precursor of the fibrils. J Chn Invest 1974;53:1054-1061. 2 Rosenthal CJ, Franklin EC. Variation with age and disease of an amyloid A protein-related serum component. J Clin Invest 1975;55:746-753. 3 McAdam KPWJ, Elin R, Sipe JD, Wolff SM. Changes in human serum amyloid A and C-reactive protein after etiocholanolone-induced inflammation. J Clin Invest 1978;61:390-394. 4 Maury CPJ, Teppo AM, Wegelius 0. Relationship between urinary sialylated saccharides, serum amyloid A protein and C-reactive protein in rheumatoid arthritis and systemic lupus erythematosus. Ann Rheum Dis 1982;41:268-271.
175 5 Chambers RE, Whither JT. Quantitative radial immunodiffusion assay for serum amyloid A protein. J Immunol Meth 1983;59:95-103. 6 Benson MD, Cohen AS. Serum amyloid A protein in amyloidosis, rheumatic, and neoplastic diseases. Arthritis Rheum 1979;22:36-42. 7 De Beer FC, Dyck RF, Pepys MB. Solid phase immunoradiometric assay for serum amyloid A protein using magnetisable cellulose particles. J Immunol Meth 1982;54:213-219. 8 Marhaug M. Three assays for the characterization and quantitation of human serum amylod A. Stand J Immunol 1983;18:329-338. 9 Doepel FM, Glorioso JC, Newcomer CE, Skinner M, Abrams CD. Enzyme-linked immunosorbent assay of serum protein SAA in rhesus monkeys with secondary amyloidosis. Lab Invest 1981;45:7-15. 10 Pras M, Schubert M, Zucker-Franklin D, Rimon A, Franklin EC. The characterization of soluble amyloid prepared in water. J Clin Invest 1968;47:924-933. 11 Glenner GG, Harada M, Isersky C. The purification of amyloid fibril proteins. Prep Biochem 1972;2:39-51. 12 Marhaug G, Husby G. Characterization of human amyloid-related protein SAA as a polymorphic protein: association with albumin and prealbumin in serum. Clin Exp Immunol 1981;45:89-106. 13 Benditt EP, Erikson N. Amyloid protein SAA is associated with high density lipoprotein from human serum. Proc Nat1 Acad Sci USA 1977;74:4025-4028. 14 Godenir NL, Jeenah MS, Coetzee GA, Van der Westhuyzen DR, Strachan AF, De Beer FC. Standardization of the quantitation of serum amyloid A protein (SAA) in human serum. J Immunol Meth 1985:83:217-225.