Klinische Wochenschrift
Klin Wochenschr (1989) 67:447451
© Springer-Verlag 1989
A Rapid Laser Immunonephelometric Assay for Serum Amyloid A (SAA) and its Application to the Diagnosis of Kidney Allograft Rejection* G. Hocke t, H. Ebel 2 K. Bittner 2 T. Miiller 2 H. Kaffarnik 3, and A. Steinmetz 3 1 Institut ffir Humangenetik und Genetische Potiklinik der Philipps-Universit/it Marburg 2 Medizinisches Zentrum ffir Innere Medizin der Philipps-Universit~it Marburg, Abteilung ffir Nephrologie 3 Medizinisches Zentrum ffir Innere Medizin der Philipps-Universit/it Marburg, Abteilung ffir Endokrinologie und Stoffwechsel
Summary. We set up a laser nephelometric assay for the quantitation of serum amyloid A (SAA) in human plasma. Therefore monospecific antibodies were raised in sheep and used in parallel to measure SAA concentrations by nephelometry and also by radial immunodiffusion, an assay usually applied for determination of SAA. The nephelometric method is precise, simple and unlike radial immunodiffusion results are obtained within an hour. The antigen concentrations determined both by laser nephelometry and radial immunodiffusion correlated highly (r=0.98). As plasma SAA concentrations were reported to be a possible marker of kidney allograft rejection, the assay was applied to measure SAA concentrations in patients after kidney transplantation. Data were compared with clinical and other biochemical parameters. The period after kidney transplantation is reported for two cases, where SAA plasma concentrations were helpful in diagnosing allograft rejection. The rapid availability of the SAA plasma concentrations by nephelometry makes them a possible additional tool to decide quickly upon antirejection therapy. Key words: Serum Amyloid A - Nephelometry Kidney Allograft Rejection
Serum amyloid A (SAA) was shown to be an apolipoprotein mainly associated with high density tipoprotein (HDL) (Benditt and Eriksen 1977). As an * Parts of this work were reported at the 21st Annual Meeting of the European Society for Clinical Investigation, Copenhagen, March 1987 Abbreviations: HDL=high density lipoprotein; IEF-isoelectric focusing; RID=radial immunodiffusion; SAA=serum amyloid A; SDS = sodium dodecylsulfate
acute phase reactant SAA is induced in a series of different disease states such as inflammation, trauma and tumor (Benson and Cohen 1979; Maury 1985). Its induction may furthermore be a helpful parameter in the diagnosis of acute renal or hepatic allograft rejection (Maury et al. 1984 and 1987). As SAA increases may even precede and thus early indicate a rejection episode (Maury 1986) a quick monitoring of SAA elevations is needed to decide on antirejection therapy. We developed a rapid immunonephelometric assay to determine SAA plasma levels within one hour.
Materials and Methods Production of Antibodies SAA was purified to homogeneity from pooled human SAA containing sera. Therefore HDL3 ( d = 1.12-1.21 g/ml) was isolated by ultracentrifugation (Havel et al. 1955). After dialysis and delipidation in acetone: ethanol (1 : 1, v:v) at --20 ° C the apoproteins were dissolved in 6 M guanidinhydrochloride and further purified by gel filtration on a Sephacryt S 200 column (2,6 x 100 cm) (Pharmacia, Freiburg, F.R.G.), equilibrated with the same buffer. The SAA containing fractions were pooled, dialyzed and lyophylized, redissolved in 8 M urea 10 m M Tris pH 8.2 and subjected to preparative isoelectric focusing in a pH range of 5-7, modifying the procedure of Radola et al. (1971) and Marcel et al. (1979). SAA was eluted and carrier ampholytes were removed by prolonged dialysis. The purified protein gave a single band of 11.500 Da upon SDS gel electrophoresis and was used for immunization. Antibodies to SAA were raised in sheep according to standardized procedures. The purity and specificity of the antiserum were checked by double
448
immunodiffusion, charge shift immunoelectrophoresis and immunoblotting. They showed no reaction with apoproteins AI, AII, AIV, E, CII and CIII.
G. Hocke et al. : Serum Amyloid A and Kidney Allograft Rejection
sion. Thus the few turbid samples that occured were cleared with Lipoclean prior to SAA determination.
Radial Immunodiffusion (RID) Assay of SAA : Immunonephelometric Assay Light scattering produced by antigen-antibody complexes was measured with a laser nephelometer (Behringwerke, Marburg, F.R.G.). SAA antiserum was diluted 5-fold in N-Reaction Buffer (Behringwerke, Marburg, F.R.G.) and filtered (Milex, pore size 0.45 ~m, Sartorius, G6ttingen, F.R.G.). Samples of serum and SAA-enriched H D L were routinely diluted 40, 60 and 80 fold with 150 m M sodium chloride. The reaction mixture containing 0.1 ml of the diluted samples and 0.2 ml of the diluted antiserum was allowed to incubate at room temperature for 1 hour. The light scatter then obtained was plotted against the SAA concentration in serial dilutions of an SAA containing standard to generate the standard curve from which the serum SAA concentration could be determined.
Plasma and standard SAA concentrations were also measured by RID following the procedure of Chambers and Whitcher (1983). Therefore 1% agarose gels were used containing 4% (w:v) PEG (6000) and 3% (v:v) antiserum either from sheep (as described above) or from a commercially available rabbit anti human SAA-antibody charge (CaP biochem, Behring Diagnostics, La Jolla, CA).
Subjects and Patients Plasma from 30 healthy blood donors from the University of Marburg blood blank was assayed for SAA content. Also recipients of renal allografts were monitored daily after transplantation at the University Hospital of Marburg. Levels of SAA were correlated to clinical signs and when available with corresponding histology.
S AA-S tandar d In plasma SAA occurs associated with lipoproteins. In purified form it is highly unstable and tends to severely aggregate. Thus standardization with purified protein seems unfeasible (Godenir et al. 1985). Furthermore laser nephelometry is measuring the light scattering caused by antigenantibody-complexes. The low molecular weight of purified SAA (11.500 Da) is not suitable to form well measurable antigen-antibody-complexes, an additional drawback of purified SAA as standard. As we found SAA most stable in complex with HDL, we used SAA-enriched H D L as standard. The SAA content of this H D L was measured by RID (see below) and also as the percentage of SAA of total protein by densitometry after analytical IEF and SDS electrophoresis.
Delipidation Turbidity of plasma samples is usually caused by serum triglyceride values exceeding 500 mg/dl. They give a high initial light scattering. These sera were thus delipidated with diisopropylether/n-Butanol 60/40 (v/v) according to Cham and Knowles (1976) or alternatively treated with Lipoclean (Behringwerke, Marburg, F.R.G.). Both methods cleared the turbidity but the diisopropylether/nButanol led to substantial losses of SAA in some cases. With Lipoclean the recovery was always more than 95% as assessed by radial immunodiffu-
Results
Immunonephelometric Assay Reaction Time To obtain the optimal reaction time for the end point determination, serial dilutions of SAA-rich H D L preparation or SAA containing sera were closely monitored over time for scattering for a period of 2 h. For SAA-rich H D L preparations and serum one hour of incubation time was sufficient to nearly complete reaction. In all subsequent measurements the light scattering was assessed after one hour of incubation. Standard Curve SAA-enriched H D L was diluted with 150 mM sodium chloride from 1:40 to 1:2560 representing SAA concentrations from 2.4 to 0.0375 mg/dl and allowed to incubate with diluted antiserum for 1 h. Thereafter the intensity of turbidity (measured in Volts) was plotted against the SAA concentration (Fig. 1). The standard curve thus obtained is nearly linear between 0.1 to 1.2 mg/dl. Plasma samples were usually diluted to reach concentrations between 0.2-1 mg/dl SAA. Correlation with RID In a series of 20 samples SAA concentrations were measured by radial immunodiffusion and corn-
G. Hocke et al. : Serum Amyloid A and Kidney Allograft Rejection
449
probably due to e.g. a minor infection. The 29 others had a mean level of 0.94+ 0.39 mg/dl_+ SD SAA. Thus values below 1.5 mg/dl were regarded as normal.
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Fig. l. Standard curve of the SAA immunoephelometric Assay, Light scattering was measured at sixty minutes after reaction of different concentrations of a SAA-rich H D L preparation with the diluted anti h u m a n SAA antibody. The standard curve is nearly linear between SAA concentrations of 0.1 to 1.2 mg/dl
pared with the results obtained by nephelometry. Both methods correlated highly (r=0.98%). In 10 samples R I D was performed with a commercially available antibody to human serum amyloid A (Calbiochem, Behring Diagnostics, La Jolla, CA, USA). The two R I D procedures gave identical results. Reproductibility Reproductibility was assessed by an intraassay coefficient of variation of 2.8% (10 times in the same assay) and an interassay coefficient of variation of 6.7% (10 consecutive assays). SAA was always fully recovered when added in form of H D L or purified SAA to plasma. The plasma levels of control persons were usually found to be lower than 1.5 mg/dl. Turbid Plasma
In a series of 30 plasma we compared SAA concentration after delipidation or Lipoclean treatment by immunonephelometry and RID. Plasma with high and low triglyceride levels were used. As the treatment with Lipoclean is a more reliable procedure to clear turbidity we used this procedure in the few cases that needed pretreatment. To circumvent the clearing procedure plasma was usually obtained in the fasting state. Plasma SAA Levels
SAA levels were determined in 30 healthy blood donors. One subject had a level above 10 mg/dl,
The assay was also applied to monitor SAA levels in patients after renal transplantation. A study is ongoing to validate the use of SAA levels in diagnosis of rejection episodes. The results obtained so far argue for a low specificity but a high sensitivity of SAA levels in the diagnosis of allograft rejection (Lange, Ebel, Mfiller, Hocke, Steinmetz unpublished observation). Two typical results of SAA monitoring in recipients of kidney allografts are shown below. In patient 1 (K.L.) SAA levels rose tremendously between day 8 and 10 after transplantation followed by other clinical parameters of a rejection episode. Successful antirejection therapy confirmed the rejection. SAA levels decreased upon therapy. Fig. 2a shows the data in this patient after transplantation. Although the kidney transplant o f patient A.B. (Fig. 2b) never functioned, SAA levels returned to and stayed at baseline after the initial SAA peak caused by the surgical trauma. The low SAA levels and the unsuccessful antirejection treatment argued against a rejection. This was confirmed by a transplant biopsy showing no signs of rejection, but a distinct tubulo-interstitial calcification.
Discussion This report describes the development and first application of a quick, sensitive and specific laser nephelometric assay for human SAA. The precision of this immunonephelometric assay fulfills the criteria required for standard procedures in clinical chemistry. Standardization of the method described was not possible with isolated purified SAA, as the isolated protein is highly unstable and also relatively small ( M W 11.500). Therefore H D L rich in SAA or H D L incubated with isolated SAA as described by Godenir et al. (1985) was used as standard. We also found SAA stable in a lipoprotein environment. The standard was kept frozen at - 2 0 ° C and was in our hands stable over at least one year. A secondary standard (SAA-rich plasma) was always used in parallel. This secondary standard was also kept frozen in aliquots at - 2 0 ° C and found to be stable during this time as well. The nephelometric assay was compared with radial immunodiffusion, a widely used assay
G. Hocke et al. : Serum Amyloid A and Kidney Allograft Rejection
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for SAA. The high correlation obtained by the two procedures establishes the validity of the new method. End point nephelometry may be disturbed by turbidity of samples mainly caused by triglycerides. Turbidity must be minimized to an extent that the light scattering of the diluted serum doesn't reach 0.5 Volt. On the other hand the procedure clearing lipids should not influence SAA concentrations. Turbidity in our samples was first tried to circumvent by using fasting plasma and, when persistent, it was cleared with Lipoclean. This procedure could be shown to effectively clear plasma with minimal influence on SAA concentrations. In future the turbidity problems may be circumvented by the adaptation of the assay to kinetic nephelometry. This may also allow an even more rapid analysis and the use on a larger scale.
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Fig. 2. SAA plasma levels during the posttransplantation periods of two renal allograft recipients. a Patient K,L. with a severe acute rejection of the kidney transplant beginning on day 8 after transplantation. b Patient A.B. with primary kidney transplant failure, baseline levels of SAA and no signs of rejection (histology), H D : Haemodialysis
20
In a cohort of apparently healthy subjects plasma levels of SAA below 1.5 mg/dl were established as normal, although an absolute normal value for an acute phase reactant seems difficult to assess. Our normal value corresponds well to those reported with R I D by Maury et al. (1985), Chambers and Whitcher (1983) and Raynes and Cooper (1983). Similar to C-reactive protein, the measurement of serum amyl0id A has a wide clinical application (Maury 1985). The rapidness of the new procedure makes it valuable in cases where levels need to be monitored quickly. There are several reports in the literature of SAA being a sensitive marker for renal allograft rejection (for review see Maury 1985). As the augmentation of S A A levels may even precede other signs of rejection and as vice versa rejection is highly unlikely when SAA levels
G. Hocke et al. : Serum Amyloid A and Kidney Allograft Rejection
don't increase (Maury et al. 1983), a frequent monitoring and rapid availability of SAA levels may be helpful in the diagnosis of a rejection episode. As indicated in the first case shown here the increase of SAA was an additional marker early available for the diagnosis of the proven rejection, whereas unchanged SAA levels in the second case made a rejection unlikely, confirming reports by Maury and colleagues (1984a). The assay is now used in a prospective study to evaluate its usefulness in diagnosing kidney allograft rejection together with other more established parameters. Acknowledgement: We thank Professor Kretschmer and his colleagues of the University of Marburg blood bank for providing the plasma samples of normal subjects, Sabine Motzny for excellent technical assistance and Silke Szarny for typing the manuscript. This work was supported by a grants from the Deutsche Forschungsgemeinschaft to A.S. and from the Kempkes Stiftung References Benditt EP, Eriksen N (1977) Amyloid protein SAA is associated with high density lipoprotein from human serum. Proc Natl Acad Sci USA 74:4025-4028 Benson MD, Cohen AS (1979) Serum amyloid A protein in amyloidosis, rheumatic, and neoplastic diseases. Arthritis Rheum 22(1): 36-42 Cham BE, Knowles BM (1976) A solvant system for delipidation of plasma or serum without protein precipitation. J Lipid Res 7:176-181 Chambers RE, Whitcher JT (1983) Quantitative radial immunodiffusion for serum amyloid-A protein. J Immunol Methods 59:95-103 Godenir NL, Jeenah MS, Coetzee GA, van der Westhuysen DR, Strachnan AF, de Beer FC (1985) Standardization of the quantitation of serum amyloid A protein (SAA) in human serum. J Immunol Methods 83:217-225 Havel RJ, Eder HA, Bragdon J (1955) The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 34:1345-1353
451 Marcel YE, Bergsetz M, Nestruck AC (1979) Preparative isoelectric focusing of apolipoprotein C and E from human very low density lipoproteins. Biochim Biophys Acta 573:175 183 Maury CPJ, Teppo AM, Eklund B, Ahonen J (1983) Serum amyloid A protein: a sensitive indicator of renal allograft rejection in humans. Transplant 36:501 504 Maury CPJ, H6ckerstedt K, Teppo AM, Lautenschlager I, Scheinin TM (1984) Changes in serum amyloid A protein and beta-2-Microglobutin in association with liver allograft rejection. Transplant 38: 551-553 Maury CPJ, Teppo AM, Ahonen J, von Willebrand E (1984a) Measurement of serum amyloid A protein concentrations as test of renal allograft rejection in patients with initially nonfunctioninggrafts. Brit Med J 288:360-361 Maury CPJ (1985) Comparative study of serum amyloid A protein and C-reactive protein in disease. Clin Sci 68:233-238 Maury CPJ, Teppo AM, Ahonen J, von Willebrand E (1986) Evaluation of serum amyloid A protein as a marker of allograft rejection in patients with initially nonfunctioningrenal transplants. Transplant Proc XVIII (1):86-87 Maury CPJ, H6ckerstedt K, Lautenschlager I and Scheinin TM (1987) Monitoring of high density lipoprotein-associated amyloid A protein after liver transplantation. Transplant Proc XIX (5) : 3825-3826 Radola BJ (1974) Isoelectric focusing in layers of granulated gels II. Preparative isoelectric focusing. Biochim Biophys Acta 386:181-195 Raynes JG, Cooper EH (1983) Comparison of serum amyloid A protein and C-reactive protein concentrations in cancer and non-malignantdisease. J Clin Pathol 36:798 803
Received : September 10, 1988 Returned for revision: January 9, 1989 Accepted: January 24, 1989
Dr. A. Steinmetz Philipps-Universit/it Marburg Zentrum fiir Innere Medizin Abteilung ffir Endokrinologie und Stoffwechsel Baldingerstrage D-3550 Marburg
