British Journal of Rheumatology 1992;31:175-178

MEASUREMENT OF HUMAN PHOSPHOLIPASE A2 IN ARTHRITIS PLASMA USING A NEWLY DEVELOPED SANDWICH ELISA BY G. M. SMITH, R. L. WARD*, L. McGUIGANf, I. A. RAJKOVIC AND K. F. SCOTT Pacific Biotechnology Ltd, 74 McLachlan Avenue, Rushcutters Bay, NSW 2011, Australia; 'Department of Haematology, St Vincents Hospital, Victoria Street, Darlinghurst, NSW 2010, Australia; tDepartment of Rheumatology, St George Hospital, Kogarah, NSW 2217, Australia

KEY WORDS:

Human non-pancreatic phospholipase A2, Sandwich ELISA, Rheumatoid arthritis.

THE enzyme phospholipase A2 (PLA 2 ) is central to the process of inflammation because of its ability to cleave phospholipids to yield the precursors of inflammatory mediators such as eicosanoids and platelet activating factor [1-3]. To date two distinct human phospholipases A2 have been isolated. One is the pancreatic or type I PLA 2 , which is synthesized primarily by the pancreas but is also present in smaller amounts in other tissues including lung, spleen and kidney [4]. The amino-acid sequence of the human pancreatic PLA 2 is similar to the pancreatic forms isolated from other mammalian species [5,6]. More recently, a non-pancreatic type II PLA 2 has been sequenced and shown to occur in platelets [7], synovial cells and fluid [7-10], ascites fluid [10] and placental tissue [9]. The human non-pancreatic PLA2 which has an amino-acid sequence similar to non-pancreatic PLA 2 found in other mammalian species [10], has recently been cloned [10]. It has been suggested that this PLA 2 may play a role in the pathogenesis of a number of disorders [11] including rheumatoid arthritis [12, 13] and septic shock [14]. PLA 2 is currently measured by enzyme activity assays, usually involving the hydrolysis of a radiolabelled phospholipid [15,16]. These activity assays have several important limitations. First, they only measure the total PLA2 catalytic activity, and thus do not identify the source(s) of the activity. Second, PLA 2 enyzme activity assays are susceptible to interference from sample components [17], thus making sample comparison difficult. Enzyme activity assays for PLA 2 have shown increased activity in the synovial fluid and serum of patients with rheumatoid arthritis [12, 13]. As a first step towards determining the role of human non-pancreatic PLA 2 in the pathogenesis of disease, we have developed a sandwich ELISA [18] using monoclonal antibodies raised against recombinant

PLA 2 . This assay has been shown to be capable of specific and sensitive quantitation of non-pancreatic PLA 2 in plasma. It has been used in the present study to evaluate plasma PLA 2 levels in patients with rheumatoid arthritis, osteoarthritis, and in healthy individuals. A small number of samples from patients with psoriatic arthritis, chronic tophaceous gout, Reiter's syndrome and ankylosing spondylitis were also measured. PATIENTS AND METHODS Antibodies Monoclonal antibodies were raised against recombinant human non-pancreatic PLA 2 which had been expressed in Chinese hamster ovary cells and purified to greater than 90% purity. Monoclonal antibodies to PLA2 were purified from hybridoma-induced ascites fluid in BALB/c mice using Sepharose CL4B-protein A (Pharmacia Fine Chemicals, Uppsala, Sweden) [19]. The monoclonal antibodies raised against PLA 2 were subtyped and were of the IgGl class. Conjugation of monoclonal antibodies to alkaline phosphatase Glutaraldehyde was used for conjugation of monoclonal antibodies to alkaline phosphatase as previously described [19]. Sandwich ELISA Microtitre plates (Immunlon 2, Dynatech, Chantilly, Virginia) were coated with the monoclonal antibody 9C1( 100 ul, 1 ug/ml) in phosphate-buffered saline (PBS) for 16 h at 4°C and then blocked with 250 u,l of 1% skimmed milk powder/0.5% bovine serum albumin (BSA) for 3h at 37°C. The plates were then washed twice with PBS containing Tween 20 (0.5% v/v) and BSA (0.5% w/v). Plates were then stored at -20°C until use. Samples diluted in PBS or 10 mM TrisHCI/150 mM NaCl were incubated in each well for 2 h at 37°C. The plates were washed twice and 100u.l of

Submitted 14 May; revised version accepted 31 July 1991. Correspondence to G. Smith, Department of Haematology, St Vincents Hospital, Victoria Street, Darlinghurst, NSW 2010, Australia. 175

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SUMMARY A sandwich enzyme-linked immunosorbent assay (ELISA) for human non-pancreatic phospholipase A2 (PLA2) was developed using monoclonal antibodies raised against purified recombinant PLA2. This assay was shown to be specific for human non-pancreatic PLA2, showing no cross-reactivity with human pancreatic PLA2 or with snake venom PLA2 (Crotalus durissus). The immunoassay showed no cross-reactivity with plasma components, and was reproducible and quantitative between 39 pmol and 2.7 nmol PLAj/1. The levels of non-pancreatic PLA2 in the plasma of patients with arthritis was measured using this immunoassay. There were significantly higher levels of PLA2 in patients with rheumatoid arthritis than in those with osteoarthritis or healthy controls. Plasma PLA2 was highest in those patients with active rheumatoid arthritis.

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PLA2 activity assay PLA2 activity was measured according to a modification of a previously published mixed micelle assay [16]. Substrate was prepared as follows: [l4C]phosphatidyl ethanolamine (0.22 nmol/assay sample) was dried under a stream of nitrogen and resuspended in 2% sodium deoxycholate (0.5 u.l/assay). The mixture was then diluted with assay buffer (100 mM Tris HC1 pH 8.0, 10 mM CaCl2, 300 ITIM NaCl) to a final volume of 25 fil/assay. Sample (25 ul/assay) was added to substrate and incubated at 37°C for 30 min. The 50 uJ reaction was terminated by the addition of 10 (j.1 100 mM ethylenediaminetetraacetic acid disodium salt (EDTA) pH 8. Thin-layer chromatography (TLC) was used to separate liberated arachidonic acid from unreacted substrate using chloroform :methanol: acetic acid (90:10:1 v/v/v) as the solvent. Liberated arachidonic acid and unreacted substrate were cut from the TLC plate, counted in scintillation fluid and PLA2 activity expressed as percentage hydrolysis of substrate. Samples were measured in duplicate. Patients The patients were recruited from the rheumatology clinic at St George Hospital, Sydney, Australia, by one of the authors (LMcG). Fifty patients fulfilled American Rheumatism Association criteria [20] for definite or classical rheumatoid arthritis (40 women aged 45-86 years, 10 men aged 50-82 years). Of these 19 were classified as having active disease, as they fulfilled one or more of the following criteria: haemoglobin50mm/h, and/or early morning stiffness lasting longer than 1 h. Osteoarthritis was diagnosed radiologically in 11 patients (six females andfivemales). The group labelled 'Other' consisted of three patients with psoriatic arthritis, three with chronic tophaceous gout, one with Reiter's syndrome and one with ankylosing spondylitis. Twenty-two healthy subjects on no medication were used as controls (15 women aged 27-77 and seven males aged 39-65). Samples Blood was collected into tubes containing EDTA-

dipotassium salt, centrifuged (2000g for 15 min) and the plasma stored at -20°C. Once all the samples had been collected the immunoassay was performed in duplicate and the results expressed as mean values. The diagnostic category of each sample was encoded to permit unbiased assay. Human pancreatic tissue was obtained at post-mortem examination from the Department of Anatomical Pathology, St Vincents Hospital, and the pancreatic PLA2 was isolated in the supernatant after tissue homogenization in 10 mM Tris-HCl pH 8.0 and centrifugation at 12 000g for 10 min. Snake venom phospholipase A2 (Crotalus durissus) was from Boehringer Mannheim, West Germany. Statistics Data were presented graphically as scatter plots with the median underlined. Comparisons between diagnostic groups was made using non-parametric methods for unpaired data (Mann-Whitney). P values less than 0.01 were considered to indicate statistical significance. RESULTS Validation of the sandwich ELISA Figure 1 shows a standard curve for the immunoassay generated with purified recombinant PLA2. The PLA2 was added at concentrations ranging from 21 pmol/1 to 44 nmol/1. The detection limit, defined as thelowest concentration giving a signal significantly different from that of the zero standard at 99% confidence, was 39 pmol/1 (0.5 ng PLA^mO.The coefficients of variation were from 1 to 6% for within-assay variation (mean = 5.3%) and from 3 to 12% (mean = 8.3%) for between-assay variation. Standard curves were routinely produced by diluting the recombinant PLA2 in PBS/0.1% BSA but other buffers could be used without any effect on the standard curve including PBS, 10 mM Tris-HCl/150 mM NaCl, 10% normal human plasma in PBS, 10% fetal calf serum in PBS and PBS containing various deter2-1

c o

in

1H

o « n

.01

.1 PLA2

1 10 (nmoles/lltre)

100

FIG. 1.—Standard curve of the assay for human non-pancreatic phospholipase A2 (PLA2). Purified recombinant PLA, wasdiluted into 10 mM Tris-HCl/150 mM NaCI/0.1% BSA and subjected to the immunoassay in triplicate. Results are expressed as mean values ± SD. without background subtraction

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monoclonal antibody 4Al/alkaline phosphatase conjugate diluted 1:500 in PBS containing 0.5% BSA was added for 30 min at 37°C. The plates were washed three times with PBS/BSA and then twice with carbonate buffer followed by the addition to each well of p-nitrophenyl phosphate (100 ul, 1 mg/ml in carbonate buffer). The plates were incubated at 37°C and the absorbance at 405 nm read after approximately 1 h by an Easy Reader SF Plus absorbance plate reader (SLTLabinstruments, Austria). Purified recombinant PLA2 was quantitated by amino-acid analysis and used to generate a standard curve. Assays were performed in triplicate wells and expressed as the mean levels. Standard curves and results were calculated using AssayZap assay calculator (Elsevier-Biosoft, Cambridge, UK).

SMITH ETAL.: SANDWICH ELISA FOR HUMAN PLA2

177

with arthritis are shown in Fig. 3. The rheumatoid arthritis group had significantly higher levels of PLA2 (median = 8.2 nmol/1) than any other group. There was no significant difference in the level of immunoreactive PLA2 between either the osteoarthritis (2.2 nmol/1, the 'other' group (2.1 nmol/1) and controls (1.1 nmol/1). Active rheumatoid arthritis was associated with significantly higher levels of PLA2 (10.6 nmol/1) than inactive disease (5.6 nmol/1).

2 -\ E c m

1 o

to

n

.01

.1 PLA2

1 10 (nmoles/lltre)

100

gents (1% TritonX-100, 0.1% deoxycholate and 0.1% sodium dodecylsulphate). One requirement was the presence of salt, at a nominal concentration of 150 mM. Samples were routinely diluted in PBS for the estimation of higher PLA2 concentrations. As shown in Fig. 2 the assay was unaffected by plasma components as identical results were obtained when recombinant PLA2 was added to 0.1% BSA or 10% normal human plasma in PBS or when PLA2 was added to neat plasma which was then diluted in PBS. The coefficient of correlation of recombinant PLA2 immunoreactivity and functional activity in PBS/0.1 % BSA buffer was 0.899. Specificity of the sandwich ELISA The specificity of the immunoassay for the human non-pancreatic PLA2 was tested by determining if the extract from human pancreatic tissue or the snake venom (C. durissus) PLA2 were immunoreactive. The results shown in Table I show that despite high levels of PLA2 activity in these samples there was no detectable signal in the immunoassay for either the human pancreatic or snake venom PLA2. In fact human pancreatic or snake venom PLA2 at concentrations up to 100-fold that are shown in Table I still gave no detectable signal in the immunoassay, indicating that the immunoassay was specific for the human non-pancreatic PLA2. PLA2 levels in arthritis patients The plasma non-pancreatic PLA2 levels in patients

40 -i

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20-

10-

TABLE I

• i

SPECIFICITY OF THE HUMAN NON-PANCREATTC PHOSPHOLIPASE A : IMMUNOASSAY

PLA, Recombinant human non-pancreatic Human pancreatic Snake venom *Mean ±SD.

Sandwich ELISA (nmol/1)*

Activity assay (% hydrolysis)

7 (±0.11)

6 (±0.8)

Not detectable Not detectable

45 (±4.9) 67 (±8.4)

1*0

RA RA active Inactive

0A

control Other

FIG. 3.—Concentration of immunoreactive PLA2 in the plasma of patients with arthritis. The median is underlined and is significantly higher in patients with rheumatoid arthritis (RA) than any other group. The highest levels were found in those patients with active RA. OA, osteoarthritis; for definition of other categories see Patients and Methods.

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FIG. 2.—The effect of human plasma on the immunoassay. Purified recombinant PLA2 was diluted into 10 mM Tris-HCl/150 mti NaCl/0.1% BSA (©); 10 mM Tris-HCl/150 mM NaCl/10% plasma ((O); and neat plasma which was then diluted with PBS (A). Samples were assayed in triplicate and expressed as the mean value without background subtraction.

DISCUSSION We have developed a rapid and reproducible immunoassay capable of detecting human non-pancreatic PLA2 in a variety of biological samples. The assay is sensitive within the range of 38pmol/l to 2.7nmol PLA^. The specificity of the assay is demonstrated by its failure to recognize the other major PLA2 in humans, namely the pancreatic enzyme. This enables us to examine the role of the non-pancreatic PLA2 in the pathology of rheumatoid arthritis and other inflammatory disorders. Immunoassays on plasma samples spiked with purified recombinant non-pancreatic PLA2 indicate that the assay is not subject to interference from plasma components. The quantitation of PLA2 measured under these conditions is consistent with that seen in buffer alone. Thus, the antibodies used in the ELISA do not cross-react with human serum proteins and provide an accurate measure of non-pancreatic PLA2 in samples containing complex mixtures of protein. There has been considerable evidence supporting the role of inflammatory eicosanoids in rheumatoid arthritis [21], and PLA2 has been shown to be the rate limiting enzyme in their production [22]. Plasma PLA2 activity has been shown to be elevated in rheumatoid arthritis [12, 13], while intra-articular injection of PLA2 reproduces synovitis in experimental animals [23]. We have demonstrated that the plasma level of the

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ACKNOWLEDGEMENTS

This work wsa supported by an Australian Government Discretionary Research Grant (no. 5209). REFERENCES

1. Larsen GL, Henson PM. Mediators of inflammation. A Rev Immunol 1983;l:335-59. 2. Irvine RF. How is the level of free arachidonic acid controlled in mammalian cells? Biochem J 1982;204:3-16. 3. Snyder F. Chemical and biochemical aspects of platelet activating factor: a novel class of acetylated ether-linked choline-phospholipids. Med Res Rev 1985;5:107. 4. Matsuda Y, Ogawa M, Shibata T, el al. Distribution of immunoreactive pancreatic phospholipase A2 (1PPL-2) in various tissues. Res Commun Chem Pathol Pharmacol 1987;58:281. 5. Verkeij HM, Westerman J, Sternby B, De Haas G. The complete primary structure of phospholipase A2 from human pancreas. Biochim Biophys Acta 1983;747:93-9. 6. Seihamer JJ, Randell TL, Yamanaka M, Johnson LK. Pancreatic phospholipase A2: isolation of the human gene and cDNAs from porcine pancreas and human lung. DNA 1986;5:519-27. 7. Kramer RM, Hession C, Johansen B, et al. Structure and properties of a human non-pancreatic phospholipase A2. J Biol Chem 1989;254:5768-75. 8. Hara S, Kudo I, Chang H, Matsuta K, Miyamoto T, Inoue K. Amino acid composition and NH3-terminal amino acid sequence of human phospholipase A2 purified from rheumatoid synovial fluid. J Biochem 1988;104:326-8.

9. Lai C, Wada K. Phospholipase A2 from human synovial fluid: purification and structural homology to the placental enzyme. Biochem Biophys Res Commun 1988; 157:488-93. 10. Seilhamer JJ, Pruzanski W, Vadas P, et al. Cloning and recombinant expression of phospholipase A2 present in rheumatoid arthritic synovial fluid. J Biol Chem 1989;264:5335-8. 11. Vadas P, Pruzanski W. Role of secretory phospholipases A2 in the pathobiology of disease. Lab Invest 1986;55:391^W4. 12. Pruzanski W, Vadas P, Stefanski E, Urowitz MB. Phospholipase A2 activity in sera and synovial fluids in rheumatoid arthritis and osteoarthritis. Its possible role as a proinflammatory enzyme. J Rheumatol 1985;12:211-16. 13. Pruzanski W, Keystone EC, Sternby B, Bombardier C, Snow HM, Vadas P. Serum phospholipase A2 correlates with disease activity in rheumatoid arthritis. J Rheumatol 1988;15:1351-5. 14. Vadas P. Plasma phospholipase A2 levels correlate with the hemodynamic and pulmonary changes in Gram negative septic shock in man. J Lab Clin Med 1984;104:873-81. 15. Van den Bosch H, Aarsman AJ. A review on methods of phospholipase A2 determination. Agents Actions 1979;9:382. 16. Vadas P, Hay JB. The release of phospholipase A2 from aggregated platelets and stimulated macrophages of sheep. Life Sci 1980;26:1721-9. 17. Davidson FF, Dennis EA, Powell M, Glenney JR. Inhibition of phospholipase A2 by 'lipocortins' and calpactins. An effect of binding to substrate phospholipids. J Biol Chem 1987;262:1698-705. 18. Voller A, Bartlett A, Bidwell DE. Enzyme immunoassays with special reference to ELISA techniques. J Clin Pathol 1978;31:507-20. 19. Harlow E, Lane D. Antibodies. A laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory, USA, 1988:310, 379. 20. Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315. 21. Bomalaski JS, Baker D, Resurreuioa NV, Clark MA. Rheumatoid arthritis synovial fluid phospholipase A2 activating protein (PLAP) stimulates human neutrophil degranulation and superoxide ion production. Agents Actions 1989;27:425. 22. Blackwell GJ, Flower RJ. Inhibition of phospholipase. Br Med Bull 1983;39:260-4. 23. Pruzanski W, Vadas P, Kim J, Fornasaier V. The proinflammatory effect of intra-articular injection of soluble human and venom phospholipase A,. Am J Pathol 1984,134:807.

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non-pancreatic PLA2, as measured by immunoassay, is significantly increased in rheumatoid arthritis patients and particularly in those with active disease. Synovial PLA2 levels were not measured in this study because of the invasive nature of this procedure and the ethical constraints in obtaining synovial fluid from normal subjects. We did not observe elevated levels of immunoreactive PLA2 in the plasma of osteoarthritis patients, in contrast to an earlier study where PLA2 activity was measured [12]. These findings have a number of important implications for the management of patients with rheumatoid arthritis. First, an obstacle in the treatment of rheumatoid arthritis lies in the difficulty in predicting and therefore treating disease progression. Serial measurement of plasma PLA2 levels may be able to predict disease progression. The high levels of plasma PLA2 in rheumatoid arthritis patients also suggests a rationale for the development of specific PLA2 antagonists which may succeed in interrupting the inflammatory process.

Measurement of human phospholipase A2 in arthritis plasma using a newly developed sandwich ELISA.

A sandwich enzyme-linked immunosorbent assay (ELISA) for human non-pancreatic phospholipase A2 (PLA2) was developed using monoclonal antibodies raised...
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