The development of an ELISA for group IVA phospholipase A2 in human red blood cells

Prostaglandins, Leukotrienes and Essential Fatty Acids 94 (2015) 43–48

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The development of an ELISA for group IVA phospholipase A2 in human red blood cells Donald J. Macdonald a,n, Rose M. Boyle a, Alastair C.A. Glen a, Christina C. Leslie b, A. Iain M. Glen 1, David F. Horrobin 2 a b

Department of Biochemistry, Victoria Infirmary, Glasgow G42 9TY, UK Department of Pediatrics, National Jewish Health, Denver, CO 80206, USA

art ic l e i nf o

a b s t r a c t

Article history: Received 3 July 2014 Received in revised form 31 August 2014 Accepted 10 November 2014

An immunoassay for IVA phospholipase A2 in human red blood cells is described. The assay is a noncompetitive sandwich assay in which increasing amounts of the measured protein produce increased luminescence. The antibodies used in the assay are directed against two unique epitopes of the molecule, which sequentially trap and detect the protein. The standard curve covers the range 0.7 ng to 23 ng/mL (0.07 to 2.3 ng/well). The intra-assay and inter-assay coefficients of variation were 9% and 12%, respectively. Evidence is presented that the assay is specific for the alpha paralog of IV PLA2. The assay allows simple and rapid quantification of IVAPLA2 in red blood cell lysates and other biological fluids. & 2014 Elsevier Ltd. All rights reserved.

Keywords: Sandwich immunoassay Alpha paralog Arachidonic acid Cell signalling Red blood cell Diagnostic marker

1. Introduction The phospholipase A2 enzymes are a large family of enzymes characterised by their ability to hydrolyse the sn-2 ester bond of phospholipids. The properties of these enzymes have been reviewed [1] and a classification provided which depends on function, structure and sequence homology. The products of sn-2 hydrolysis of phospholipids are a fatty acid and lysophospholipid, both of which are potentially biologically active molecules. Early interest in phospholipase was in diagnosis of pancreatitis when substrate assays detected a low molecular weight enzyme in plasma. A high molecular weight phospholipase A2, an intracellular enzyme, highly selective for phospholipid with arachidonate in the sn-2 position, was first recognised in human neutrophils [2]. This protein, cytosolic phospholipase A2, has been sequenced [3], purified [4] and its tertiary structure resolved [5]. Cytosolic phospholipase A2 is now classed as Group IVA phospholipase A2 (IVAPLA2). Its arachidonate selectivity derives from the unusual serine-aspartate dyad configuration at its active site [6].

n

Corresponding author. E-mail address: [email protected] (D.J. Macdonald). 1 A Iain M Glen formerly of The Ness Foundation, died on February 14, 2013. 2 David F. Horrobin formerly of Laxdale Ltd, died on April 1, 2003.

http://dx.doi.org/10.1016/j.plefa.2014.11.003 0952-3278/& 2014 Elsevier Ltd. All rights reserved.

The enzyme is widely distributed and is expressed in all human cells examined with the exception of mature T and B lymphocytes [7]. Its selective release of arachidonic acid provides a regulator of diverse cellular functions and a precursor for biosynthesis of potent inflammatory lipids such as eicosanoids, including prostaglandins, thromboxanes, leukotrienes and lipoxins [7]. The release of arachidonic acid can itself function directly in cell signal transduction [8]. Studies of the specificity of the enzyme reported in 1995 by Clark [9] show that membrane phospholipid with the fatty acid eicosapentaenoic in the sn-2 position is the next most favoured substrate after arachidonic acid for the enzyme which is recognised for its part in dopamine receptors and neurotransmission [10]. Our group became interested in IVAPLA2 when the membrane phospholipid hypothesis of schizophrenia was postulated by the late Professor Horrobin [11] as an explanation for the reduced levels of arachidonic acid in the red blood cell (RBC) membranes of patients with schizophrenia [12]. In order for the membrane phospholipid hypothesis to be tenable we proposed that the enzyme IVAPLA2 must be present in the RBC. We were able to demonstrate the presence of IVAPLA2 in human RBC haemolysates by affinity chromatography and Western blot detection at a level of 0.16 fg/cell [13]. In developing an assay for the IVAPLA2 protein, we chose an enzyme linked immunosorbent assay (ELISA) which could be made specific for this protein by the choice of capture and

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detection antibodies as a simple method of analysis that would avoid the limitations of the enzyme activity measurements and the Western Blot analysis presently available. Other PLA2 enzymes are present in human blood. A secretory PLA2, sPLA2 human type IIA is present in the plasma and a commercial Enzyme Immunoassay Kit is available to measure this enzyme [14]. There is also a calcium independent PLA2 (iPLA2) present in the plasma and cells, first described and measured by Gattaz [15] using a flurometric assay and by a radioenzymatic method described by Ross [16]. IVA PLA2 can be measured by substrate assay in the presence of inhibitors of the other phospholipases utilising the hydrolysis of radiolabeled arachidonic acid from the sn-2 position of phophatidylcholine in unilamellar vesicles [17]. The group IV PLA2 proteins exist as six paralogs and their properties and amino acid homology have been reviewed [18]. Some paralogs of IVPLA2 are ubiquitous in human cells; others have a more limited distribution. An ELISA has been developed for IVAPLA2 in human eosinophils [19] but used antibodies to different epitopes to those reported in the present study. Our ELISA method allows large numbers of samples to be assayed easily in population studies. The ELISA described here was originally reported as an abstract [20] and was used in a study of IVAPLA2 in the RBCs of patients with schizophrenia previously reported in this journal [21]. Neither of these two reports provided the detail required for others to perform the assay for themselves. We have rectified this in the present article and provided additional information to assist others in the replication of the assay.

purified using the immunogen and stored as 2 mg/mL aliquots in PBS at  80 1C. This preparation was used to provide an IgGalkaline phosphatase conjugate by adapting the method of Duncan et al. [22] and involves generating free SH groups in the IgG molecule which then react with maleimide activated alkaline phosphatase. The conjugation was performed as follows. S-Acetylthioglycolic Acid N-Hydroxysuccinimide (SATA) 4 mg was dissolved in 1 mL of dimethylformamide and 20 mL added to 2 mg/mL of affinity purified IgG. The solution was incubated at room temperature for 30 min then de-acetylated by the addition of 100 mL de-acetylation solution. The de-acetylation solution contained 50 mg/mL of hydroxylamine hydrochloride in PBS and 0.93 g of di-sodium ethylenediaminetetra-acetic acid (EDTA) per 100 mL; solid di-sodium hydrogen orthophosphate dodecahydrate was added to pH the solution to 7.4. The solution was mixed at room temperature for 2 h. The acetylation reagents were separated from the IgG by chromatography on a 43  1 cm Sephadex G100 column, at a flow rate of 0.5 mL/min and equilibrated with PBS, containing 1 mMol/L di-sodium EDTA with di-sodium hydrogen orthophosphate dodecahydrate added to bring the pH to 7.4. The excluded peak, containing about 1.5 mg of protein, was added to 6 mg of polymerised and maleimide activated alkaline phosphatase dissolved in 3 mL of PBS and mixed at room temperature for 30 min. The solution was diluted in saline and titred in the assay so that the top standard gave a luminescence reading of 3000 relative light units (RLU) when the luminometer is set to maximum amplification. After titreing, the solution is diluted into 1 mL aliquots and stored at 80 1C.

2. Materials and methods

2.3. U 937 cell culture

2.1. Materials

U937 cells were provided by the European Collection of Cell Cultures, Salisbury, Wiltshire, SP4 0JG, UK. U937 cells were cultured in roller bottles to a total volume of 25 L and 4  1010 cells. A cytosol was prepared as described by Clark et al. [4] without the addition of ATP and stored as aliquots at  80 1C. Preparation of insect Sf9 cells expressing recombinant IVAPLA2 The baculovirus-insect cell expression system was used to produce human recombinant IVAPLA2 in Sf9 cells and lysates were prepared as described [23]. The cytosolic fraction was stored in aliquots at 20 1C.

Polymerised and maleimide activated alkaline phosphatase, human albumin (lyophilized powder), bovine serum albumin (BSA) 3,30 diaminobenzidine (DAB) tablets and capsules of phosphate–citrate buffer with sodium perborate were from Sigma, Dorset, BH12 4QH, UK. Complete™ protease inhibitor cocktail tablets with EDTA from Roche, East Sussex, BN7 1LG, UK. Phosphate Buffered Saline tablets [PBS] pH 7.4 and saline tablets from Oxoid Limited, Hampshire, RG24 8PW UK. Roti-block from Rotech Scientific, Milton Keynes, MK12 5QL, UK. Alkaline phosphatase luminescence reagent from Beckman Coulter™ High Wycombe, HP 12 4LJ, UK. Nunc C 96 white maxisorp fluorogenic microtitre plates from VWR International, Lutterworth, LE17 4XN, UK. Luminoskan Ascent, Wellwash Ascent and ELISA incubator from ThermoQuest, Basingstoke, Hampshire, RG21 6YH, UK. Electrophoresis: 4–12% gradient Bis–Tris gels, 3-(N-morpholino) sulphonic acid [MOPS], SDS running buffer and Simply Blue™ protein stain from Invitrogen Ltd, Paisley PA4 9RF, UK. Antibodies for Western Blots were from Santa Cruz Biotechnology Inc., 2145 Delaware Avenue, Santa Cruz, California 95060, USA and the Binding Site Group Ltd, Birmingham B15, UK. Secondary antibodies for Western blotting from Dako A/S, Denmark. 2.2. Antibodies and enzyme conjugate The primary (capture) antibody was sheep IgG, raised against amino acid sequence 725–749 and used as obtained from The Binding Site Group Ltd, Birmingham, UK. The second (detection) antibody was raised in sheep by Diagnostics Scotland Ltd, Edinburgh EH17 7QT, UK, against amino acid sequence 241–260 of the IVAPLA2 protein conjugated to Keyhole Limpet Haemocyanin. The IgG fraction was prepared by protein A chromatography, affinity

2.4. SDS-PAGE and Western blots Samples from Sf9 and U937 cell lysates were diluted in Laemmli sample buffer, separated by SDS-PAGE on 4–12% Bis–Tris gradient gels using MOPS running buffer. The protein gels were electrophoresed for 2 h at 4 1C then stained for 1 h with Simply Blue™ protein stain and the gel washed for 1 h with distilled water. For Western blots the gels were electrophoresed for 50 min at room temperature, transferred to nitrocellulose membrane then blocked for 1 h in blocking solution (10% Roti-block in PBS). The membranes were then probed with 10 mg of mouse anti IVAPLA2 raised against a sequence within the 1–216 amino acids of the protein (Santa Cruz) or 30 mL of sheep anti IVAPLA2 (Binding Site) in 30 mL of blocking solution. After washing the membranes, the appropriate secondary antibodies i.e. rabbit anti mouse-HRP or rabbit anti sheep-HRP were added as a 1 in 1000 dilution in 30 mL blocking solution. The blots were visualised by incubating the membranes in a 100 mL of a solution containing one capsule of phosphate–citrate buffer with sodium perborate and one tablet of DAB. The reaction was stopped by washing with tap water.

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2.5. Sample preparation K/EDTA samples of venous blood (approximately 5 mL) were centrifuged for 5 min at 1500g, the plasma, buffy coat and the top 2 mm of red cells were aspirated. The packed RBCs were frozen without any further manipulation in liquid nitrogen within 60 min of collecting the blood sample and stored at  80 1C until analysed. Immediately prior to analysis the packed RBCs were thawed, centrifuged at 10,500g and diluted to give both a 1 in 40 and a 1 in 80 dilution with sample diluent, with both dilutions being assayed. Sample diluent was prepared as follows: 3.0 g of Tris, 0.74 g disodium EDTA, 0.37 g KCl and two saline tablets were dissolved in 800 mL of de-ionised water; pH to 7.4 with HCl and made up to 1litre. To 200 mL of this solution was added 0.2 g human albumin and 1 tablet Complete™. This solution was used as sample diluent, stored at 4 1C and made up fresh every week. 2.6. ELISA procedure A Nunc white microtitre plate was coated overnight at 4 1C with a 100 μL per well of a 1 in 480 dilution of the Binding Site antibody in 0.1 M carbonate–bicarbonate buffer pH 9.6. After four washes with TBSS (tris buffered saline þ0.5 M sodium chloride adjusted to pH 7.4 with HCl) on the Wellwasher the wells were blocked with 300 μL per well of 50% Roti-Block in TBSS for 1.5 h at 30 1C with shaking in the ELISA incubator after which the plate was washed eight times with TBSS. Duplicate wells were incubated with 100 μL of standard or sample RBC lysate for 1.5 h at 30 1C with shaking in the ELISA incubator. The plate was then washed eight times with TBSS. The previously prepared 1 mL antibody–enzyme conjugate was added to: 5 mL TBSS þ6 mL RotiBlockþ 0.2 mL of 1 M sodium citrate (pH 7.4) þ0.012 g HSA and 100 μL added to each well then incubated for 1.5 h at 30 1C with shaking in the ELISA incubator and the plate washed eight times with TBSS. The luminometer added 100 μL of alkaline phosphatase luminescence reagent to each well, incubated with shaking at room temperature for 10 min and the luminescence in each well read as relative light units (RLUs). The standard curve was fitted by a four parameter logistic curve fit and the IVAPLA2 content of the samples expressed as ng IVAPLA2/mL.

Fig. 1. Western blot of U937 and Sf9 lysate probed with mouse anti IVAPLA2 and rabbit anti mouse-HRP. Lane 1: molecular weight markers, lane 2: 13 μg of U937 lysate and lane 3: 1.3 μg Sf9 lysate.

2.7. Haemoglobin measurement To eliminate the variation in RBC numbers between subjects all the red cell IVAPLA2 results were related to the amount of haemoglobin (Hb) present in the sample and expressed as ng IVAPLA2/g Hb. The haemoglobin content of the RBC lysate was measured [24] by adding 20 μL of RBC lysate into 4 mL of 0.04% ammonia (SG ¼0.880) and read against a blank solution of ammonia at 540 nm. The haemoglobin assay was standardised against a Beckman Coulters standard. The assay results were then reported as mg IVAPLA2/g Hb.

3. Results 3.1. ELISA standard curve The amount of IVAPLA2 in the insect cell preparation and the U937 cell preparation was compared by western blot as shown in Fig. 1. Inspection of the blot showed that the insect cell preparation had substantially more IVAPLA2, at least ten times more than the U937 preparation. The assay was calibrated as follows. The IVAPLA2 protein in the baculovirus-insect cell cytosol was identified by PAGE after staining with Simply Blue™ as a band at 90 kDa present in the infected Sf9 insect cell cytosol which was not

Fig. 2. Electrophoresis on 4–12% Bis–Tris gel for 2 h at 4 1C and staining for protein with Simply Blue™. Lane 1 molecular weight markers, lane 2 (2.3 μg) uninfected Sf9 cytosol, lane 3 (5.8 μg) Sf9 cytosol showing the IVAPLA2 as a band at 90 kDa, lanes 4 and 5 (2.6, 1.3 μg) U937 cytosol and lanes 6 to 10 (31.2, 62.5, 125, 250, 500 ng) BSA.

present in the uninfected Sf9 cytosol (Fig. 2). The IVAPLA2 protein in the infected insect cell cytosol was also quantified on the same gel using dilutions of BSA as a protein standard. We established that the infected insect cell cytosol contained 100 μg IVAPLA2 by visually comparing the protein band densities of both the BSA and infected

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cell cytosol. The working standard was prepared by comparing the IVAPLA2 content of the U937 cytosol with that of the infected Sf9 insect cell cytosol by Western blot. The U937 cytosol does not contain sufficient IVAPLA2 protein to be visualised in the PAGE system by protein staining (Fig. 2). The IVAPLA2 protein content of the U937 cytosol was estimated in the following way. The insect cell cytosol containing 100 μg IVAPLA2/mL was double diluted to give a standard curve between 100 and 3.1 μg/mL. The U937 cytosol was double diluted to give three dilutions. All the dilutions from the SF9 insect cell cytosol and the dilutions from the U937 cytosol were compared for protein content using Western blots [not shown]. The blots were probed with sheep anti IVAPLA2 (Binding Site) and rabbit anti sheep-HRP. The preparation of U937 cytosol used was found to contain 9.4 μg IVAPLA2/mL. For use in the ELISA the U937 cytosol was diluted 1 in 408 in sample diluent to give a level of 23.0 ng/mL. This preparation was then stored at  80 1C in 1 mL aliquots. When the assay was run an aliquot was removed from the freezer and double diluted to give a standard curve from 23.0 to 0.7 ng/mL. Fig. 3 illustrates a standard curve for the ELISA derived from four separate assays covering the measurement range of 0.7 to 23.0 ng/mL (0.07 to 2.3 ng/well). 3.2. Detection limit The 23.0 ng/mL standard was double diluted ten times and eight replicates of each standard together with eight replicates of the zero standard were assayed for IVAPLA2 on the same plate. The mean RLU and standard deviation was calculated for each standard. It was found that the 0.7 ng/mL standard was significantly greater than the zero standard (po 0.0001) and three standard deviations above the zero standard. This is the detection limit of the assay. 3.3. Precision The within batch precision was estimated by preparing a haemolysate from a recently expired unit of donated blood. This was aliquoted, stored at  80 1C and assayed as 40 duplicate samples on a microtitre plate. The assay mean of this preparation was 2.60 μg/g Hb (SD 70.22) and a CV of 9%. Between batch precision was estimated by assaying the same preparation in 25 different assays. The assay mean was 2.59 μg/g Hb (SD 70.30) and a CV of 12%. 3.4. Specificity As the Group IV PLA2 proteins are known to exist as paralogs and we tested the cross reaction of the alpha, epsilon, gamma and delta paralogs in the ELISA as follows. These four paralogs were kindly gifted by Professor M. Gelb, University of Washington,

Relative light units (RLU)

3500 3000 2500 2000 1500 1000 500 0 0

5

10

15

20

25

IVAPLA2 (ng/ml)

Fig. 3. The standard curve for the IVAPLA2. Each data point represents the mean 7standard error of the mean (SEM) for the ELISA from 4 assays. Luminometer relative light units in the ordinate.

Fig. 4. Dilution of the alpha paralog IV PLA2 compared to the standard curve for IVA PLA2. The ordinate shows the relative light units in the ELISA assay. There is parallelism between these two sources of IVAPLA2.

Table 1 Cross reaction of Type IV PLA2 paralogs and and calcium independent PLA2 in the ELISA for IVAPLA2. Protein tested

Protein concentration range (ng/mL)

Measured IVA PLA2 (ng/mL)

Epsilon PLA2 Gamma PLA2 Delta PLA2 Calcium independent PLA2

3.5–225 2.7–700 1–1031 1.25–648

o 0.7, o 0.7, o 0.7, o 0.7,

Undetected Undetected Undetected Undetected

Seattle. The alpha paralog Group IV PLA2 was diluted to give a level of 17 ng/mL, then double diluted and analysed in the ELISA. The results are shown in Fig. 4 and confirm that the alpha paralog cross reacts in the assay as expected since the alpha paralog is the IVAPLA2 protein. The alpha paralog was observed to parallel our standard curve prepared from U937cytosol. We tested the cross reactivity of the other Group IV PLA2 paralogs in the ELISA as follows. The epsilon, gamma and delta paralogs were each diluted over a wider concentration range than the standard curve for the ELISA and assayed in the ELISA. The results are summarised in Table 1 and show that these three paralogs did not cross react in the ELISA over the concentration range measured. TypeVIPLA2 (calcium independent PLA2) was kindly gifted by Dr. Haowei Song, Washington University in St. Louis. Any cross reaction by the calcium independent PLA2 enzyme TypeVI PLA2 in the ELISA was tested as for the paralogs. The results shown in Table 1 confirm that the Type VI PLA2 does not cross react in the ELISA at the range of concentration tested. Human plasma contains secretory sPLA2 enzyme (human Type IIA) and we would not expect this enzyme to cross react in the assay, as there is no homology with the Group IV PLA2s. However, we excluded the possibility of any cross reaction as follows. Eighteen volunteers recruited after local ethical permission donated a sample of venous blood collected as a potassium EDTA sample. The sample was centrifuged, the plasma separated and stored at  80 1C. The RBCs were washed three times with saline to remove any trace of plasma sPLA2 human Type IIA protein from the RBCs then stored at  80 1C. Both the plasma and RBC haemolysates were diluted 1 in 40 and analysed using a commercial immunoassay for sPLA2 human Type IIA protein [14]. The plasma samples had a mean of 3.1 ng/mL with a range of 1.2 to 7.5 ng/mL. All the RBC samples had levels of less than the detection

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limit (15.6 pg/mL) of the commercial assay. We concluded from this experiment that the sPLA2 (human Type IIA) is not present in detectable amounts in the RBC and is unlikely to interfere in our ELISA. In order to test and ensure that there was no IVAPLA2 in plasma we measured IVAPLA2 in samples of centrifuged plasma from humans, ox, rabbit and sheep. All the plasma samples had IVAPLA2 levels below the detection limit of the assay. 3.5. Recovery Recovery was assessed by diluting an RBC haemolysate 1:20 and adding increasing amounts of IVAPLA2 from the cytosol of a diluted U937 solution to the haemolysate and measuring the IVAPLA2. Results are shown in Table 2 confirming adequate recovery in the ELISA procedure. 3.6. Effect of storage on RBC IVAPLA2 We observed that storage of RBCs at  20 1C over 35 weeks showed a reduction of 81% in the measured IVAPLA2 while RBCs stored at  80 1C showed a reduction of 16% over the same period We therefore recommend that the RBC samples for IVAPLA2 measurement should be stored either in liquid nitrogen or at 80 1C, analysed as soon as practical and always within six months of collection. 3.7. IVAPLA2 in healthy Subjects We measured the IVAPLA2 in 51 healthy adult volunteers (27 males, mean age 31 7 8.3 and 24 females, mean age 437 11.4). The sampling was performed as described in the sample preparation section above. The level we observed (mean72SD) was 1.50 70.62 μg IVAPLA2/g Hb. No gender difference was observed. In a study of the fatty acid composition of erythrocytes in children [25] the red cell IVAPLA2 was measured but not reported. The IVAPLA2 level (mean72SD) in 41 normal children was 1.57 71.54 μg IVAPLA2/g Hb.

4. Discussion With the exception of the measurements on eosinophils (18), previous quantitation of IVAPLA2 has been based on enzymic activity measurements involving substrates, including radio labelled substrates, in the presence of suitable inhibitors to eliminate possible interference from other PLA2 enzymes. Inhibitors must be carefully chosen to ensure that the selective inhibition is effective [26]. Apart from the general protease inhibitors specified in the method section the ELISA does not require any specific inhibitors.

Table 2 Recovery of known amounts of IVAPLA2 from U937 lysate added to a diluted haemolysate and assayed for IVAPLA2. IVAPLA2 (ng/mL)

Recovery (%)

Added

Expected

Measured

0 4.2 8.4 16.8

9.5 13.7 22.1

5.3 10.4 14.0 21.0

109 102 95

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The ELISA reported here allows a specific, rapid, precise and convenient assay of IVAPLA2 that can be completed in the working day and allows measurement of the protein in RBC haemolysates that contain a complex mixture of proteins. Attention to detail in sample collection protocol and the pre-treatment required before storage is considered essential for all measurement of this protein in whatever cell type is being studied. It should be noted that this enzyme is potentially sensitive to oxidation [17]. Western blot methods with subsequent band density analysis have the limitation that relatively large amounts of protein are required for this type of analysis. The recovery data is satisfactory over the 4.0 to 16 ng/mL range without any apparent interference from RBC lysate. In our laboratory we routinely make two dilutions of all RBC samples to ensure that elevated levels of IVAPLA2 fall into this range and are readily measured. The processing of stored, frozen samples for this assay is simple and rapid, lending to ease of assay of blood specimens in large numbers. The specificity studies are important for confidence in the ELISA. The specificity of the assay depends on the capture and detection antibodies used in the assay, each against different epitopes of the IVAPLA2 molecule. There is no detectable interference from either the calcium independent type VI or the secretory type II PLA2 found in the plasma. We are not aware of a sequence homology with other proteins at the epitope chosen for the capture antibody, amino acid sequence 725 to 749. For the detection antibody, amino acids 241 to 260, there are six instances of matching amino acids that IVAPLA2 shares with its alpha, beta, delta, epsilon and zeta paralogs (IVAPLA2, IVBPLA2, IVDPLA2, IVEPLA2, IVFPLA2 [18]. These paralogs share six single identical amino acids that are separated by at least one non-identical amino acid. The areas of homology of the gamma paralog IVCPLA2 are not at the epitopes used to raise antibodies for the ELISA. The specificity studies using Group IV paralogs (Table 1) demonstrate that the assay measures only the alpha paralog of IV PLA2. Clark and Colleagues [9] have examined the similarity of IVAPLA2 to other proteins in terms of amino acid sequence homology, detailing shared sequences with other proteins in the calcium binding domain at the N-terminal end of the molecule and other limited homology sequences, in Phospholipase B of Pencillium Notatum and a pulmonary surfactant, Protein C. The epitopes of IVAPLA2 that we have chosen to raise the antibodies for both capture and detection in this ELISA avoid these known shared sequences. The antibodies were prepared before the IVPLA2 paralogs were identified and we have presented our evidence (Table 1) for the absence of cross reactivity by testing purified paralog proteins in the ELISA. The absence of detectable IVAPLA2 in measurements of human plasma gives confidence that the method has no detectable cross reactivity with the wide range of proteins present there. The demonstrated parallelism between purified recombinant IVAPLA2 and the U937 cytosol together with the lack of reactivity with any of the paralog proteins adds confidence in the assay. Assays measuring activity can be expected to give different results to assays measuring enzyme protein. However, the specificity of the ELISA confers an advantage to this assay in comparison to other assay methods. It is presented as an alternative method for assessing IVAPLA2. The lack of stability of IVAPLA2 at  20 1C is surprising and was prompted by the observation that the same RBC preparation stored at 20 1C for 1 week had lost a third of the ELISA measurable IVAPLA2 compared to paired samples stored at  80 1C. Since  20 1C is likely to be above the eutectic point for protein solutions within the red cell it is possible that limited

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proteolysis occurs at this temperature. Our recommendation that the RBC samples should be initially cooled in liquid nitrogen and then stored at  80 1C will provide the safest method of securing reliable results for measurement of RBC IVAPLA2. We have also shown that measurements on RBCs in storage at 80 1C show a gradual deterioration and emphasise the importance of freezer storage protocol. Our Western blots show IVAPLA2 running as a 90 kDa protein although the calculated molecular weight of this protein is 85 kDa. The anomaly is recognised by others as a feature of Western blots of this protein [4]. In our previous report [21] we observed that the IVAPLA2 concentration was significantly increased in RBCs that were washed with buffer. This phenomenon could be reversed if the washed RBCs were washed with their own plasma or plasma that had been excluded on a G25 Sephadex column. We have not investigated this observation further, but consider it merits further study. Our protocol for the RBC sample preparation does not include a washing step. The IVAPLA2 protein levels in the red cells of the healthy adults and children reported in this paper are for guidance only and would advise that any study using this assay should generate normal reference ranges from the population being studied. In conclusion this assay provides a simple method that may be useful for measuring the IVAPLA2 protein in red cells, other cell preparations and in disease states. Acknowledgments We acknowledge the generous support of NHS Greater Glasgow and Clyde and Dr. A.S. Hutchison Consultant Clinical Biochemist and Head of Department in offering research facilities in the laboratory. We thank Drs L Bence, M Chambers and D Fatori of Diagnostics Scotland for their immense help. We acknowledge the advice of Dr J. D. White, A. Reid, H. Oliphant and A. Kyle and the excellent technical assistance of all the BMS staff in our department. We are indebted to Professor Michael Gelb for his gifts of IVPLA2 paralogs. Professor Michael H. Gelb, Departments of Chemistry and Biochemistry, Campus Box 351700, 36 Bagley Hall, Univ. of Washington, Seattle, WA 98195, USA. We also thank Dr. Haowei Song for his kind gift of calcium independent PLA2. Dr. Haowei Song, Division of Endocrinology, Metabolism and Lipid Research, Washington University in St. Louis, School of Medicine, Box 8127660S. Euclid Ave. St. Louis, MO 63110314-362-8195, USA. We are particularly grateful for the advice, encouragement and past financial support for this work provided by the late Professor Horrobin through his company Laxdale Ltd. References [1] D.A. Six, E.A. Dennis, The expanding superfamily of phospholipase A2 enzymes: classification and characterisation, Biochim. Biophys. Acta 1488 (2000) 1–19. [2] F. Alonso, P.M. Henson, C.C. Leslie, A cytosolic phospholipase in human neutrophils that hydrolyzes arachidonyl-containing phosphatidylcholine, Biochim. Biophys. Acta 878 (1986) 273–280.

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