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Human atrial natriuretic factor (ANF)
Journal of Immunological Methods, 118 (1989) 91-100 Elsevier
91
JIM05094
H u m a n atrial natriuretic factor ( A N F ) Characterisation of a monoclonal antibody panel and its use in radioimmunoassay C. Prowse 3, E. George 1, L.R. M i c k l e m 1, V. H o r n s e y 3, j. Brown 2 a n d K. James Departments of 1 Surgery and 2 Clinical Pharmacology, Edinburgh University, Edinburgh, U.K., and "~Edinburgh and South-East Scotland Blood Transfusion Service, Royal Infirmary, Edinburgh, U.K.
(Received 8 July 1988, revised received 28 October 1988, accepted 31 October 1988)
The production of nine monoclonal antibodies to human atrial natriuretic factor (ANF 1 - 2 8 ) i s described. All possible combinations of two antibodies failed to reveal any which could simultaneously bind ANF. Studies with ANF analogues and the antibodies having the three highest affinity values (K D = 5, 25 and 21 pM) indicated that the antibodies are directed to the central portion of the antigen molecule. The highest affinity antibody was able to replace polyclonal antisera in the radioimmunoassay of ANF in extracts of plasma. Key words: Atrial natriuretic factor; Monoclonal antibody; Radioimmunoassay
Introduction
Since its discovery in 1981, there has been great interest in atrial natriuretic factor, ANF (Flynn and Davies, 1985; De Bold, 1986; Gutkowska et al., 1986b; Baxter et al., 1988), but the actions of this peptide hormone in regulating water balance, sodium levels and blood pressure are still incompletely understood. Such understanding as exists has been aided by the use of synthetic ANF peptides and by the development of assays for the
Correspondence to: C. Prowse, Blood Transfusion Centre, Royal Infirmary, Edinburgh, EH3 9HB, U.K. Abbreviations: ANF, atrial natriuretic factor; RIA, radioimmunoassay; BSA, bovine serum albumin; MAb, monoclonal antibody; KLH, keyhole limpet haemocyanin; BTG, bovine thyroglobulin; SAM, sheep anti-mouse antiserum; DAR, donkey anti-rabbit antiserum; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; BUH, Buccinum undatum haemocyanin; KIU, kallikrein inhibitor units.
hormone. The latter have largely been classical radioimmunoassays using rabbit antisera (Andersson et al., 1986; Hartter et al., 1986; Marumo et al., 1986; Tikkanen et al., 1985; Wilson et al., 1986; Gutkowska et al., 1985, 1986a; Juppner et al., 1986; Rosmalen et al., 1987; Cernacek et al., 1988) and usually require the extraction and concentration of plasma samples. Alternative receptor binding assays for A N F have been described which are of narrower specificity than most radioimmunoassays but are more rapid (Sagnella et al., 1987). Recently three murine MAbs to human ANF (John et al., 1986; Naomi et al., 1987; Stasch et al., 1987) and one to rat ANF (Milne et al., 1987) have been described. Initial results with these have suggested that they might be useful in increasing our understanding of the functions of ANF and in investigating its histological location. In this study, the production of a panel of MAbs to ANF and their potential use in an immunoassay for human A N F in plasma is described.
0022-1759/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
92 14
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mine cyanogen bromide method of Kohn and Wilchek (1982). The subclass of the monoclonal antibodies was determined by immunodiffusion using antisera from Serotec (obtained through Uniscience, Cambridge, England) or the University of Birmingham Research Institute (University of Birmingham, England). All other reagents were of at least Analar Grade.
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Fig. 1. Structure of ANF. In rat, mouse and rabbit ANF isoleucine 12 replacesmethionine12 of the human (and canine) peptide. Despite the recent recommendations of Dzau et al. (1987) on ANF nomenclature, the alpha human ANF has, for convenience, been described in this paper as ANF (1-28) and analogues of the peptide described using numbering which permits direct comparison of homologous residues (Fig. 1).
Materials
and methods
Synthetic human ANF (1-28, batch 380A or 220C) and rat atriopeptin II (5-27, batch 188B) were obtained from Bachem (Saffron Waldon, England). Rat atriopeptin I (5-25, batch 122), human ANF (7-28, batch 098) and ANF fragment 13-28 (batch 129) were from Cambridge Research Biochemicals (Cambridge, England). 1desamino-8-D-arginine vasopressin (DDAVP) was purchased from Ferring (Feltham, Middlesex, U.K.). [3-125I]iodotyrosyl 28 human A N F (1-28) (IM-187, 2000 C i / m m o l ) was from Amersham I n t e r n a t i o n a l ( A m e r s h a m , England). 25% glutaraldehyde, KLH, BTG, BSA, anti-mouse IgG-horseradish peroxidase conjugate (A2028) and EDC were purchased from Sigma. BUH was purified and supplied by Dr. D. Pepper. SAM and DAR antisera were obtained from the Scottish Antibody Production Unit (Law Hospital, Carluke, Lanarkshire). Immunoglobulins and antisera were immobilised on Sephacryl S-1000 (Pharmacia) by the low temperature triethanola-
Human A N F (1-28) was coupled to carrier proteins in 50 mM phosphate 0.1 M NaC1 pH 7.2 using two basic procedures. For EDC coupling, 8-10 mg of protein in 4 ml were mixed with 6 mg EDC and added dropwise to 1 mg A N F in 1 ml 0.15 M NaC1 with stirring for 15 min. Following overnight incubation at 4 ° C in the dark a further 6 mg EDC were added with stirring and, after 2 h at 20°C, the reaction mixture was extensively dialysed against 0.15 M NaC1 and frozen in aliquots. For glutaraldehyde cross-linking 8-10 mg of carrier protein in 4 ml phosphate buffer were mixed with 1 mg A N F in 1 ml 0.15 M NaC1 and 25% glutaraldehyde was added to a final concentration of 0.2% w / v . After 3 h incubation at 2 0 ° C in the dark, the mixture was dialysed, aliquoted and frozen as above. Alternatively, the reaction was performed overnight at 4 ° C with a final concentration of 0.05% w / v glutaraldehyde.
Immunisation and fusion The more relevant immunisation procedures are shown in Table I. Immunisations performed in mice with free ANP (s.c.) BUH-glutaraldehyde, B S A - g l u t a r a l d e h y d e and cellulose-periodate (Gurvich and Korukova, 1986) conjugates were unsuccessful. Two rabbits were also immunised with B T G - E D C conjugate, being given 6 weekly boosts of 1 mg conjugate as two s.c. injections after priming with 4 mg in complete Freund's adjuvant at multiple intramuscular sites. Antiserum from one of these (titre 1/10000) was used as a positive control in the screening assays. The MAbs described below were obtained from one fusion using spleen cells from mouse 2.1 which was boosted i.p. with 50 ~tg of BTG-EDC conjugate in saline 49 days after priming. The fusion
93 TABLE I IMMUNISATION OF MICE Groups of five or six mice were immunised with 50 /tg of various ANF-protein conjugates (i.e., about 5 /Lg ANF) with an equal volume of Freund's complete adjuvant by the subcutaneous (s.c.) or intraperitoneal (i.p.) routes. Unless otherwise indicated boosts were given with alum and test bleeds taken 7 days after the last boost. Titres were determined as the average dilution, in responding mice, to give 50% binding in the RIA. The best responses (titre 1/10000) were seen in mice 3.5 and 4.3. The BTG-EDC conjugates for groups 2/3 and 4 were separate preparations. Group
Carrier
Conjugate
Route
Responders
Titre
i.p. s.c.
Boosting (days) 42 (BUH-glut) 9, 20, 28 (no alum)
1 2
BSA BTG
Glutaraldehyde EDC
1/6 6/6
i.p. i.p. i.p. i.p.
42 42 42 42
5/5 4/6 3/5 0/5
1/10 1/500 (at 42 days) 1/1000 1/500 1/200 < 1/5
3 4 5 6
BTG BTG BTG KLH
EDC EDC Glutaraldehyde EDC
was performed 3 days later using 50% polyethylene glycol 4000 (Fisons, Loughborough, England) at p H 8.5-9, NS-0 myeloma cells (Clarke et al., 1980) at a ratio of 1 : 1 and selection medium containing hypoxanthine-aminopterin-thymidine as described previously (Dawes et al., 1984) but using thymocyte-conditioned medium in place of feeder cells (Micklem et al., 1987). Hybrids producing specific MAb were cloned by limiting dilution and grown in culture and as ascites. Immunoglobulin was purified from ten-fold concentrated culture supernatant by sodium sulphate precipitation (Wright and Hunter 1982) or by chromatography on protein A-Sephacryl S-1000.
0.1 ml substrate was added and colour development stopped after 15 min with 0.05 ml 2.5 M H2SO 4. Optical densities were then read at 450 nm on a Twinreader (Flow Laboratories, Ayr, Scotland). Substrate was prepared just before use by mixing 20 ml 0.1 M acetate-citrate p H 6, 0.2 ml 10 m g / m l tetramethyl benzidine (Aldrich) in dimethyl sulphoxide and 0.3 ml of 1% w / v urea hydrogen peroxide. Samples were tested in duplicate, using a 1/1000 dilution of pooled immune mouse serum as control (O1)450 n m about 1.0). Assays on culture medium, buffer, non-immune mouse serum or in the absence of A N F coating always gave an absorption below 0.1, usually below 0.04.
Screening assays Radioimmunoassay Enzyme-linked immunosorption assay (ELISA) The ELISA was performed at room temperature throughout using 96 well microtitre trays (Nunc, Immulon II) coated overnight with 0.1 ml/well of 100 n g / m l human A N F (1-28) in PBS (0.1 M phosphate, 0.15 M NaC1, p H 7.5). After three washes with PBS, wells were blocked with 0.4 ml PBS, 1% BSA, 0.1% lactose for 1 h prior to washing and the addition of 0.1 ml test antibody, diluted as necessary in TB (0.25 M Tris, 0.1% Tween 20, p H 8). After a further hour and wash, 0.1 ml of a 1/200 dilution of Sigma anti-mouse I g G peroxidase in PBS, 1% BSA, 0.1% Tween 20 was added for 1 h. After three washes with PBS,
0.05 ml test antibody, 0.05 ml dilute radiolabelled human A N F (1-28) and 0.1 ml assay buffer (50 m M Tris, 0.1 M NaC1, 10 m M EDTA, 3 m M N a N 3, 0.1% Triton X-100, 0.25% BSA, p H 7.35) were incubated overnight at room temperature. Bound and free tracer were then separated by the addition of 0.05 ml SAM or D A R (as appropriate) immobilised on Sephacryl S-1000, shaking for 1 h and sedimenting through buffer containing 10 % sucrose (Hornsey et al., 1985). Assays were performed in duplicate using immune rabbit or mouse serum diluted to give about 50% binding as a positive control. Non-specific binding was 3-4% and maximal binding in excess of 95%.
94 For some assays BSA was replaced with gelatin. Antibody titre was defined as the dilution necessary to allow 50% maximum binding and varied from 1/107 to 1/108 for ascites from clones 4, 5 and 9.
ml removed, using sheep anti-mouse immunoglobulin gel, showed that more than 90% of tracer was bound by each of the fluid-phase antibodies after overnight incubation with the chosen antibody concentration.
Antibody characterisation
Plasma assays
Antibody isotype was determined by immunodiffusion of ten-fold concentrated culture supernatant against subclass-specific antisera and confirmed, in some cases, by the p H required to elute MAb from protein A. Antibody affinity was determined, as the dissociation constant, by Scatchard analysis of data obtained by a modification of the above RIA. MAbs were diluted to allow 50-60% binding of tracer alone and the percent binding of tracer determined after substitution of various amounts of unlabelled human A N F (1-28) diluted in assay buffer for the 0.1 ml of buffer. The ability of MAb to bind various A N F analogues was determined in a similar assay using carefully prepared stock solutions (1 m g / m l in 0.1 M acetic acid), further diluted in assay buffer. The percentage reactivity was defined as
10 ml blood samples were collected by clean venepuncture, mixed with 0.1 ml of a solution containing 15 mg E D T A and 1000 K I U aprotinin (Trasylol, Bayer) and immediately cooled on melting ice. Plasma obtained by centrifugation was either stored at - 4 0 ° C or was extracted on a C-18 Sep-pak column (Waters Associates, Northwich, Cheshire, U.K.), and the extract stored below - 2 0 o C. All assays were performed within 6 months of sample collection.
molarity of h ANF (1-28) to half displace tracer × 100 molarity of analogue to half displace tracer Reduced h A N F (1-28) was prepared by incubation of 10 /~g/ml A N F in nitrogen saturated 50 m M Tris p H 8.0 with 2 m M dithiothreitol for 30 min, followed by the addition of iodoacetamide to 4 mM. After appropriate dilution, control reaction mixture (no ANF) did not interfere in the assay. Competition between antibodies for binding to A N F (1-28) was performed essentially as described previously (Dawes et al., 1984). Briefly, 0.2 ml of labelled A N F with or without excess fluidphase antibody was incubated overnight and 0.05 ml of sufficient distinct antibody immobilised on Sephacryl S-1000 (usually 1/16 of 1 mg I g G / m l settled gel) to allow 60-80% binding of tracer in the absence of fluid-phase antibody was added. After shaking for 2 h and settling, 0.05 ml of supernatant was removed and bound tracer determined after sedimentation through 10% sucrose. If fluid-phase and immobilised antibodies competed for the same site, the amount of bound tracer was reduced. Control studies, on the 0.05
Direct assay Acid heat treatment (Iinuma et al., 1987). Duplicate 0.5 ml samples of freshly thawed plasma were mixed with 0.5 ml 0.1 M acetic acid in screw top polyethylene 2 ml tubes, heated to 85 ° C for 10 min in a water bath and centrifuged at 10000 × g for 5 min. The supernatant was assayed directly, diluting where necessary in dilution buffer (0.1 M Na2HPOa-HC1, 10 m M EDTA, 3 m M N a N 3 0.25% BSA, 0.1% Triton X-100, p H 7.4). This buffer was also used to prepare dilutions of standard human A N F (1-28). All other reagents were prepared in assay buffer (as dilution buffer but containing 0.2 M phosphate). 0.1 ml of test or standard was incubated for 18-20 h at room temperature with 0.1 ml MAb ESA 4 (diluted to allow about 35% tracer binding in the absence of added ANF). 0.05 ml of labelled A N F (10 000 cpm) was then added and 3 h later 0.05 ml of a 1 / 1 6 suspension of SAM-Sephacryl S-1000. After a further hour of shaking, bound and free tracer were separated by sedimentation through 10% sucrose. Centricon 10 separation (Cernacek et al. 1988). 2 ml freshly thawed plasma was filtered through a Centricon 10 (Amicon) by centrifugation at 1600 × g and 4 ° C for 90 min. The resultant filtrate (0.2-0.3 ml) was assayed as described above. lndirect assay This was performed on extracted plasma samples essentially as described by Andersson et al.
95 TA B LE II
(1986). Briefly, antibody (0.1 ml of 1/11000 of rabbit antiserum or 1/22 000 of sodium sulphate concentrate of MAb ESA 4) and test antigen or A N F standard (0.1 ml) were incubated in a final volume of 0.8 ml for 24 h at 4 ° C in 0.1 M phosphate, 0.25% ovalbumin p H 7.4. Tracer (0.1 ml) was then added and after a further 20 h at 4 ° C, free and bound tracers were separated using dextran-coated charcoal (0.5 ml 1.6% Norit charcoal, 0.16% dextran M r 81000 in assay buffer).
C H A R A C T E R I S T I C S O F N I N E M O N O C L O N A L ANTIBODIES TO A N F
Results
Table I shows the results of immunising mice by various schedules and reveals that immunity was successfully achieved with BTG-ANP con-
Clone
Hybrid supernatant ELISA (OD)
Clone supernatant R I A (titre) a
Isotype
KD (pM)
ESA ESA ESA ESA ESA ESA ESA ESA ESA PAb
2.38 1.77 2.07 1.81 0.38 1.56 1.18 2.40 2.60 ( > 1.20) b
0.1 5 0.2 1 2 0.2 5 0.5 0.5 1
G2a G2a G2b G2a G1 G2a G1 G2a G2a -
270 5 25 40 45 108 21 208 111 - 31
a
2 4 5 6 7 8 9 11 12
X104.
b Pooled mouse antiserum diluted 1/500.
TABLE III CROSS-REACTIVITY OF SELECTED M O N O C L O N A L A N T I B O D I E S F O R A N F A N A L O G U E S Numbering of human (h) and rat (r) peptides is according to that for h A N F (1-28). Cross reactivity is defined relative to the analogue marked *. Stasch et al. (1987) have recently described a MAb, 23M-D9, of very similar specificity to l l A - A l l following immunisation with h A N F (1-28). Reference John, 1986
Milne, 1987
Naomi, 1987
Present study
Immunogen
r5-27
r3-28
hl-28
hl-28
Screening (1) Antigen (2) Assay
r5-28 RIA
r3-28 IRMA
hl-28/7-28 RIA
hl-28 ELISA + R IA
Code
llA.All
2H2
13A.1; 10.B1
ESA 4
ESA 5
ESA
Isotype
G1
G1
G1
G2a
G2b
G1
Cross-reactivity (%) hl-28 h l - 2 8 (reduced) rl-28 r3-28 r3-25 r3-23 r 4- 28 r5-28 (AT III) r5-27 (AT II) r5-25 (AT I) r 6- 28 r 7- 28 h7-28 r 8- 27 13-28 18-28 DDAVP
100 * 90 100 74 48 < 0.01 < 0.01 <1
10 100 100 * 10 80 30 0 0 0 0 0 -
100
*
100
*
100
*
100
9
*
82
59
14
89 55
115 101
1 1
54
99
45
69 <10
~< 0.01
< 0.7
< 0.03
< 0.003
< 0.002
< 0.05
< 0.005
< 0.01
96
jugates prepared using two different cross-linking procedures with subcutaneous or intraperitoneal administration. Prior to the successful fusion described below, fusions were performed with NS-0 cells and spleen cells from a slightly immune mouse (1.6; 45 hybrids), non-immune spleen cells stimulated with ANP in vitro (457 hybrids) and with node cells from an immune rabbit (621 hybrids from two fusions) but none yielded antibodies of interest. The fusion performed with mouse 2.1 spleen cells yielded 431 hybrids. ELISA screening showed 16 of these to be secreting antibody specific for ANF. This was confirmed by RIA and nine of these were successfully cloned. Table II shows that these were of IgG1, IgG2a and IgG2b isotypes and exhibited dissociation constants, ranging from 5 to 270 pM. Immuno-
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Fig. 3. Comparison of monoclonal immunoassays with RIA using polyclonal antiserum on extracted plasma samples. A: monoclonal RIA on extracted plasma samples• n =12, r = 0.9942. The regression line is shown (slope 1.027, intercept -4.05). B: monoclonal RIA on acid-treated plasma samples. n = 52, r = 0•7256. The regression line is indicated by - (slope 0•561, intercept 112) and the line of identity by - - - - - -
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ANF (pg/ml] Fig. 2. Standard curves for A N F assays• A: standard curve for assay format used on extracted plasma samples; (O O) MAb ESA 4 diluted 1/22000, ( z x - - A) polyclonal antisera diluted 1/11000. B: standard curves for assay format used on acid treated or filtered plasma samples using MAb ESA 4 diluted 1/50 000.
globulin was partially purified from each of these by sodium sulphate precipitation and used in competitive-binding assays. These studies showed that any of the nine MAb effectively inhibited binding of ANF to all the other MAbs. Furthermore, surprisingly, each MAb also inhibited binding of ANF to immobilised polyclonal rabbit immunoglobulin (not shown).
97 The three highest affinity MAb (ESA 4, 5 and 9) were chosen for further study. These were purified from concentrated culture supernatant by chromatography on protein A and exhibited single heavy and light chain bands after SDS-polyacrylamide gel electrophoresis (Laemmli, 1970). The binding of these three MAb to different analogues of A N F is shown, together with results on other described MAbs, in Table III. Besides human ANF (1-28), all bound human A N F (7-28), but not A N F (13-28) or DDAVP. In addition ESA 4 and 5 also bound rat A N F (5-27) and (5-25), and human ANF (1-28) after reduction of the intramolecular disulphide bridge, while ESA 9 had a lower affinity for the reduced hormone. Initial studies showed that binding of iodinated A N F to these MAb assessed by RIA, was minimally affected by changing pH in the range 6.8-8.0, but that increased ionic strength reduced binding to some extent, especially for ESA 9. These studies also showed no difference between results with phosphate or Tris buffer, with BSA or gelatin or with and without aprotinin addition. For ESA 4 inclusion of EDTA improved assay consistency marginally. Inclusion of whole plasma drastically interfered with A N F binding of all three MAb. Assays on ANF diluted in buffer indicated that only ESA 4 was of sufficient affinity to possibly allow direct assay of basal plasma levels of ANF, variously reported to be between 10 and 100 p g / m l . Representative standard curves are shown in Fig. 2 for the formats eventually chosen for direct RIA of plasma A N F (Fig. 2B) and RIA on C-18 Sep-pak extracts of plasma (Fig. 2A). In assessing the suitability of ESA 4 for assay of A N F in plasma we initially tested its ability to substitute for polyclonal antisera in assays on Sep-pak extracts of plasma (indirect assay). The results (Figs. 2A and 3A) showed that it was suitable for this purpose. Since this assay involves a costly and time-consuming extraction of each plasma sample, more direct routes for assay were also assessed. As neat plasma interfered with binding of ESA 4 to ANF, the approach of Iinuma et al. (1987), whereby samples are treated with an equal volume of 0.1 M acetic acid at 85°C, was tried. For these studies it was found necessary to use strong phosphate buffers (see materials and methods section) in the assay to neutralise the
TABLE IV RADIOIMMUNOASSAY OF PLASMA TO WHICH KNOWN AMOUNTS OF HUMAN ANF (1-28) WERE ADDED PRIOR TO ACID TREATMENT Results are mean+ SD from three experiments on a normal plasma containing 21 pg/ml ANF. This basal level was subtracted prior to making the calculations below. The regression line for the following data was: observed= 0.85 expected + 15.4 (r = 0.9995). Expected (pg/ml) 500 200 50 20
Observed (pg/ml) 439 + 58 189+ 24 64 + 16 24+ 4
% Recovery 88 95 128 120
acidic supernatant (pH 4.5). By delaying addition of the iodinated tracer, this format yielded an assay with a significant dose-response in the range of 30-600 p g / m l , after allowing for the 1 / 2 dilution resulting from the acid treatment step. Experiments, in which plasma containing basal levels of A N F was spiked with 20-500 p g / m l of synthetic ANF, showed recoveries of at least 85% (Table IV). This procedure was applied to a range of plasma samples from patients and from normal volunteers before and after ANF infusion. Comparison of the results of such assays with those obtained on the same samples after Sep-pak extraction (Fig. 3B) indicated the variable presence of interfering substance(s) in the supernatants from the acid treatment step. As an alternative approach to processing A N F from plasma prior to assay, we also assessed centrifugal filtration, as suggested by Cernacek et al. (1988). This yielded results ranging from undetectable to 36 p g / m l for 38 samples shown to contain between 20 and 674 p g / m l by the indirect assay.
Discussion In view of the fact that human and canine ANF (1-28) only differ from rodent A N F by the substitution of methionine for isoleucine at residue 12 it is perhaps not surprising that some difficulty was encountered in hyperimmunising mice with
98 human ANF (1-28). This was, however, achieved using two different forms of BTG conjugate. The fusion described here led to the isolation of nine MAbs to human A N F (1-28) of IgG1, IgG2a and IgG2b isotypes with differing affinities. The inability of any pair of these antibodies to bind A N F simultaneously shows that the epitopes to which this panel of MAb bind are all close to each other. In view of this it seems unlikely that this panel of MAbs could be used to assay A N F in a two-site enzyme-linked immunoadsorbent assay, although it might be useful in a competitive ELISA (McLaughlin et al., 1987), or a direct A N F sandwich ELISA, such as that described by Hashida et al. (1988). Evidence from this and other studies suggest that there are at least four distinct epitopes on A N F (see below) although steric hindrance may prevent simultaneous MAb binding to adjacent epitopes. In this study MAb ESA 4 and 5 were shown to bind human A N F (1-28), (7-28) and rat ANF (5-25) as well as the reduced parent (1-28) peptide, but not the (13-28) fragment. It thus seems likely that these two antibodies are directed, at least in part, to the region of the molecule between residues 7 and 12, although they are insensitive to the isoleucine/methionine difference at residue 12 between rodent and human hormones. This appears to be a similar specificity to the MAb described by John et al. (1986) (see Table III), which inhibits the natriuretic and hypotensive effects of ANF (5-27) following infusion in rats, as well as the diuretic effect of blood infusion in the same model (Hirth et al., 1986). The recently described MAb of Stasch et al. (1987) is also of similar specificity. MAb ESA 9, in contrast, recognises human (7-28) but not rat (5-28) or reduced human (1-28) and thus appears to bind a portion of the central loop of ANF which includes residue 12. Of the other MAbs described in the literature, one also inhibits the natriuretic effect of rat ANF, shows little binding to human ANF and binds an epitope involving residues 3, 4 and 12 (Milne et al., 1987), while that described by Naomi et al. (1987) binds to an epitope involving the first two residues of human and rat A N F (1-28) and inhibits binding of ANF to renal receptors. It has also been shown that polyclonal antisera may exhibit selectivity for
N- and C-terminal peptides, as well as species specificity (see Table II in Naomi et al., 1987; Iinuma et al., 1987). Thus studies with both polyclonal and monoclonal antibodies have demonstrated the existence of epitopes at the N and C termini of ANF, as well as at least two within the disulphide loop, one involving residue 12. Inhibition studies with MAbs have shown that binding of antibodies to three of these epitopes can inhibit A N F activities. Such studies have yet to be performed with the MAbs described in this study. Sudoh et al. (1988) have recently described a peptide in extracts of brain with homologous structure and activity to ANF. We have yet to test the ability of our MAb panel to bind this BNP peptide. Similarly, it would be of interest to examine binding of the MAbs to analogues of A N F resistant to inactivation resulting from cleavage between residues 12 and 13 (Condra et al., 1988). Direct plasma RIAs for A N F have been described but have often suffered from interference from plasma components or have been of insufficient sensitivity to permit assay of human plasma (Gutkowska et al., 1985; John et al., 1986; Juppner et al., 1986; Yandle et al., 1986; Richards et al., 1987; Cernacek et al., 1988). Most groups have therefore resorted to plasma extraction on C-18 Sep-pak columns prior to RIA (Gutkowska et al., 1986) or the faster receptor assay (Sagnella et al., 1987). Extraction not only removes interfering substances but also concentrates samples. The MAb ESA 4 can substitute for polyclonal antisera in RIA on such extracts. Iinuma et al. (1987) and Cernacek et al. (1988) have reported using acid extraction or filtration, in place of the established Sep-pak extraction of plasma samples, prior to immunoassay of ANF. Using the ESA 4 monoclonal antibody we were unable to develop radioimmunoassays for plasma A N F using either of these approaches. Processing by the former method yielded samples containing variable levels of interfering material, as has previously been reported for unprocessed samples (see above). Buckley et al. (1987) have also reported that acid treatment may increase A N F immunoreactiviy in extracts of human plasma. In our hands the filtration approach to separating A N F from plasma resulted in very low recoveries. In summary, this study has resulted in the
99
production of a panel of monoclonal antibodies to ANF. The most avid of these provides an alternative to polyclonal antisera in the established immunoassays for plasma ANF which should be easier to produce consistently and may have cost benefits.
Acknowledgements We would like to thank the University of Edinburgh Quantum Fund for financial support; D. Sampson, S. Smith, G. Willis, J. Gardner, B. Garden and D. Pepper for help and advice; J. Lim for manuscript preparation; and Dr. D.B.L. McClelland, Professor M. Lee and Professor G.D. Chisholm for support.
Note added in proof Mukoyama et al. (1988) also describe a MAb to the N terminal of hANF (Biochem. Biophys. Res. Commun. 151, 1277-1284).
References Andersson, J.V., Christofides, N.D., Vinas, P., Wharton, J., Varndell, I.M., Polak, J.M. and Bloom, S.R. (1986) Radioimmunoassay of alpha rat atrial natriuretic peptide. Neuropeptides 7, 159-173. Baxter, J.D., Lewicki, J.A. and Gardner, D.G. (1988) Atrial natriuretic peptide. Biotechnology 6, 529-546. Buckley, M.G., Sagnella, G.A. and MacGregor, G.A. (1987) Plasma acidification increases atrial natriuretic peptide as measured by radioimmunoassay. Clin. Chem. 33, 1104-1105. Cernacek, P., Crawhall, J.C. and Levy, M. (1988) Atrial natriuretic peptide: Blood levels in human disease and their measurement. Clin. Biochem. 21, 5-17. Clarke, M., Wright, B.W. and Milstein, C. (1981) Preparation of monoclonal antibodies: strategies and procedures. In: J.J. Langone and H. Van Vunakis (Eds.), Methods in Enzymology, Vol. 73. Academic Press, New York, pp. 3-47. Condra, C.L., Leidy, E.A., Bunting, P., Colton, C.D., Nutt, R.F., Rosenblatt, M. and Jacobs, J.W. (1988) Clearance and early hydrolysis of atrial natriuretic factor in vivo: structural analysis of cleavage sites and design of an analogue that inhibits hormone cleavage. J. Clin. Invest. 81, 1348-1354. Dawes, J., James, K., Micklem, L.R., McClelland, D.B.L.,
Pepper, D.S. and Prowse, C.V. (1984) Monoclonal antibodies directed against human thrombin and the thrombin-antithrombin complex. Thromb. Res. 36, 397-409. De Bold, A.J. (1986) Atrial natriuretic factor; an overview. Fed. Proc. 45, 2081-2085. Donckier, J., Anderson J.V., Yeo, T. and Bloom, S.R. (1986) Diurnal rhythm in the plasma concentration of atrial natriuretic peptide. New Engl. J. Med. 315, 710-711. Dzau, V.J. et al. (1987) Nomenclature of atrial peptides. Lancet ii, 1278-1279. Flynn, T.G. and Davies, P.L. (1985) The biochemistry and molecular biology of atrial natriuretic factor. Biochem. J. 232, 313-321. Gurvich, A.E. and Korukova, A. (1986) Introduction of abundant antibody formation with a protein cellulose complex in mice. J. Immunol. Methods 87, 161-167. Gutkowska, J., Bonan, R., Roy, D., Thibault, G., Garcia, R., Cantin, M. and Genest, J. (1985) Immunoreactive atrial natriuretic factor in human plasma. Biochem. Biophys. Res. Commun. 128, 1350-1357. Gutkowska, J., Bonan, R., Roy, D., Bourassa, M., Garcia, R., Thibault, G., Genest, J. and Cantin, M. (1986a) Atrial natriuretic factor in human plasma. Biochem. Biophys. Res. Commun. 139, 287-295. Gutkowska, J., Horky, K., Shiffrin, E.L., Thibault, G., Garcia, R., De Leon, A., Hamet, P., Tremblay, J., Anand-Srivastava, M.B., Januszewicz, P., Genest, A. and Cantin, M. (1986b) Atrial natriuretic factor: Radioimmunoassay and effects on adrenal and pituitary glands. Fed. Proc. 45, 2101-2105. Hartter, E., Woloszczuk, W. and Stummvol, L.H. (1986) Radioimmunoassay of atrial natriuretic peptides in human plasma. Clin. Chem. 32, 441-445. Hashida, S., Ishikawa, E., Nakao, K., Mukoyama, M. and Imura, H. (1988) Enzyme immunoassay for alpha-human atrial natriuretic polypeptide: direct measurement of plasma levels. Clin. Chim. Acta 175, 11-18. Hirth, C., Stasch, J.-P., John, A., Kazda, S., Morich, F., Neuset, D. and Wohlfeil, S. (1986) The renal response to acute hypervolemia is caused by atrial natriuretic peptides. J. Cardiovasc. Pharmacol. 2, 268-275. Hornsey, V., Micklem, L.R., McCann, M.C., James, K., Dawes, J., McClelland, D.B.L. and Prowse, C.V. (1985) Enhancement of Factor VIII-von Willebrand factor ristocetin cofactor activity by monoclonal antibodies. Thromb. Haemostasis 54, 510-514. Iinuma, K., Ikeda, I., Ogihara, T., Hara, H., Shima, J., Kurata, K. and Yumahara, Y. (1987) Radioimmunoassay of atrial natriuretic polypeptide in heat treated human plasma. Clin. Chem. 33, 674-676. Itoh, H., Nakao, K., Shiono, S., Mukoyama, M., Morii, N., Sugawara, A., Yamada, T., Saito, Y., Arai, H., Kambayashi, Y., Inouye, K. and Imura, H. (1987) Conversion of fl-human atrial polypeptide into a-human atrial natriuretic polypeptide in human plasma in vitro. Biochem. Biophys. Res. Commun. 143, 560-569. John, A., Stasch, J.-P., Neuser, D., Hirth, C. and Morich, F.J. (1986) The use of a monoclonal antibody to measure plasma atriopeptins in rat. Life Sci. 38, 1991-1997.
100 Juppner, H., Brabant, G., Kapteina, U., Kirschner, M., Klein, H. and Hesch, R.D. (1986) Direct radioimmunoassay for human atrial natriuretic peptide (h ANP) and its clinical evaluation. Biochem. Biophys. Res. Commun. 39, 1215-1223. Kohn, J. and Wilchek, M. (1982) A new approach (cyanotransfer) for cyanogen bromide activation of Sepharose at neutral pH, which yields activated resins, free of interfering nitrogen derivatives. Biochem. Biophys. Res. Commun. 107, 878-884. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. McLaughlin, L.L., Wei, Y., Stockman, P.T., Leahy, K.M. and Needleman, P. (1987) Development, validation and application of an enzyme immunoassay (EIA) of atriopeptin. Biochem. Biophys. Res. Commun. 144, 469-476. Marumo, F., Sakamoto, H., Ando, K., Ishigami, T. and Kawakami, M. (1986) A highly sensitive radioimmunoassay of atrial natriuretic peptide in human plasma and urine. Biochem. Biophys. Res. Commun. 137, 231-236. Micklem, L.R., McCann, M.C. and James, K. (1987) The use of rat mixed-thymocyte culture-conditioned medium for hybridoma production, cloning and revival. J. Immunol. Methods 104, 81-86. Milne, R., Gutkowska, J., Thibault, G., Schiller, P., Charbonneau, C., Genest, J. and Cantin, M. (1987) A murine monoclonal antibody against rat atrial natriuretic factor (ANF) which cross-reacts with mouse ANF. Mol. Immunol. 24, 127-132. Naomi, S., Umeda, T., Sato, T., Harada, N., Tominaga, A. and Takatsu, K. (1987) Production and characterisation of monoclonal antibodies against amino-terminus of human a-atrial natriuretic polypeptide. Hybridoma 6, 433-440. Richards, A.M., Tonolo, G., Mclntyre, G.D., Leckie, B.J. and
Robertson, J.l. (1987) Radio-immunoassay for plasma alpha human atrial natriuretic peptide: a comparison of direct and pre-extracted methods. J. Hypertension 5, 227-236. Rosmalen, F.M.A., Tan, A.C.I.T.L., Tan, H.S. and Benraad, T.J. (1987) A sensitive radioimmunoassay of atrial natriuretic peptide in human plasma using a tracer with an immobilised glycouril agent. Clin. Chim. Acta 165, 331-340. Sagnella, G.A., Buckley, M.G., Markandu, N.D. and MacGregor, G.A. (1987) Atrial natriuretic peptide in human plasma: Comparison of radioreceptor versus radioimmunoassay. Clin. Chim. Acta 166, 37-44. Stasch, J.P., Neuser, D., John, A., Hein, D., Heuck, K. and Morich, F.-J. (1987) The use of a monoclonal antibody for the determination of atrial natriuretic peptides in human plasma. Klin. Wochenschr. 65, 533-537. Sudoh, T., Kangawa, K., Minamino, N. and Matsuo, H. (1988) A new natriuretic peptide in porcine brain. Nature 332, 78-81. Tikkanen, I., Fyhrquist, F., Metsarinne, K. and Leidenius, R. (1985) Plasma atrial natriuretic peptide in cardiac disease and during infusion in healthy volunteers. Lancet ii, 65-69. Wilson, N., Ledsome, J.R., Keeler, R., Rankin, A.J., Wade, J.P. and Courneya, C.A. (1986) Heterologous radioimmunoassay of atrial natriuretic polypeptide in dog and rabbit plasma. J. Immunoassay 7, 73-96. Wright, J.F. and Hunter, W.M. (1982) A convenient replacement for cyanogen bromide - Activated solid phases in immunoradiometric assays. J. Immunol. Methods 48, 311-325. Yandle, T.G., Espiner, E.A., Nicholls, M.G. and Duff, H. (1986) Radioimmunoassay and characterization of atrial natriuretic peptide in human plasma. J. Clin. Endocrinol. Metab. 63, 72-79.
91
JIM05094
H u m a n atrial natriuretic factor ( A N F ) Characterisation of a monoclonal antibody panel and its use in radioimmunoassay C. Prowse 3, E. George 1, L.R. M i c k l e m 1, V. H o r n s e y 3, j. Brown 2 a n d K. James Departments of 1 Surgery and 2 Clinical Pharmacology, Edinburgh University, Edinburgh, U.K., and "~Edinburgh and South-East Scotland Blood Transfusion Service, Royal Infirmary, Edinburgh, U.K.
(Received 8 July 1988, revised received 28 October 1988, accepted 31 October 1988)
The production of nine monoclonal antibodies to human atrial natriuretic factor (ANF 1 - 2 8 ) i s described. All possible combinations of two antibodies failed to reveal any which could simultaneously bind ANF. Studies with ANF analogues and the antibodies having the three highest affinity values (K D = 5, 25 and 21 pM) indicated that the antibodies are directed to the central portion of the antigen molecule. The highest affinity antibody was able to replace polyclonal antisera in the radioimmunoassay of ANF in extracts of plasma. Key words: Atrial natriuretic factor; Monoclonal antibody; Radioimmunoassay
Introduction
Since its discovery in 1981, there has been great interest in atrial natriuretic factor, ANF (Flynn and Davies, 1985; De Bold, 1986; Gutkowska et al., 1986b; Baxter et al., 1988), but the actions of this peptide hormone in regulating water balance, sodium levels and blood pressure are still incompletely understood. Such understanding as exists has been aided by the use of synthetic ANF peptides and by the development of assays for the
Correspondence to: C. Prowse, Blood Transfusion Centre, Royal Infirmary, Edinburgh, EH3 9HB, U.K. Abbreviations: ANF, atrial natriuretic factor; RIA, radioimmunoassay; BSA, bovine serum albumin; MAb, monoclonal antibody; KLH, keyhole limpet haemocyanin; BTG, bovine thyroglobulin; SAM, sheep anti-mouse antiserum; DAR, donkey anti-rabbit antiserum; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; BUH, Buccinum undatum haemocyanin; KIU, kallikrein inhibitor units.
hormone. The latter have largely been classical radioimmunoassays using rabbit antisera (Andersson et al., 1986; Hartter et al., 1986; Marumo et al., 1986; Tikkanen et al., 1985; Wilson et al., 1986; Gutkowska et al., 1985, 1986a; Juppner et al., 1986; Rosmalen et al., 1987; Cernacek et al., 1988) and usually require the extraction and concentration of plasma samples. Alternative receptor binding assays for A N F have been described which are of narrower specificity than most radioimmunoassays but are more rapid (Sagnella et al., 1987). Recently three murine MAbs to human ANF (John et al., 1986; Naomi et al., 1987; Stasch et al., 1987) and one to rat ANF (Milne et al., 1987) have been described. Initial results with these have suggested that they might be useful in increasing our understanding of the functions of ANF and in investigating its histological location. In this study, the production of a panel of MAbs to ANF and their potential use in an immunoassay for human A N F in plasma is described.
0022-1759/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
92 14
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mine cyanogen bromide method of Kohn and Wilchek (1982). The subclass of the monoclonal antibodies was determined by immunodiffusion using antisera from Serotec (obtained through Uniscience, Cambridge, England) or the University of Birmingham Research Institute (University of Birmingham, England). All other reagents were of at least Analar Grade.
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Fig. 1. Structure of ANF. In rat, mouse and rabbit ANF isoleucine 12 replacesmethionine12 of the human (and canine) peptide. Despite the recent recommendations of Dzau et al. (1987) on ANF nomenclature, the alpha human ANF has, for convenience, been described in this paper as ANF (1-28) and analogues of the peptide described using numbering which permits direct comparison of homologous residues (Fig. 1).
Materials
and methods
Synthetic human ANF (1-28, batch 380A or 220C) and rat atriopeptin II (5-27, batch 188B) were obtained from Bachem (Saffron Waldon, England). Rat atriopeptin I (5-25, batch 122), human ANF (7-28, batch 098) and ANF fragment 13-28 (batch 129) were from Cambridge Research Biochemicals (Cambridge, England). 1desamino-8-D-arginine vasopressin (DDAVP) was purchased from Ferring (Feltham, Middlesex, U.K.). [3-125I]iodotyrosyl 28 human A N F (1-28) (IM-187, 2000 C i / m m o l ) was from Amersham I n t e r n a t i o n a l ( A m e r s h a m , England). 25% glutaraldehyde, KLH, BTG, BSA, anti-mouse IgG-horseradish peroxidase conjugate (A2028) and EDC were purchased from Sigma. BUH was purified and supplied by Dr. D. Pepper. SAM and DAR antisera were obtained from the Scottish Antibody Production Unit (Law Hospital, Carluke, Lanarkshire). Immunoglobulins and antisera were immobilised on Sephacryl S-1000 (Pharmacia) by the low temperature triethanola-
Human A N F (1-28) was coupled to carrier proteins in 50 mM phosphate 0.1 M NaC1 pH 7.2 using two basic procedures. For EDC coupling, 8-10 mg of protein in 4 ml were mixed with 6 mg EDC and added dropwise to 1 mg A N F in 1 ml 0.15 M NaC1 with stirring for 15 min. Following overnight incubation at 4 ° C in the dark a further 6 mg EDC were added with stirring and, after 2 h at 20°C, the reaction mixture was extensively dialysed against 0.15 M NaC1 and frozen in aliquots. For glutaraldehyde cross-linking 8-10 mg of carrier protein in 4 ml phosphate buffer were mixed with 1 mg A N F in 1 ml 0.15 M NaC1 and 25% glutaraldehyde was added to a final concentration of 0.2% w / v . After 3 h incubation at 2 0 ° C in the dark, the mixture was dialysed, aliquoted and frozen as above. Alternatively, the reaction was performed overnight at 4 ° C with a final concentration of 0.05% w / v glutaraldehyde.
Immunisation and fusion The more relevant immunisation procedures are shown in Table I. Immunisations performed in mice with free ANP (s.c.) BUH-glutaraldehyde, B S A - g l u t a r a l d e h y d e and cellulose-periodate (Gurvich and Korukova, 1986) conjugates were unsuccessful. Two rabbits were also immunised with B T G - E D C conjugate, being given 6 weekly boosts of 1 mg conjugate as two s.c. injections after priming with 4 mg in complete Freund's adjuvant at multiple intramuscular sites. Antiserum from one of these (titre 1/10000) was used as a positive control in the screening assays. The MAbs described below were obtained from one fusion using spleen cells from mouse 2.1 which was boosted i.p. with 50 ~tg of BTG-EDC conjugate in saline 49 days after priming. The fusion
93 TABLE I IMMUNISATION OF MICE Groups of five or six mice were immunised with 50 /tg of various ANF-protein conjugates (i.e., about 5 /Lg ANF) with an equal volume of Freund's complete adjuvant by the subcutaneous (s.c.) or intraperitoneal (i.p.) routes. Unless otherwise indicated boosts were given with alum and test bleeds taken 7 days after the last boost. Titres were determined as the average dilution, in responding mice, to give 50% binding in the RIA. The best responses (titre 1/10000) were seen in mice 3.5 and 4.3. The BTG-EDC conjugates for groups 2/3 and 4 were separate preparations. Group
Carrier
Conjugate
Route
Responders
Titre
i.p. s.c.
Boosting (days) 42 (BUH-glut) 9, 20, 28 (no alum)
1 2
BSA BTG
Glutaraldehyde EDC
1/6 6/6
i.p. i.p. i.p. i.p.
42 42 42 42
5/5 4/6 3/5 0/5
1/10 1/500 (at 42 days) 1/1000 1/500 1/200 < 1/5
3 4 5 6
BTG BTG BTG KLH
EDC EDC Glutaraldehyde EDC
was performed 3 days later using 50% polyethylene glycol 4000 (Fisons, Loughborough, England) at p H 8.5-9, NS-0 myeloma cells (Clarke et al., 1980) at a ratio of 1 : 1 and selection medium containing hypoxanthine-aminopterin-thymidine as described previously (Dawes et al., 1984) but using thymocyte-conditioned medium in place of feeder cells (Micklem et al., 1987). Hybrids producing specific MAb were cloned by limiting dilution and grown in culture and as ascites. Immunoglobulin was purified from ten-fold concentrated culture supernatant by sodium sulphate precipitation (Wright and Hunter 1982) or by chromatography on protein A-Sephacryl S-1000.
0.1 ml substrate was added and colour development stopped after 15 min with 0.05 ml 2.5 M H2SO 4. Optical densities were then read at 450 nm on a Twinreader (Flow Laboratories, Ayr, Scotland). Substrate was prepared just before use by mixing 20 ml 0.1 M acetate-citrate p H 6, 0.2 ml 10 m g / m l tetramethyl benzidine (Aldrich) in dimethyl sulphoxide and 0.3 ml of 1% w / v urea hydrogen peroxide. Samples were tested in duplicate, using a 1/1000 dilution of pooled immune mouse serum as control (O1)450 n m about 1.0). Assays on culture medium, buffer, non-immune mouse serum or in the absence of A N F coating always gave an absorption below 0.1, usually below 0.04.
Screening assays Radioimmunoassay Enzyme-linked immunosorption assay (ELISA) The ELISA was performed at room temperature throughout using 96 well microtitre trays (Nunc, Immulon II) coated overnight with 0.1 ml/well of 100 n g / m l human A N F (1-28) in PBS (0.1 M phosphate, 0.15 M NaC1, p H 7.5). After three washes with PBS, wells were blocked with 0.4 ml PBS, 1% BSA, 0.1% lactose for 1 h prior to washing and the addition of 0.1 ml test antibody, diluted as necessary in TB (0.25 M Tris, 0.1% Tween 20, p H 8). After a further hour and wash, 0.1 ml of a 1/200 dilution of Sigma anti-mouse I g G peroxidase in PBS, 1% BSA, 0.1% Tween 20 was added for 1 h. After three washes with PBS,
0.05 ml test antibody, 0.05 ml dilute radiolabelled human A N F (1-28) and 0.1 ml assay buffer (50 m M Tris, 0.1 M NaC1, 10 m M EDTA, 3 m M N a N 3, 0.1% Triton X-100, 0.25% BSA, p H 7.35) were incubated overnight at room temperature. Bound and free tracer were then separated by the addition of 0.05 ml SAM or D A R (as appropriate) immobilised on Sephacryl S-1000, shaking for 1 h and sedimenting through buffer containing 10 % sucrose (Hornsey et al., 1985). Assays were performed in duplicate using immune rabbit or mouse serum diluted to give about 50% binding as a positive control. Non-specific binding was 3-4% and maximal binding in excess of 95%.
94 For some assays BSA was replaced with gelatin. Antibody titre was defined as the dilution necessary to allow 50% maximum binding and varied from 1/107 to 1/108 for ascites from clones 4, 5 and 9.
ml removed, using sheep anti-mouse immunoglobulin gel, showed that more than 90% of tracer was bound by each of the fluid-phase antibodies after overnight incubation with the chosen antibody concentration.
Antibody characterisation
Plasma assays
Antibody isotype was determined by immunodiffusion of ten-fold concentrated culture supernatant against subclass-specific antisera and confirmed, in some cases, by the p H required to elute MAb from protein A. Antibody affinity was determined, as the dissociation constant, by Scatchard analysis of data obtained by a modification of the above RIA. MAbs were diluted to allow 50-60% binding of tracer alone and the percent binding of tracer determined after substitution of various amounts of unlabelled human A N F (1-28) diluted in assay buffer for the 0.1 ml of buffer. The ability of MAb to bind various A N F analogues was determined in a similar assay using carefully prepared stock solutions (1 m g / m l in 0.1 M acetic acid), further diluted in assay buffer. The percentage reactivity was defined as
10 ml blood samples were collected by clean venepuncture, mixed with 0.1 ml of a solution containing 15 mg E D T A and 1000 K I U aprotinin (Trasylol, Bayer) and immediately cooled on melting ice. Plasma obtained by centrifugation was either stored at - 4 0 ° C or was extracted on a C-18 Sep-pak column (Waters Associates, Northwich, Cheshire, U.K.), and the extract stored below - 2 0 o C. All assays were performed within 6 months of sample collection.
molarity of h ANF (1-28) to half displace tracer × 100 molarity of analogue to half displace tracer Reduced h A N F (1-28) was prepared by incubation of 10 /~g/ml A N F in nitrogen saturated 50 m M Tris p H 8.0 with 2 m M dithiothreitol for 30 min, followed by the addition of iodoacetamide to 4 mM. After appropriate dilution, control reaction mixture (no ANF) did not interfere in the assay. Competition between antibodies for binding to A N F (1-28) was performed essentially as described previously (Dawes et al., 1984). Briefly, 0.2 ml of labelled A N F with or without excess fluidphase antibody was incubated overnight and 0.05 ml of sufficient distinct antibody immobilised on Sephacryl S-1000 (usually 1/16 of 1 mg I g G / m l settled gel) to allow 60-80% binding of tracer in the absence of fluid-phase antibody was added. After shaking for 2 h and settling, 0.05 ml of supernatant was removed and bound tracer determined after sedimentation through 10% sucrose. If fluid-phase and immobilised antibodies competed for the same site, the amount of bound tracer was reduced. Control studies, on the 0.05
Direct assay Acid heat treatment (Iinuma et al., 1987). Duplicate 0.5 ml samples of freshly thawed plasma were mixed with 0.5 ml 0.1 M acetic acid in screw top polyethylene 2 ml tubes, heated to 85 ° C for 10 min in a water bath and centrifuged at 10000 × g for 5 min. The supernatant was assayed directly, diluting where necessary in dilution buffer (0.1 M Na2HPOa-HC1, 10 m M EDTA, 3 m M N a N 3 0.25% BSA, 0.1% Triton X-100, p H 7.4). This buffer was also used to prepare dilutions of standard human A N F (1-28). All other reagents were prepared in assay buffer (as dilution buffer but containing 0.2 M phosphate). 0.1 ml of test or standard was incubated for 18-20 h at room temperature with 0.1 ml MAb ESA 4 (diluted to allow about 35% tracer binding in the absence of added ANF). 0.05 ml of labelled A N F (10 000 cpm) was then added and 3 h later 0.05 ml of a 1 / 1 6 suspension of SAM-Sephacryl S-1000. After a further hour of shaking, bound and free tracer were separated by sedimentation through 10% sucrose. Centricon 10 separation (Cernacek et al. 1988). 2 ml freshly thawed plasma was filtered through a Centricon 10 (Amicon) by centrifugation at 1600 × g and 4 ° C for 90 min. The resultant filtrate (0.2-0.3 ml) was assayed as described above. lndirect assay This was performed on extracted plasma samples essentially as described by Andersson et al.
95 TA B LE II
(1986). Briefly, antibody (0.1 ml of 1/11000 of rabbit antiserum or 1/22 000 of sodium sulphate concentrate of MAb ESA 4) and test antigen or A N F standard (0.1 ml) were incubated in a final volume of 0.8 ml for 24 h at 4 ° C in 0.1 M phosphate, 0.25% ovalbumin p H 7.4. Tracer (0.1 ml) was then added and after a further 20 h at 4 ° C, free and bound tracers were separated using dextran-coated charcoal (0.5 ml 1.6% Norit charcoal, 0.16% dextran M r 81000 in assay buffer).
C H A R A C T E R I S T I C S O F N I N E M O N O C L O N A L ANTIBODIES TO A N F
Results
Table I shows the results of immunising mice by various schedules and reveals that immunity was successfully achieved with BTG-ANP con-
Clone
Hybrid supernatant ELISA (OD)
Clone supernatant R I A (titre) a
Isotype
KD (pM)
ESA ESA ESA ESA ESA ESA ESA ESA ESA PAb
2.38 1.77 2.07 1.81 0.38 1.56 1.18 2.40 2.60 ( > 1.20) b
0.1 5 0.2 1 2 0.2 5 0.5 0.5 1
G2a G2a G2b G2a G1 G2a G1 G2a G2a -
270 5 25 40 45 108 21 208 111 - 31
a
2 4 5 6 7 8 9 11 12
X104.
b Pooled mouse antiserum diluted 1/500.
TABLE III CROSS-REACTIVITY OF SELECTED M O N O C L O N A L A N T I B O D I E S F O R A N F A N A L O G U E S Numbering of human (h) and rat (r) peptides is according to that for h A N F (1-28). Cross reactivity is defined relative to the analogue marked *. Stasch et al. (1987) have recently described a MAb, 23M-D9, of very similar specificity to l l A - A l l following immunisation with h A N F (1-28). Reference John, 1986
Milne, 1987
Naomi, 1987
Present study
Immunogen
r5-27
r3-28
hl-28
hl-28
Screening (1) Antigen (2) Assay
r5-28 RIA
r3-28 IRMA
hl-28/7-28 RIA
hl-28 ELISA + R IA
Code
llA.All
2H2
13A.1; 10.B1
ESA 4
ESA 5
ESA
Isotype
G1
G1
G1
G2a
G2b
G1
Cross-reactivity (%) hl-28 h l - 2 8 (reduced) rl-28 r3-28 r3-25 r3-23 r 4- 28 r5-28 (AT III) r5-27 (AT II) r5-25 (AT I) r 6- 28 r 7- 28 h7-28 r 8- 27 13-28 18-28 DDAVP
100 * 90 100 74 48 < 0.01 < 0.01 <1
10 100 100 * 10 80 30 0 0 0 0 0 -
100
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100
*
100
*
100
9
*
82
59
14
89 55
115 101
1 1
54
99
45
69 <10
~< 0.01
< 0.7
< 0.03
< 0.003
< 0.002
< 0.05
< 0.005
< 0.01
96
jugates prepared using two different cross-linking procedures with subcutaneous or intraperitoneal administration. Prior to the successful fusion described below, fusions were performed with NS-0 cells and spleen cells from a slightly immune mouse (1.6; 45 hybrids), non-immune spleen cells stimulated with ANP in vitro (457 hybrids) and with node cells from an immune rabbit (621 hybrids from two fusions) but none yielded antibodies of interest. The fusion performed with mouse 2.1 spleen cells yielded 431 hybrids. ELISA screening showed 16 of these to be secreting antibody specific for ANF. This was confirmed by RIA and nine of these were successfully cloned. Table II shows that these were of IgG1, IgG2a and IgG2b isotypes and exhibited dissociation constants, ranging from 5 to 270 pM. Immuno-
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pg/ml [POLVELOHAL RIA ON [XTRACTEO PLASNA]
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Fig. 3. Comparison of monoclonal immunoassays with RIA using polyclonal antiserum on extracted plasma samples. A: monoclonal RIA on extracted plasma samples• n =12, r = 0.9942. The regression line is shown (slope 1.027, intercept -4.05). B: monoclonal RIA on acid-treated plasma samples. n = 52, r = 0•7256. The regression line is indicated by - (slope 0•561, intercept 112) and the line of identity by - - - - - -
25
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ANF (pg/ml] Fig. 2. Standard curves for A N F assays• A: standard curve for assay format used on extracted plasma samples; (O O) MAb ESA 4 diluted 1/22000, ( z x - - A) polyclonal antisera diluted 1/11000. B: standard curves for assay format used on acid treated or filtered plasma samples using MAb ESA 4 diluted 1/50 000.
globulin was partially purified from each of these by sodium sulphate precipitation and used in competitive-binding assays. These studies showed that any of the nine MAb effectively inhibited binding of ANF to all the other MAbs. Furthermore, surprisingly, each MAb also inhibited binding of ANF to immobilised polyclonal rabbit immunoglobulin (not shown).
97 The three highest affinity MAb (ESA 4, 5 and 9) were chosen for further study. These were purified from concentrated culture supernatant by chromatography on protein A and exhibited single heavy and light chain bands after SDS-polyacrylamide gel electrophoresis (Laemmli, 1970). The binding of these three MAb to different analogues of A N F is shown, together with results on other described MAbs, in Table III. Besides human ANF (1-28), all bound human A N F (7-28), but not A N F (13-28) or DDAVP. In addition ESA 4 and 5 also bound rat A N F (5-27) and (5-25), and human ANF (1-28) after reduction of the intramolecular disulphide bridge, while ESA 9 had a lower affinity for the reduced hormone. Initial studies showed that binding of iodinated A N F to these MAb assessed by RIA, was minimally affected by changing pH in the range 6.8-8.0, but that increased ionic strength reduced binding to some extent, especially for ESA 9. These studies also showed no difference between results with phosphate or Tris buffer, with BSA or gelatin or with and without aprotinin addition. For ESA 4 inclusion of EDTA improved assay consistency marginally. Inclusion of whole plasma drastically interfered with A N F binding of all three MAb. Assays on ANF diluted in buffer indicated that only ESA 4 was of sufficient affinity to possibly allow direct assay of basal plasma levels of ANF, variously reported to be between 10 and 100 p g / m l . Representative standard curves are shown in Fig. 2 for the formats eventually chosen for direct RIA of plasma A N F (Fig. 2B) and RIA on C-18 Sep-pak extracts of plasma (Fig. 2A). In assessing the suitability of ESA 4 for assay of A N F in plasma we initially tested its ability to substitute for polyclonal antisera in assays on Sep-pak extracts of plasma (indirect assay). The results (Figs. 2A and 3A) showed that it was suitable for this purpose. Since this assay involves a costly and time-consuming extraction of each plasma sample, more direct routes for assay were also assessed. As neat plasma interfered with binding of ESA 4 to ANF, the approach of Iinuma et al. (1987), whereby samples are treated with an equal volume of 0.1 M acetic acid at 85°C, was tried. For these studies it was found necessary to use strong phosphate buffers (see materials and methods section) in the assay to neutralise the
TABLE IV RADIOIMMUNOASSAY OF PLASMA TO WHICH KNOWN AMOUNTS OF HUMAN ANF (1-28) WERE ADDED PRIOR TO ACID TREATMENT Results are mean+ SD from three experiments on a normal plasma containing 21 pg/ml ANF. This basal level was subtracted prior to making the calculations below. The regression line for the following data was: observed= 0.85 expected + 15.4 (r = 0.9995). Expected (pg/ml) 500 200 50 20
Observed (pg/ml) 439 + 58 189+ 24 64 + 16 24+ 4
% Recovery 88 95 128 120
acidic supernatant (pH 4.5). By delaying addition of the iodinated tracer, this format yielded an assay with a significant dose-response in the range of 30-600 p g / m l , after allowing for the 1 / 2 dilution resulting from the acid treatment step. Experiments, in which plasma containing basal levels of A N F was spiked with 20-500 p g / m l of synthetic ANF, showed recoveries of at least 85% (Table IV). This procedure was applied to a range of plasma samples from patients and from normal volunteers before and after ANF infusion. Comparison of the results of such assays with those obtained on the same samples after Sep-pak extraction (Fig. 3B) indicated the variable presence of interfering substance(s) in the supernatants from the acid treatment step. As an alternative approach to processing A N F from plasma prior to assay, we also assessed centrifugal filtration, as suggested by Cernacek et al. (1988). This yielded results ranging from undetectable to 36 p g / m l for 38 samples shown to contain between 20 and 674 p g / m l by the indirect assay.
Discussion In view of the fact that human and canine ANF (1-28) only differ from rodent A N F by the substitution of methionine for isoleucine at residue 12 it is perhaps not surprising that some difficulty was encountered in hyperimmunising mice with
98 human ANF (1-28). This was, however, achieved using two different forms of BTG conjugate. The fusion described here led to the isolation of nine MAbs to human A N F (1-28) of IgG1, IgG2a and IgG2b isotypes with differing affinities. The inability of any pair of these antibodies to bind A N F simultaneously shows that the epitopes to which this panel of MAb bind are all close to each other. In view of this it seems unlikely that this panel of MAbs could be used to assay A N F in a two-site enzyme-linked immunoadsorbent assay, although it might be useful in a competitive ELISA (McLaughlin et al., 1987), or a direct A N F sandwich ELISA, such as that described by Hashida et al. (1988). Evidence from this and other studies suggest that there are at least four distinct epitopes on A N F (see below) although steric hindrance may prevent simultaneous MAb binding to adjacent epitopes. In this study MAb ESA 4 and 5 were shown to bind human A N F (1-28), (7-28) and rat ANF (5-25) as well as the reduced parent (1-28) peptide, but not the (13-28) fragment. It thus seems likely that these two antibodies are directed, at least in part, to the region of the molecule between residues 7 and 12, although they are insensitive to the isoleucine/methionine difference at residue 12 between rodent and human hormones. This appears to be a similar specificity to the MAb described by John et al. (1986) (see Table III), which inhibits the natriuretic and hypotensive effects of ANF (5-27) following infusion in rats, as well as the diuretic effect of blood infusion in the same model (Hirth et al., 1986). The recently described MAb of Stasch et al. (1987) is also of similar specificity. MAb ESA 9, in contrast, recognises human (7-28) but not rat (5-28) or reduced human (1-28) and thus appears to bind a portion of the central loop of ANF which includes residue 12. Of the other MAbs described in the literature, one also inhibits the natriuretic effect of rat ANF, shows little binding to human ANF and binds an epitope involving residues 3, 4 and 12 (Milne et al., 1987), while that described by Naomi et al. (1987) binds to an epitope involving the first two residues of human and rat A N F (1-28) and inhibits binding of ANF to renal receptors. It has also been shown that polyclonal antisera may exhibit selectivity for
N- and C-terminal peptides, as well as species specificity (see Table II in Naomi et al., 1987; Iinuma et al., 1987). Thus studies with both polyclonal and monoclonal antibodies have demonstrated the existence of epitopes at the N and C termini of ANF, as well as at least two within the disulphide loop, one involving residue 12. Inhibition studies with MAbs have shown that binding of antibodies to three of these epitopes can inhibit A N F activities. Such studies have yet to be performed with the MAbs described in this study. Sudoh et al. (1988) have recently described a peptide in extracts of brain with homologous structure and activity to ANF. We have yet to test the ability of our MAb panel to bind this BNP peptide. Similarly, it would be of interest to examine binding of the MAbs to analogues of A N F resistant to inactivation resulting from cleavage between residues 12 and 13 (Condra et al., 1988). Direct plasma RIAs for A N F have been described but have often suffered from interference from plasma components or have been of insufficient sensitivity to permit assay of human plasma (Gutkowska et al., 1985; John et al., 1986; Juppner et al., 1986; Yandle et al., 1986; Richards et al., 1987; Cernacek et al., 1988). Most groups have therefore resorted to plasma extraction on C-18 Sep-pak columns prior to RIA (Gutkowska et al., 1986) or the faster receptor assay (Sagnella et al., 1987). Extraction not only removes interfering substances but also concentrates samples. The MAb ESA 4 can substitute for polyclonal antisera in RIA on such extracts. Iinuma et al. (1987) and Cernacek et al. (1988) have reported using acid extraction or filtration, in place of the established Sep-pak extraction of plasma samples, prior to immunoassay of ANF. Using the ESA 4 monoclonal antibody we were unable to develop radioimmunoassays for plasma A N F using either of these approaches. Processing by the former method yielded samples containing variable levels of interfering material, as has previously been reported for unprocessed samples (see above). Buckley et al. (1987) have also reported that acid treatment may increase A N F immunoreactiviy in extracts of human plasma. In our hands the filtration approach to separating A N F from plasma resulted in very low recoveries. In summary, this study has resulted in the
99
production of a panel of monoclonal antibodies to ANF. The most avid of these provides an alternative to polyclonal antisera in the established immunoassays for plasma ANF which should be easier to produce consistently and may have cost benefits.
Acknowledgements We would like to thank the University of Edinburgh Quantum Fund for financial support; D. Sampson, S. Smith, G. Willis, J. Gardner, B. Garden and D. Pepper for help and advice; J. Lim for manuscript preparation; and Dr. D.B.L. McClelland, Professor M. Lee and Professor G.D. Chisholm for support.
Note added in proof Mukoyama et al. (1988) also describe a MAb to the N terminal of hANF (Biochem. Biophys. Res. Commun. 151, 1277-1284).
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Robertson, J.l. (1987) Radio-immunoassay for plasma alpha human atrial natriuretic peptide: a comparison of direct and pre-extracted methods. J. Hypertension 5, 227-236. Rosmalen, F.M.A., Tan, A.C.I.T.L., Tan, H.S. and Benraad, T.J. (1987) A sensitive radioimmunoassay of atrial natriuretic peptide in human plasma using a tracer with an immobilised glycouril agent. Clin. Chim. Acta 165, 331-340. Sagnella, G.A., Buckley, M.G., Markandu, N.D. and MacGregor, G.A. (1987) Atrial natriuretic peptide in human plasma: Comparison of radioreceptor versus radioimmunoassay. Clin. Chim. Acta 166, 37-44. Stasch, J.P., Neuser, D., John, A., Hein, D., Heuck, K. and Morich, F.-J. (1987) The use of a monoclonal antibody for the determination of atrial natriuretic peptides in human plasma. Klin. Wochenschr. 65, 533-537. Sudoh, T., Kangawa, K., Minamino, N. and Matsuo, H. (1988) A new natriuretic peptide in porcine brain. Nature 332, 78-81. Tikkanen, I., Fyhrquist, F., Metsarinne, K. and Leidenius, R. (1985) Plasma atrial natriuretic peptide in cardiac disease and during infusion in healthy volunteers. Lancet ii, 65-69. Wilson, N., Ledsome, J.R., Keeler, R., Rankin, A.J., Wade, J.P. and Courneya, C.A. (1986) Heterologous radioimmunoassay of atrial natriuretic polypeptide in dog and rabbit plasma. J. Immunoassay 7, 73-96. Wright, J.F. and Hunter, W.M. (1982) A convenient replacement for cyanogen bromide - Activated solid phases in immunoradiometric assays. J. Immunol. Methods 48, 311-325. Yandle, T.G., Espiner, E.A., Nicholls, M.G. and Duff, H. (1986) Radioimmunoassay and characterization of atrial natriuretic peptide in human plasma. J. Clin. Endocrinol. Metab. 63, 72-79.