159
Journal of Immunological Methods, 137 (1991) 159-166 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 A D O N I S 002217599100105F
JIM 05852
Production and characterisation of monoclonal antibodies to parathyroid hormone (1-34) F.C. Logue, B. Perry, E.M. Biggart *, R.S. C h a p m a n and G . H . Beastall Institute of Biochemistry, Royal Infirmary, Glasgow G40SF, U.K. (Received 25 June 1990, revised received 18 September 1990, accepted 12 November 1990)
Monoclonal antibodies to the biologically active N terminal region of parathyroid hormone (PTH) suitable for use in the measurement of circulating PTH concentrations have proved difficult to produce. In this study, no serum PTH antibody titres could be detected in mice using synthetic human PTH (1-34) (free or coupled to albumin) or PTH (1-10) (coupled to keyhole limpet haemocyanin) as immunogen. A consistent response to PTH (1-34) peptide was obtained in DA rats. We have produced five monoclonal antibodies to PTH (1-34) derived from the fusion of DA rat spleen cells and the mouse myeloma line X63 Ag.8.653. Bulk production of the antibodies was achieved using congenitally athymic mice for ascites production. Antibody assessment studies revealed the antibodies to be sensitive to the oxidation state of the methionine residues in PTH (1-34). Two of the antibodies, 3B3 and 6E3, were shown to be of potential use in measuring circulating PTH (1-84) when used in combination with available antibodies to C terminal PTH. A third antibody, 4G3, which failed to recognise PTH (1-84) when used in combination with 3B3, formed the basis of a specific assay for PTH (1-34). Key words: Monoclonal antibody; Parathyroid hormone; Oxidation; Two-site immunometric assay
Introduction
Parathyroid hormone (PTH), an 84 amino acid peptide, is a major factor in the homeostasis of calcium metabolism. Acting on its target tissues, kidney and bone, PTH prevents hypocalcaemia and protects a variety of metabolic functions that depend on a normal concentration of ionised calcium in the extracellular fluid. Since the first
Correspondence to: F.C. Logue, Institute of Biochemistry, Royal Infirmary, Glasgow G 4 0 S F , U.K. * Current address: Department of Molecular Endocrinology, Middlesex Hospital, London, U.K. Abbreviations: PTH, parathyroid hormone; KLH, keyhole limpet haemocyanin; Mab, monoclonal antibody; BSA, bovine serum albumin.
development of a radioimmunoassay (Berson and Yalow, 1963), the measurement of PTH has been central to the laboratory investigation of patients with disorders of calcium metabolism (Boyd and Ladenson, 1984). However, the metabolism of PTH both within the parathyroid gland itself and also in peripheral tissues, such as liver and kidney, generates large amounts of biologically inactive C terminal fragments together with some N terminal fragments (Silverman and Yalow, 1973). This molecular heterogeneity in the presence of low circulating concentrations of intact PTH (1-84) has limited the clinical utility of PTH immunoassays (Armitage, 1986). Two-site immunometric assays have the sensitivity and specificity to measure circulating intact PTH (1-84) (Brown et al., 1987; Nussbaum et al.,
160 1987; Logue et al., 1989). However, large quantities of N terminal and C terminal specific antisera are required to sustain such assays. To date, these assays have been based mainly on affinity purified polyclonal antisera directed against N terminal determinants. The aim of this project was to produce monoclonal antibodies (Mab) reacting with the N terminal region (1-34) of PTH which would be of use in two-site immunometric assays for PTH (1-84).
Materials and methods
PTH peptides Synthetic human PTH (1-34) and PTH (1-84) peptides were purchased from Peninsula Laboratories Europe (Merseyside, U.K.) and Universal Biologicals (Cambridge, U.K.). Preparations of oxidised and reduced PTH (1-34) and PTH (1-84) were prepared from these by the method of Tashijian et al. (1964).
Imrnunogens Synthetic human PTH (1-34) peptide was coupled to bovine serum albumin by the carbodiimide method of Orth (1979). Synthetic human PTH (1-10) was custom synthesised by Cambridge Research Biochemicals (Cambridge, U.K.) and then conjugated to keyhole limpet haemocyanin, using a bisdiazotised tolidene linkage that produces a C terminally bound peptide.
Immunisations For all primary immunisations, a solution of peptide or conjugate was emulsified in Freund's complete adjuvant. For subsequent secondary immunisations, Freund's incomplete adjuvant was employed. Mice. Groups of six female mice of six different strains (BALB/c, NZB, B A L B / c / N Z B F 1 hybrids, SWR, SJL and CBA) were given primary immunisations of peptide or conjugate intraperitoneally (i.p.) followed by up to four secondary immunisations (also i.p.) at intervals of 2 weeks. Immunisations with unconjugated PTH (1-34) peptide were carried out in BALB/c mice initially
using 10 /~g PTH (1-34) for both primary and secondary immunisations. The immunisation experiments in BALB/c mice were repeated at 20 and 50 /~g PTH (1-34) for both primary and secondary immunisations. For all other mouse strains, 50 /~g PTH (1-34) was used for both primary and secondary immunisations. Immunisations with the PTH (1-34)-BSA conjugate were performed in BALB/c mice at 100/~g and at 200 btg (equivalent to 11/zg and 22 ktg PTH (1-34) peptide, respectively) for both primary and secondary immunisations. For all other strains, 200 /~g PTH (1-34)-BSA conjugate was used for both the primary and secondary immunisations. Immunisation with PTH (1-10)-KLH conjugate was carried out at 100/~g conjugate (equivalent to 30/~g PTH (1-34) peptide) for both primary and secondary immunisations in all strains. Rats. Groups of six female rats (DA or Lou) were given a primary immunisation of 50 ttg PTH (1-34) followed by up to five secondary immunisations, also of 50/~g PTH (1-34), at intervals of 2 weeks. Immunisations were administered subcutaneously using the multi-site method (Vaitukaitis et al., 1971). An intravenous dose (50 ~g) of PTH (1-34) was also administered into the jugular vein of the rats 3 days prior to fusion. Sheep. Groups of two sheep (Suffolk wethers) were given a primary immunisation followed by up to six secondary immunisations at monthly intervals (50 btg PTH (1-34) peptide or 100 ~tg PTH (1-34)-BSA conjugate). Immunisations were administered subcutaneously and intra-muscularly using the multi-site method (Vaitukaitis et al., 1971).
Hybridoma production All cell culture reagents were purchased from Flow Laboratories (lrvine, Scotland, U.K.) except where stated. The fusion procedure was based on the method of Fazekas de St. Groth and Scheidegger (1980). Splenic lymphocytes from the immunised rats were fused with X63Ag8.653 mouse myeloma cells (Kearney et al., 1979) using 50% polyethylene glycol 1500 (BDH, Poole, U.K.). Each fusion experiment consisted of three separate fusion events, the spleen cells recovered being split into three aliquots of 50 x 1 0 6 cells. The products of each of the fusion events were plated-
161 out into five microtitre plates (96 X 0.2 ml wells) on mouse peritoneal macrophages as feeder cells in H A T selection medium.
Results
Iodination method Peptides and monoclonal antibodies were iodinated by the lactoperoxidase (solid-phased) enzymatic method of Karonen et al. (1975). To an aliquot of either peptide (2 /~g) or Mab (25 /~g) was added sodium phosphate buffer, p H 7.4 (20 ffl, 0.5 M), carrier-free 12Siodine (10 /~1, 0.5 mCi) and solid-phased lactoperoxidase (20 ffl, 200 ng). The reaction was started by the addition of hydrogen peroxide solution (10/tl, 0.01% ( w / v ) in distilled water). After 30 min the reaction was quenched by the addition of sodium phosphate buffer, pH 7.4 (200 #l, 0.05 M containing NaC1 0.9% ( w / v ) and bovine serum albumin 0.1% (w/v)). The iodinated peptides were then purified on a Biogel P10 column (1.6 x 30 cm) and the monoclonal antibodies on a Sepharose 6B column (1.6 x 30 cm).
Response to immunisation Mice. No serum titres of PTH antibodies were detected in any of the mouse strains immunised with PTH (1-34), PTH (1-34)-BSA or P T H (110)-KLH. Rats. DA and Lou rats were immunised with PTH (1-34). Lou rats failed to show a detectable PTH (1-34) antibody titre. However, a consistent response was detected in DA rats, 6 / 6 responding with serum titres of 1 : 3000-1 : 5000 at one month past primary immunisation Maximum titres of 1 : 50,000 were detected after the primary and five secondary immunisations. Sheep. There was a brisk response in Suffolk wethers to immunisation with P T H (1-34). Antibody titres were detected in 2 / 2 sheep at 1 month post primary immunisation. Maximum titres of 1:64,000 were detected after a primary and five secondary immunisations. Similar results in sheep were obtained using PTH (1-34)-BSA as immunogen.
Antibody screening assay Serum or culture fluid (100 ffl) diluted in 0.05 M barbitone buffer p H 8.6, 0.5% BSA, was incubated with radiolabelled PTH (1-34) (5000 cpm) overnight at 4 ° C. Separation was by incubation with second antibody (either anti-sheep or antir a t / m o u s e IgG) coupled to Sepharose CL-4B for 2 h at room temperature, followed by two centrifugation and wash cycles in 0.9% saline/Tween 20 (0.2%, v/v).
Fusion experiments Three fusion experiments were carried out using the mouse myeloma line X63Ag8.653 and spleen cells from DA rats found to have serum titres of PTH (1-34) antibodies. In each of the fusion experiments, growing hybrids were seen in greater than 70% (1050) of the wells (1440). A total of 39 wells were identified as positive for PTH (1-34) antibodies. From these five hybridoma lines were stabilised and remained secreting PTH (1-34) antibodies through cloning ( x 2 ) by limiting dilution.
Bulk production of monoclonal antibodies For each hybridoma line, six nude mice were pristane primed and inoculated with 5 x 10 6 cells intraperitoneally. Mab IgG was purified from ascitic fluid by an n-octanoic acid precipitation method (Steinbuch and Audran, 1969). Protein was measured by the method of Schacterle and Pollack (1973). Rat monoclonal antibody isotyping kits (Serotec, Oxford, U.K.) were used for Mab class and subclass determinations.
Initial assessment of hybridoma lines Culture fluid from the five hybridoma fines was used for isotype analysis and initial displacement studies using synthetic PTH (1-34) and purified human PTH (1-84) (MRC 75/549) (Table I). Of the five cell lines, four were producing IgG Mab and one was producing an IgM Mab. The greatest displacement with PTH (1-34) was observed with 9E3 followed by 4G3, 6E3, 3B3 and 1D1, respectively. The displacement experiments with the human PTH (1-84) preparation ( M R C / 7 5 9 ) showed 1D1 and 9E3 to be specific for PTH (1-34). Both
162 TABLE I ISOTYPE ANALYSIS A N D I N I T I A L D I S P L A C E M E N T STUDIES ON CULTURE FLUID FROM THE M O U S E / R A T H Y B R I D O M A LINE Hybridoma line
Isotype of Mab
Concentration of PTH (1-34) at 50% displacement (nM)
Relative displacement with h u m a n PTH (1-34)
1DI 3B3 4G3 6E3 9E3
IgG 2b IgG 2b IgG 2a IgG 2a IgM
> 77.0 38.0 1.2 9.7 0.6
0.96 0.43 0.90
3B3 and 6E3 also recognised PTH (1-84), with the displacements observed being equivalent to those obtained with PTH (1-34) peptide (Table I). Antibody 4G3 also appeared to recognise the PTH (1-84) in this preparation, although less well than the PTH (1-34) peptide. However, subsequent experiments with purified PTH (1-84) gave conflicting results for 4G3, leading to the conclusions that 4G3 was specific for PTH (1-34) and that MRC/759 contained N terminal fragments of PTH (see paragraph characterisation of monoclonal antibodies below).
Bulk production of monoclonal antibodies On the basis of the displacement experiments, those cell lines which recognised PTH (1-84) (3B3, 4G3 and 6E3) were chosen for bulk production of Mab. Since the hybridoma lines were mouse/rat hybrids and not histocompatible with BALB/c mice, congenitally athymic ( n u / n u , nude) mice were used for the production of ascitic fluid. All the nude mice inoculated (6/6) responded with ascites production. Taps of ascitic fluid showed PTH (1-34) antibody titres ranging from 1 : 103 at 20 days to > 10 t° at 40 days post inoculation. Total protein concentrations of the ascitic fluids were in the range 64.3-75.2 mg/ml with 20.4 31.1% of protein being recovered as IgG. Characterisation of monoclonal antibodies The methionine residues found within the N terminal sequence of PTH peptides are known to oxidise on storage (Tashijian et al., 1964) and therefore both oxidised (Met[O]-) and reduced
PTH peptides were used in the Mab characterisation experiments. Figs. 1-3 show the displacement curves obtained for 3B3, 4G3 and 6E3 Mabs with oxidised and reduced synthetic human PTH (1-34) and PTH (1-84) peptides. Greater than 60% of radioactivity in the peptide labels was bound by the Mab's under conditions of maximal binding. For the displacement curves, dilutions of the Mab's were selected to give approximately 20% of label counts bound in the absence of unlabelled peptide. For 3B3 and 6E3, the results confirmed the initial displacement analysis suggesting that both these Mabs recognised PTH (1-34) and PTH (184), essentially equipotently. However, Mab 4G3 showed no displacement with PTH (1-84) and
Assessment of Mab 3B3 with PTH peptides 30-
%Bound el ',..
.tit....--it...
20
.~
i
i "--'O--
1 84
Ox~ISed
I
1 34 OxidiSed
...... • ....
1 84 R e d u c e d
........ t i ......
1 34 R e d u c e d
10
0
........
i
........
i
10
........
100
PTH
i 1000
........
i ~ 0000
pmolll
Fig. 1. Displacement curves obtained with Mab 3B3 using oxidised and reduced PTH (1-34) and PTH (1-84) peptides. The Mab was diluted in 0.05 M barbitone buffer, p H 8.6, 0.5% BSA and incubated with radiolabelled P T H (1-34) and the relevant PTH peptide overnight at room temperature. Sheep a n t i - m o u s e / r a t IgG (on the solid phase) was then added. After incubation for 2 h with shaking, the assays were separated by centrifugation and washing ( × 2) in 0.9% s a l i n e / T w e e n (0.2%,
v/v).
163 appeared to be specific for P T H (1-34). This was c o n t r a r y to the initial displacement experiments (see above) and was most likely explained by the fact that the biological preparation of h u m a n P T H ( 1 - 8 4 ) ( M R C 7 5 / 5 4 9 ) also contained P T H ( 1 - 3 4 ) fragments. All three M a b s reacted preferentially with oxidised P T H peptides to differing degrees. This effect was most m a r k e d with 3B3 which showed a ten-fold preference for oxidised P T H peptides. Scatchard analysis of the data for oxidised P T H ( 1 - 3 4 ) peptide gave avidity constants of 5.9 x 109, 3.0 x 101° and 1.1 x 109 L / M for 3B3, 4G3 and 6E3, respectively.
30
Assessment of Mab 6E3 with PTH peptides 2o %Bound
15 . .A. . ... / "
""~.
10
Assessment of Mab 4G3 with PTH peptides 5 ¸
%Bound
....-" ~.
......
10
1o
.....
1o0
.....
,o'00 . . . .
;0000
PTH pmol/I
20
'\
Jt
1-34
!
1-34ieduced
'~i
1 84 Reduced ........ i lo
Fig. 3. Displacement curves obtained with Mab 6E3 using oxidised and reduced PTH (1-34) and PTH (1-84) peptides. The Mab was diluted in 0.05 M barbitone buffer, pH 8.6, 0.5% BSA and incubated with radiolabelled PTH (1-34) and the relevant PTH peptide overnight at room temperature. Sheep anti-mouse/rat IgG (on the solid phase) was added. After incubation for 2 h with shaking, the assays were separated by centrifugation and washing (x 2) in 0.9% saline/Tween (0.2%, v/v).
N o displacement was observed for any of M a b s with the P T H ( 1 - 1 0 ) peptide.
~
k"...=....-~R,,.~ ........ i ........ , loo ~ooo 1000o . . . . . . . .
i
PTH pmol/I
Fig. 2. Displacement curves obtained with Mab 4G3 using oxidised and reduced PTH (1-34) and PTH (1-84) peptides. The Mab was diluted in 0.05 M barbitone buffer, pH 8.6, 0.5% BSA and incubated with radiolabelled PTH (1-34) and the relevant PTH peptide overnight at room temperature. Sheep anti-mouse/rat IgG (on the solid phase) was added. After incubation for 2 h with shaking, the assays were separated by centrifugation and washing (x 2) in 0.9% saline/Tween (0.2%, v/v).
Application to two-site immunometric assays T w o of the antibodies, 3B3 and 6E3, were used in the development of two-site i m m u n o m e t r i c assays for P T H (1-84). Fig. 4 shows the standard curves for (a) radiolabelled 3B3 in c o m b i n a t i o n with a C terminal specific Mab, ESQ1 (produced by Dr. K. James, University of Edinburgh, and s u p p l i e d b y N o v o BioLabs, Cambridge, U . K . ) , and (b) 6E3 in c o m b i n a t i o n with an affinity purified C terminal specific polyclonal antibody,
164 C A G 3 (supplied by IDS, N e w c a s t l e - u p o n - T y n e , U.K.). Th es e assays h a v e been s h o w n to have the sensitivity to m e a s u r e P T H ( 1 - 8 4 ) in n o r m a l subj e c t s an d to be u n a f f e c t e d by excess C t e r m i n a l fr a gme n t s ( L o g u e et al., 1988; G r e i g et al., 1990). Fig. 5 shows the s t a n d a r d c u r v e for a two-site i m m u n o m e t r i c assay for P T H ( 1 - 3 4 ) using radiolabelled 3B3 in c o m b i n a t i o n with 4G3. T h e assay
A s s e s s m e n t of 3B3/4G3 combination ~n a two-site immunometric assay for PTH(1-34) 100
%Bound
10
Assessment of Mabs 3B3 and 6E3 in a two-site immunometric assay for PTH(1-84) 100 7
%Bound
10
PTH(1-34)
PTH(1-84)
01 I
3B3in comi~nationwithESQ1 •
01
J 1
........
i 1
........
6E3tncombinationwithCAG3
i
........
i
10
100
PTH(1-84)
pmol/I
........
i 1000
Fig. 4. PTH (1-84) standard curves in two-site immunometric assay, a: 3B3 in combination with C terminal specific Mab ESQ1. Radiolabelled 3B3 (100,000 cpm/tube) in 0.05 M phosphate buffer, pH 7.0, 0.5% BSA was incubated with PTH (1-84) peptide overnight at room temperature. ESQ1 (coupled to Sepharose solid phase) was added, incubated for 2 h with shaking, and the assays separated by centrifugation and washing (×2) in 0.9% saline/Tween 20 (0.2%, v/v). b: 6E3 in combination with the C terminal specific affinity purified polyclonal antibody CAG3. PTH (1-84) peptide in 0.05 M phosphate buffer, pH 7.4, 0.5% BSA, was incubated overnight at room temperature in tubes coated with 6E3. Radiolabelled CAG3 (200,000 cpm/tube) was added, Incubated for 2 h at room temperature, and the assays separated by aspiration and washing ( × 2) in 0.9% saline/Tween 20 (0.2%, v/v).
10
100
1000
pmol/I Fig. 5. PTH (1-34) and PTH (1-84) standard curves using 4G3 in combination with 3B3 in a two-site immunometric assay for PTH (1-34). PTH (1-34) or PTH (1-84) peptides in 0.05 M phosphate buffer, pH 7.4, 0.5% BSA, were incubated in tubes coated with 4G3. RadiolabeUed 3B3 (100,000 cpm/tube) was added, incubated for 2 h at room temperature, and the assays separated by aspiration and washing (x2) in 0.9% saline/Tween (0.2%, v/v). shows less than 0.5% c r o s s - r e a c t i o n with P T H ( 1 - 8 4 ) . It should be n o t e d f r o m b o t h Figs. 4 a n d 5 that, u n d e r the c o n d i t i o n s of assay, a p p r o x i m a t e l y 60% of the respective labelled M a b s are b o u n d at a P T H c o n c e n t r a t i o n of 500 p m o l / 1 with n o evid en ce of a h o o k effect.
Discussion A n t i b o d i e s to the biologically active N t e r m i n a l region ( 1 - 3 4 ) of P T H w h i ch h a v e the specificity
165 and avidity to be of use in the measurement of circulating PTH, without prior affinity purification, have proven difficult to produce. A previous attempt to obtain Mabs to N terminal PTH, using purified bovine PTH (1-84) as immunogen, produced only low avidity IgM antibodies and suggested that B A L B / c mice were poor responders to PTH immunogens (Nussbaum et al., 1981). In a more detailed study on the genetic control of the immune response to synthetic PTH (1-34) peptide in mice, Nussbaum et al. (1985) confirmed that B A L B / c mice were genetically poor responders to P T H (1-34). The immune response was found to be under the control of Ir genes and associated with the IA and IE subregions of the major histocompatibility complex of the mouse. The CBA mouse strain was predicted to be a high responder to PTH (1-34). The results of the immunisations in this study, with 6 / 6 DA rats and 2 / 2 sheep positive and all mice and Lou rats negative, are consistent with a species and strain specific response. However, we did not detect the predicted response in CBA mice. The methionine residues at positions 8 and 18 of P T H (1-34) have been shown to be susceptible to oxidation on storage under acid conditions (Tashijian et al., 1964) and during purification by H P L C (Bennett et al., 1981). It has also been shown that the spectrum of biological activities of P T H peptides is altered depending on the oxidation state of the peptide (Pang et al., 1983). Furthermore, polyclonal antisera to N terminal PTH have been found to be sensitive to the oxidation state of PTH (Tashijian et al., 1964). Recent studies using circular dichroism have confirmed that oxidation of the methionine residue, at position 8 in particular, produces substantial changes in the secondary structure of P T H (1-34). These changes alter the conformation of the PTH (1-34) peptide, resulting in a reduced affinity for receptors (Zull et al., 1990). The peptides used in this study were stored in the main under acid conditions and the Mabs produced showed a preference to various degrees for oxidised PTH (1-34) peptide. It is not possible to fully evaluate the effect of oxidation from the results of this study, but it is probable that the oxidation state affects both the immunogenicity of
PTH peptides and the ability of screening assays to detect an antibody response. We would recommend that, for PTH (1-34) and for other peptides containing methionine residues, the oxidation state be taken into consideration both when assessing the immunogenicity of a peptide and also in the design of antibody screening assays. Using spleen cells from the DA rats and the mouse myeloma line X63Ag8.653, five hybridoma lines secreting anti-PTH (1-34) antibodies were established. This is a low yield, representing 0.1% of the wells screened, compared to mouse-mouse fusions for highly immunogenic proteins, such as TSH, LH, FSH and AFP (Siddle and Soos, 1981; Stevenson et al., 1987), where positive rates of 70-80% can be achieved. However, it is consistent with the results obtained with mouse-mouse fusions for the poorly immunogenic peptide hormone A C T H (1-39) (White et al., 1985). Since the early work of Lerner et al. (1982) and Schmitz et aI. (1983) it is now well established that small peptide sequences can be used to produce antibodies to the intact protein of which they form a part. However, this approach frequently produces peptide specific antibodies which fail to react with the native molecule (Lerner, 1984). This study is no exception, with three of the five Mabs raised to synthetic PTH (1-34) failing to recognise the intact PTH (1-84) molecule. However, two of the Mabs, 3B3 and 6E3, have been shown to be of use in a two-site immunometric assay for PTH (1-84). A third, 4G3, in combination with 6E3, forms the basis of a two-site immunometric assay specific for PTH (1-34). The generation of useful Mabs to biologically active regions of PTH, using synthetic PTH (1-34) peptide as immunogen, is important, since in vivo PTH is not stored in large quantities within the parathyroid gland, and therefore the availability of human preparations of P T H for use as immunogen is limited.
Acknowledgements We are grateful to Dr. Angus Munro of the Scottish Antibody Production Unit for undertaking the sheep immunisations. We thank Novo BioLabs and IDS for the supply of ESQ1 and CAG3 antibodies respectively. We are grateful
166 also for the technical assistance of Steven Greig and Jacqueline McDade and the expert secretarial assistance of Myra Ogilvie.
References Armitage, E.K. (1986) Parathyrin (parathyroid hormone): metabolism and methods for assay. Clin. Chem. 32, 418. Bennett, H.P.J., Solomon, S. and Goltzman, D. (1981) Isolation and analysis of human parathyrin in parathyroid tissue and plasma. Biochem. J. 197, 391. Berson, S.A., Yalow, R.S., Aurbach, G.D. and Potts, J.T. (1963) Immunoassay of bovine and human parathyroid hormone. Proc. Natl. Acad. Sci. U.S.A. 49, 613. Boyd, J.C. and Ladenson, J.H. (1984) Value of laboratory tests in the differential diagnosis of hypercalcaemia. Am. J. Med. 77, 863. Brown, R.C., Aston, J.P., Weeks, I. and Woodhead, J.S. (1987) Circulating intact parathyroid hormone measured by a two-site immunochemiluminometric assay. J. Clin. Endocrinol. Metab. 65, 407. Fazekas de St.Groth, S. and Scheidegger, D. (1980) Production of monoclonal antibodies: strategy and tactics. J. Immunol. Methods 35, 1. Greig, S., Logne, F.C., Beastall, G.H. and Duggan, R.G. (1990) A prototype IRMA for PTH (1-84) utilising the preferred configuration of antibodies to exclude C terminal fragments [abstract]. Proceedings of the National Meeting, Brighton: Association of Clinical Biochemists, May, 1990, C43. Karonen, S.L., Morsky, P., Seren, M. and Seuderling, U. (1975) An enzymatic method for trace iodination of proteins and peptides with 125-iodine. Ann. Biochem. 67, 1. Kearney, J.F., Radbruch, A., Liesegang, B. and Rajewsky, K. (1979) A new mouse myeioma cell line that has lost immunoglobin expression but permits the construction of antibody-secreting hybrid cell lines. J. Immunol. 123, 1548. Lerner, R.A. (1982) Tapping the immunological repertoire to produce antibodies of predetermined specificity. Nature 299, 592. Lerner, R.A. (1984) Antibodies of predetermined specificity in biology and medicine. Adv. Immunol. 36, 1. Logue, F.C., Perry, B., Chapman, R.S., James, K. and Beastall, G.H. (1988) The development of a two-site immunometric assay (IRMA) for intact 1-84 human parathyroid hormone using monoclonal antibodies [abstract]. J. Endocrinol. 117, 67. Nussbaum, S.R., Rosenblatt, M., Mudgett-Hunter, M. and Potts, Jr., J.T. (1981) Monoclonal antibodies directed against the biologically active regions of parathyroid hormone. In: R. Fellow and G. Eisenearth (Eds.), Monoclonal Antibodies in Endocrine Research. Raven Press, New York, p. 181.
Nussbaum, S.R., Lin, C.S., Potts, J.T., Rosenthal, A.S. and Rosenblatt, M. (1985) Development of monoclonal antibodies against parathyroid hormone: genetic control of the immune response to human PTH. Methods Enzymol. 109, 625. Nussbaum, S.R., Zaharadnik, R.J., Lavigne, J.R. et al., (1987) Highly sensitive two-site immunoradiometric assay of parathyrin and its clinical utility in evaluating patients with hypercalcaemia. Clin. Chem. 33, 1364. Orth, D.N. (1979) Adrenocorticotrophin (ACTH). In: B.M. Jaffe and H.R. Behrman (Eds.), Methods of Radioimmunoassay. Academic Press, New York, p. 245. Pang, P.K.T., Yang, M.C.M., Keutmann, H.T. and Kenny, A.D. (1983) Structure activity relationships of parathyroid hormone: separation of the hypotensive and the hypercalcaemic properties. Endocrinology 112, 284. Schacterle, G.R. and Pollack, R.L. (1973) A simplified method for the quantitative assay of small amounts of protein in biological materials. Ann. Biochem. 51,654. Schmitz, H.E., Atassi, M. and Atassi, M.Z. (1983) Production of monoclonal antibodies with preselected submolecular binding specificities to protein antigenic sites. Antibodies to sperm whale myoglobin. Mol. Immunol. 20, 719. Siddle, K. and Soos, M. (1981) Monoclonal antibodies for human pituitary glycoprotein hormones and rabbit immunoglobulin. In: A. Albertini and R. Ekins (Eds.), Monoclonal Antibodies and Developments in Immunoassay. Elsevier/North Holland, Amsterdam, p. 53. Silverman, R. and Yalow, R.S. (1973) Heterogeneity of parathyroid hormone. Clinical and physiological implications. J. Clin. Invest. 52, 1958. Steinbuch, M. and Audran, R. (1969) The isolation of IgG from mammalian sera with the aid of caprylic acid. Arch. Biochem. Biophys. 134, 279. Stevenson, J.D., Chapman, R.S., Perry, B. and Logue, F.C. (1987) Evaluation and clinical application of a two-site immunoradiometric assay for alpha-l-foetoprotein using readily available reagents. Ann. Clin. Biochem. 24, 411. Tashijian, A.H., Ontjes, D.A. and Munson, P.L. (1964) Alkylation and oxidation of methionine in bovine parathyroid hormone: effects on hormonal activity and antigenicity. Biochemistry 3, 1175. Vaitukaitis, J., Robbins, J.B. and Nieschlag, E. (1971) A method for producing specific antisera with small doses of immunogen. J. Clin. Endocrinol. Metab. 33, 988. White, A., Gray, C. and Ratcliffe, J.G. (1985) Characterisation of monoclonal antibodies to adrenocorticotrophin. J. Immunol. Methods 79, 185. Zull, E.S., Smith, S.K. and Wiltshire, R. (1990) Effect of methionine oxidation and deletion of aminoterminal residues on the conformation or parathyroid hormone. J. Biol. Chem. 265, 5671.
Journal of Immunological Methods, 137 (1991) 159-166 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 A D O N I S 002217599100105F
JIM 05852
Production and characterisation of monoclonal antibodies to parathyroid hormone (1-34) F.C. Logue, B. Perry, E.M. Biggart *, R.S. C h a p m a n and G . H . Beastall Institute of Biochemistry, Royal Infirmary, Glasgow G40SF, U.K. (Received 25 June 1990, revised received 18 September 1990, accepted 12 November 1990)
Monoclonal antibodies to the biologically active N terminal region of parathyroid hormone (PTH) suitable for use in the measurement of circulating PTH concentrations have proved difficult to produce. In this study, no serum PTH antibody titres could be detected in mice using synthetic human PTH (1-34) (free or coupled to albumin) or PTH (1-10) (coupled to keyhole limpet haemocyanin) as immunogen. A consistent response to PTH (1-34) peptide was obtained in DA rats. We have produced five monoclonal antibodies to PTH (1-34) derived from the fusion of DA rat spleen cells and the mouse myeloma line X63 Ag.8.653. Bulk production of the antibodies was achieved using congenitally athymic mice for ascites production. Antibody assessment studies revealed the antibodies to be sensitive to the oxidation state of the methionine residues in PTH (1-34). Two of the antibodies, 3B3 and 6E3, were shown to be of potential use in measuring circulating PTH (1-84) when used in combination with available antibodies to C terminal PTH. A third antibody, 4G3, which failed to recognise PTH (1-84) when used in combination with 3B3, formed the basis of a specific assay for PTH (1-34). Key words: Monoclonal antibody; Parathyroid hormone; Oxidation; Two-site immunometric assay
Introduction
Parathyroid hormone (PTH), an 84 amino acid peptide, is a major factor in the homeostasis of calcium metabolism. Acting on its target tissues, kidney and bone, PTH prevents hypocalcaemia and protects a variety of metabolic functions that depend on a normal concentration of ionised calcium in the extracellular fluid. Since the first
Correspondence to: F.C. Logue, Institute of Biochemistry, Royal Infirmary, Glasgow G 4 0 S F , U.K. * Current address: Department of Molecular Endocrinology, Middlesex Hospital, London, U.K. Abbreviations: PTH, parathyroid hormone; KLH, keyhole limpet haemocyanin; Mab, monoclonal antibody; BSA, bovine serum albumin.
development of a radioimmunoassay (Berson and Yalow, 1963), the measurement of PTH has been central to the laboratory investigation of patients with disorders of calcium metabolism (Boyd and Ladenson, 1984). However, the metabolism of PTH both within the parathyroid gland itself and also in peripheral tissues, such as liver and kidney, generates large amounts of biologically inactive C terminal fragments together with some N terminal fragments (Silverman and Yalow, 1973). This molecular heterogeneity in the presence of low circulating concentrations of intact PTH (1-84) has limited the clinical utility of PTH immunoassays (Armitage, 1986). Two-site immunometric assays have the sensitivity and specificity to measure circulating intact PTH (1-84) (Brown et al., 1987; Nussbaum et al.,
160 1987; Logue et al., 1989). However, large quantities of N terminal and C terminal specific antisera are required to sustain such assays. To date, these assays have been based mainly on affinity purified polyclonal antisera directed against N terminal determinants. The aim of this project was to produce monoclonal antibodies (Mab) reacting with the N terminal region (1-34) of PTH which would be of use in two-site immunometric assays for PTH (1-84).
Materials and methods
PTH peptides Synthetic human PTH (1-34) and PTH (1-84) peptides were purchased from Peninsula Laboratories Europe (Merseyside, U.K.) and Universal Biologicals (Cambridge, U.K.). Preparations of oxidised and reduced PTH (1-34) and PTH (1-84) were prepared from these by the method of Tashijian et al. (1964).
Imrnunogens Synthetic human PTH (1-34) peptide was coupled to bovine serum albumin by the carbodiimide method of Orth (1979). Synthetic human PTH (1-10) was custom synthesised by Cambridge Research Biochemicals (Cambridge, U.K.) and then conjugated to keyhole limpet haemocyanin, using a bisdiazotised tolidene linkage that produces a C terminally bound peptide.
Immunisations For all primary immunisations, a solution of peptide or conjugate was emulsified in Freund's complete adjuvant. For subsequent secondary immunisations, Freund's incomplete adjuvant was employed. Mice. Groups of six female mice of six different strains (BALB/c, NZB, B A L B / c / N Z B F 1 hybrids, SWR, SJL and CBA) were given primary immunisations of peptide or conjugate intraperitoneally (i.p.) followed by up to four secondary immunisations (also i.p.) at intervals of 2 weeks. Immunisations with unconjugated PTH (1-34) peptide were carried out in BALB/c mice initially
using 10 /~g PTH (1-34) for both primary and secondary immunisations. The immunisation experiments in BALB/c mice were repeated at 20 and 50 /~g PTH (1-34) for both primary and secondary immunisations. For all other mouse strains, 50 /~g PTH (1-34) was used for both primary and secondary immunisations. Immunisations with the PTH (1-34)-BSA conjugate were performed in BALB/c mice at 100/~g and at 200 btg (equivalent to 11/zg and 22 ktg PTH (1-34) peptide, respectively) for both primary and secondary immunisations. For all other strains, 200 /~g PTH (1-34)-BSA conjugate was used for both the primary and secondary immunisations. Immunisation with PTH (1-10)-KLH conjugate was carried out at 100/~g conjugate (equivalent to 30/~g PTH (1-34) peptide) for both primary and secondary immunisations in all strains. Rats. Groups of six female rats (DA or Lou) were given a primary immunisation of 50 ttg PTH (1-34) followed by up to five secondary immunisations, also of 50/~g PTH (1-34), at intervals of 2 weeks. Immunisations were administered subcutaneously using the multi-site method (Vaitukaitis et al., 1971). An intravenous dose (50 ~g) of PTH (1-34) was also administered into the jugular vein of the rats 3 days prior to fusion. Sheep. Groups of two sheep (Suffolk wethers) were given a primary immunisation followed by up to six secondary immunisations at monthly intervals (50 btg PTH (1-34) peptide or 100 ~tg PTH (1-34)-BSA conjugate). Immunisations were administered subcutaneously and intra-muscularly using the multi-site method (Vaitukaitis et al., 1971).
Hybridoma production All cell culture reagents were purchased from Flow Laboratories (lrvine, Scotland, U.K.) except where stated. The fusion procedure was based on the method of Fazekas de St. Groth and Scheidegger (1980). Splenic lymphocytes from the immunised rats were fused with X63Ag8.653 mouse myeloma cells (Kearney et al., 1979) using 50% polyethylene glycol 1500 (BDH, Poole, U.K.). Each fusion experiment consisted of three separate fusion events, the spleen cells recovered being split into three aliquots of 50 x 1 0 6 cells. The products of each of the fusion events were plated-
161 out into five microtitre plates (96 X 0.2 ml wells) on mouse peritoneal macrophages as feeder cells in H A T selection medium.
Results
Iodination method Peptides and monoclonal antibodies were iodinated by the lactoperoxidase (solid-phased) enzymatic method of Karonen et al. (1975). To an aliquot of either peptide (2 /~g) or Mab (25 /~g) was added sodium phosphate buffer, p H 7.4 (20 ffl, 0.5 M), carrier-free 12Siodine (10 /~1, 0.5 mCi) and solid-phased lactoperoxidase (20 ffl, 200 ng). The reaction was started by the addition of hydrogen peroxide solution (10/tl, 0.01% ( w / v ) in distilled water). After 30 min the reaction was quenched by the addition of sodium phosphate buffer, pH 7.4 (200 #l, 0.05 M containing NaC1 0.9% ( w / v ) and bovine serum albumin 0.1% (w/v)). The iodinated peptides were then purified on a Biogel P10 column (1.6 x 30 cm) and the monoclonal antibodies on a Sepharose 6B column (1.6 x 30 cm).
Response to immunisation Mice. No serum titres of PTH antibodies were detected in any of the mouse strains immunised with PTH (1-34), PTH (1-34)-BSA or P T H (110)-KLH. Rats. DA and Lou rats were immunised with PTH (1-34). Lou rats failed to show a detectable PTH (1-34) antibody titre. However, a consistent response was detected in DA rats, 6 / 6 responding with serum titres of 1 : 3000-1 : 5000 at one month past primary immunisation Maximum titres of 1 : 50,000 were detected after the primary and five secondary immunisations. Sheep. There was a brisk response in Suffolk wethers to immunisation with P T H (1-34). Antibody titres were detected in 2 / 2 sheep at 1 month post primary immunisation. Maximum titres of 1:64,000 were detected after a primary and five secondary immunisations. Similar results in sheep were obtained using PTH (1-34)-BSA as immunogen.
Antibody screening assay Serum or culture fluid (100 ffl) diluted in 0.05 M barbitone buffer p H 8.6, 0.5% BSA, was incubated with radiolabelled PTH (1-34) (5000 cpm) overnight at 4 ° C. Separation was by incubation with second antibody (either anti-sheep or antir a t / m o u s e IgG) coupled to Sepharose CL-4B for 2 h at room temperature, followed by two centrifugation and wash cycles in 0.9% saline/Tween 20 (0.2%, v/v).
Fusion experiments Three fusion experiments were carried out using the mouse myeloma line X63Ag8.653 and spleen cells from DA rats found to have serum titres of PTH (1-34) antibodies. In each of the fusion experiments, growing hybrids were seen in greater than 70% (1050) of the wells (1440). A total of 39 wells were identified as positive for PTH (1-34) antibodies. From these five hybridoma lines were stabilised and remained secreting PTH (1-34) antibodies through cloning ( x 2 ) by limiting dilution.
Bulk production of monoclonal antibodies For each hybridoma line, six nude mice were pristane primed and inoculated with 5 x 10 6 cells intraperitoneally. Mab IgG was purified from ascitic fluid by an n-octanoic acid precipitation method (Steinbuch and Audran, 1969). Protein was measured by the method of Schacterle and Pollack (1973). Rat monoclonal antibody isotyping kits (Serotec, Oxford, U.K.) were used for Mab class and subclass determinations.
Initial assessment of hybridoma lines Culture fluid from the five hybridoma fines was used for isotype analysis and initial displacement studies using synthetic PTH (1-34) and purified human PTH (1-84) (MRC 75/549) (Table I). Of the five cell lines, four were producing IgG Mab and one was producing an IgM Mab. The greatest displacement with PTH (1-34) was observed with 9E3 followed by 4G3, 6E3, 3B3 and 1D1, respectively. The displacement experiments with the human PTH (1-84) preparation ( M R C / 7 5 9 ) showed 1D1 and 9E3 to be specific for PTH (1-34). Both
162 TABLE I ISOTYPE ANALYSIS A N D I N I T I A L D I S P L A C E M E N T STUDIES ON CULTURE FLUID FROM THE M O U S E / R A T H Y B R I D O M A LINE Hybridoma line
Isotype of Mab
Concentration of PTH (1-34) at 50% displacement (nM)
Relative displacement with h u m a n PTH (1-34)
1DI 3B3 4G3 6E3 9E3
IgG 2b IgG 2b IgG 2a IgG 2a IgM
> 77.0 38.0 1.2 9.7 0.6
0.96 0.43 0.90
3B3 and 6E3 also recognised PTH (1-84), with the displacements observed being equivalent to those obtained with PTH (1-34) peptide (Table I). Antibody 4G3 also appeared to recognise the PTH (1-84) in this preparation, although less well than the PTH (1-34) peptide. However, subsequent experiments with purified PTH (1-84) gave conflicting results for 4G3, leading to the conclusions that 4G3 was specific for PTH (1-34) and that MRC/759 contained N terminal fragments of PTH (see paragraph characterisation of monoclonal antibodies below).
Bulk production of monoclonal antibodies On the basis of the displacement experiments, those cell lines which recognised PTH (1-84) (3B3, 4G3 and 6E3) were chosen for bulk production of Mab. Since the hybridoma lines were mouse/rat hybrids and not histocompatible with BALB/c mice, congenitally athymic ( n u / n u , nude) mice were used for the production of ascitic fluid. All the nude mice inoculated (6/6) responded with ascites production. Taps of ascitic fluid showed PTH (1-34) antibody titres ranging from 1 : 103 at 20 days to > 10 t° at 40 days post inoculation. Total protein concentrations of the ascitic fluids were in the range 64.3-75.2 mg/ml with 20.4 31.1% of protein being recovered as IgG. Characterisation of monoclonal antibodies The methionine residues found within the N terminal sequence of PTH peptides are known to oxidise on storage (Tashijian et al., 1964) and therefore both oxidised (Met[O]-) and reduced
PTH peptides were used in the Mab characterisation experiments. Figs. 1-3 show the displacement curves obtained for 3B3, 4G3 and 6E3 Mabs with oxidised and reduced synthetic human PTH (1-34) and PTH (1-84) peptides. Greater than 60% of radioactivity in the peptide labels was bound by the Mab's under conditions of maximal binding. For the displacement curves, dilutions of the Mab's were selected to give approximately 20% of label counts bound in the absence of unlabelled peptide. For 3B3 and 6E3, the results confirmed the initial displacement analysis suggesting that both these Mabs recognised PTH (1-34) and PTH (184), essentially equipotently. However, Mab 4G3 showed no displacement with PTH (1-84) and
Assessment of Mab 3B3 with PTH peptides 30-
%Bound el ',..
.tit....--it...
20
.~
i
i "--'O--
1 84
Ox~ISed
I
1 34 OxidiSed
...... • ....
1 84 R e d u c e d
........ t i ......
1 34 R e d u c e d
10
0
........
i
........
i
10
........
100
PTH
i 1000
........
i ~ 0000
pmolll
Fig. 1. Displacement curves obtained with Mab 3B3 using oxidised and reduced PTH (1-34) and PTH (1-84) peptides. The Mab was diluted in 0.05 M barbitone buffer, p H 8.6, 0.5% BSA and incubated with radiolabelled P T H (1-34) and the relevant PTH peptide overnight at room temperature. Sheep a n t i - m o u s e / r a t IgG (on the solid phase) was then added. After incubation for 2 h with shaking, the assays were separated by centrifugation and washing ( × 2) in 0.9% s a l i n e / T w e e n (0.2%,
v/v).
163 appeared to be specific for P T H (1-34). This was c o n t r a r y to the initial displacement experiments (see above) and was most likely explained by the fact that the biological preparation of h u m a n P T H ( 1 - 8 4 ) ( M R C 7 5 / 5 4 9 ) also contained P T H ( 1 - 3 4 ) fragments. All three M a b s reacted preferentially with oxidised P T H peptides to differing degrees. This effect was most m a r k e d with 3B3 which showed a ten-fold preference for oxidised P T H peptides. Scatchard analysis of the data for oxidised P T H ( 1 - 3 4 ) peptide gave avidity constants of 5.9 x 109, 3.0 x 101° and 1.1 x 109 L / M for 3B3, 4G3 and 6E3, respectively.
30
Assessment of Mab 6E3 with PTH peptides 2o %Bound
15 . .A. . ... / "
""~.
10
Assessment of Mab 4G3 with PTH peptides 5 ¸
%Bound
....-" ~.
......
10
1o
.....
1o0
.....
,o'00 . . . .
;0000
PTH pmol/I
20
'\
Jt
1-34
!
1-34ieduced
'~i
1 84 Reduced ........ i lo
Fig. 3. Displacement curves obtained with Mab 6E3 using oxidised and reduced PTH (1-34) and PTH (1-84) peptides. The Mab was diluted in 0.05 M barbitone buffer, pH 8.6, 0.5% BSA and incubated with radiolabelled PTH (1-34) and the relevant PTH peptide overnight at room temperature. Sheep anti-mouse/rat IgG (on the solid phase) was added. After incubation for 2 h with shaking, the assays were separated by centrifugation and washing (x 2) in 0.9% saline/Tween (0.2%, v/v).
N o displacement was observed for any of M a b s with the P T H ( 1 - 1 0 ) peptide.
~
k"...=....-~R,,.~ ........ i ........ , loo ~ooo 1000o . . . . . . . .
i
PTH pmol/I
Fig. 2. Displacement curves obtained with Mab 4G3 using oxidised and reduced PTH (1-34) and PTH (1-84) peptides. The Mab was diluted in 0.05 M barbitone buffer, pH 8.6, 0.5% BSA and incubated with radiolabelled PTH (1-34) and the relevant PTH peptide overnight at room temperature. Sheep anti-mouse/rat IgG (on the solid phase) was added. After incubation for 2 h with shaking, the assays were separated by centrifugation and washing (x 2) in 0.9% saline/Tween (0.2%, v/v).
Application to two-site immunometric assays T w o of the antibodies, 3B3 and 6E3, were used in the development of two-site i m m u n o m e t r i c assays for P T H (1-84). Fig. 4 shows the standard curves for (a) radiolabelled 3B3 in c o m b i n a t i o n with a C terminal specific Mab, ESQ1 (produced by Dr. K. James, University of Edinburgh, and s u p p l i e d b y N o v o BioLabs, Cambridge, U . K . ) , and (b) 6E3 in c o m b i n a t i o n with an affinity purified C terminal specific polyclonal antibody,
164 C A G 3 (supplied by IDS, N e w c a s t l e - u p o n - T y n e , U.K.). Th es e assays h a v e been s h o w n to have the sensitivity to m e a s u r e P T H ( 1 - 8 4 ) in n o r m a l subj e c t s an d to be u n a f f e c t e d by excess C t e r m i n a l fr a gme n t s ( L o g u e et al., 1988; G r e i g et al., 1990). Fig. 5 shows the s t a n d a r d c u r v e for a two-site i m m u n o m e t r i c assay for P T H ( 1 - 3 4 ) using radiolabelled 3B3 in c o m b i n a t i o n with 4G3. T h e assay
A s s e s s m e n t of 3B3/4G3 combination ~n a two-site immunometric assay for PTH(1-34) 100
%Bound
10
Assessment of Mabs 3B3 and 6E3 in a two-site immunometric assay for PTH(1-84) 100 7
%Bound
10
PTH(1-34)
PTH(1-84)
01 I
3B3in comi~nationwithESQ1 •
01
J 1
........
i 1
........
6E3tncombinationwithCAG3
i
........
i
10
100
PTH(1-84)
pmol/I
........
i 1000
Fig. 4. PTH (1-84) standard curves in two-site immunometric assay, a: 3B3 in combination with C terminal specific Mab ESQ1. Radiolabelled 3B3 (100,000 cpm/tube) in 0.05 M phosphate buffer, pH 7.0, 0.5% BSA was incubated with PTH (1-84) peptide overnight at room temperature. ESQ1 (coupled to Sepharose solid phase) was added, incubated for 2 h with shaking, and the assays separated by centrifugation and washing (×2) in 0.9% saline/Tween 20 (0.2%, v/v). b: 6E3 in combination with the C terminal specific affinity purified polyclonal antibody CAG3. PTH (1-84) peptide in 0.05 M phosphate buffer, pH 7.4, 0.5% BSA, was incubated overnight at room temperature in tubes coated with 6E3. Radiolabelled CAG3 (200,000 cpm/tube) was added, Incubated for 2 h at room temperature, and the assays separated by aspiration and washing ( × 2) in 0.9% saline/Tween 20 (0.2%, v/v).
10
100
1000
pmol/I Fig. 5. PTH (1-34) and PTH (1-84) standard curves using 4G3 in combination with 3B3 in a two-site immunometric assay for PTH (1-34). PTH (1-34) or PTH (1-84) peptides in 0.05 M phosphate buffer, pH 7.4, 0.5% BSA, were incubated in tubes coated with 4G3. RadiolabeUed 3B3 (100,000 cpm/tube) was added, incubated for 2 h at room temperature, and the assays separated by aspiration and washing (x2) in 0.9% saline/Tween (0.2%, v/v). shows less than 0.5% c r o s s - r e a c t i o n with P T H ( 1 - 8 4 ) . It should be n o t e d f r o m b o t h Figs. 4 a n d 5 that, u n d e r the c o n d i t i o n s of assay, a p p r o x i m a t e l y 60% of the respective labelled M a b s are b o u n d at a P T H c o n c e n t r a t i o n of 500 p m o l / 1 with n o evid en ce of a h o o k effect.
Discussion A n t i b o d i e s to the biologically active N t e r m i n a l region ( 1 - 3 4 ) of P T H w h i ch h a v e the specificity
165 and avidity to be of use in the measurement of circulating PTH, without prior affinity purification, have proven difficult to produce. A previous attempt to obtain Mabs to N terminal PTH, using purified bovine PTH (1-84) as immunogen, produced only low avidity IgM antibodies and suggested that B A L B / c mice were poor responders to PTH immunogens (Nussbaum et al., 1981). In a more detailed study on the genetic control of the immune response to synthetic PTH (1-34) peptide in mice, Nussbaum et al. (1985) confirmed that B A L B / c mice were genetically poor responders to P T H (1-34). The immune response was found to be under the control of Ir genes and associated with the IA and IE subregions of the major histocompatibility complex of the mouse. The CBA mouse strain was predicted to be a high responder to PTH (1-34). The results of the immunisations in this study, with 6 / 6 DA rats and 2 / 2 sheep positive and all mice and Lou rats negative, are consistent with a species and strain specific response. However, we did not detect the predicted response in CBA mice. The methionine residues at positions 8 and 18 of P T H (1-34) have been shown to be susceptible to oxidation on storage under acid conditions (Tashijian et al., 1964) and during purification by H P L C (Bennett et al., 1981). It has also been shown that the spectrum of biological activities of P T H peptides is altered depending on the oxidation state of the peptide (Pang et al., 1983). Furthermore, polyclonal antisera to N terminal PTH have been found to be sensitive to the oxidation state of PTH (Tashijian et al., 1964). Recent studies using circular dichroism have confirmed that oxidation of the methionine residue, at position 8 in particular, produces substantial changes in the secondary structure of P T H (1-34). These changes alter the conformation of the PTH (1-34) peptide, resulting in a reduced affinity for receptors (Zull et al., 1990). The peptides used in this study were stored in the main under acid conditions and the Mabs produced showed a preference to various degrees for oxidised PTH (1-34) peptide. It is not possible to fully evaluate the effect of oxidation from the results of this study, but it is probable that the oxidation state affects both the immunogenicity of
PTH peptides and the ability of screening assays to detect an antibody response. We would recommend that, for PTH (1-34) and for other peptides containing methionine residues, the oxidation state be taken into consideration both when assessing the immunogenicity of a peptide and also in the design of antibody screening assays. Using spleen cells from the DA rats and the mouse myeloma line X63Ag8.653, five hybridoma lines secreting anti-PTH (1-34) antibodies were established. This is a low yield, representing 0.1% of the wells screened, compared to mouse-mouse fusions for highly immunogenic proteins, such as TSH, LH, FSH and AFP (Siddle and Soos, 1981; Stevenson et al., 1987), where positive rates of 70-80% can be achieved. However, it is consistent with the results obtained with mouse-mouse fusions for the poorly immunogenic peptide hormone A C T H (1-39) (White et al., 1985). Since the early work of Lerner et al. (1982) and Schmitz et aI. (1983) it is now well established that small peptide sequences can be used to produce antibodies to the intact protein of which they form a part. However, this approach frequently produces peptide specific antibodies which fail to react with the native molecule (Lerner, 1984). This study is no exception, with three of the five Mabs raised to synthetic PTH (1-34) failing to recognise the intact PTH (1-84) molecule. However, two of the Mabs, 3B3 and 6E3, have been shown to be of use in a two-site immunometric assay for PTH (1-84). A third, 4G3, in combination with 6E3, forms the basis of a two-site immunometric assay specific for PTH (1-34). The generation of useful Mabs to biologically active regions of PTH, using synthetic PTH (1-34) peptide as immunogen, is important, since in vivo PTH is not stored in large quantities within the parathyroid gland, and therefore the availability of human preparations of P T H for use as immunogen is limited.
Acknowledgements We are grateful to Dr. Angus Munro of the Scottish Antibody Production Unit for undertaking the sheep immunisations. We thank Novo BioLabs and IDS for the supply of ESQ1 and CAG3 antibodies respectively. We are grateful
166 also for the technical assistance of Steven Greig and Jacqueline McDade and the expert secretarial assistance of Myra Ogilvie.
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