1ournalo[hamunologicaIMethods. 146(1992) 33-42

33

© 1992 ElsevierScience Publishers B.V. All rights reserved 0022-1759/q2/$05.00

JIM 06151

Production and characterisation of monoclonal antibodies to the mid-region 37-67 sequence of parathyroid hormone-related protein W . A . Ratcliffe, S.J. B o w d e n , J. Emly, S. H u g h e s and J.G. Ratcliffe Wolfson Research Laboratories. Department of Clinical Chemistry. Queen Elizabeth Medical Centre. Birmingham, BI5 2TH. U.K.

(Received3 June 1991, revised received31 July 1991, accepted 28 August Iqql)

The production and characterisation of monoclonal antibodies (MAb) to the mid-region sequence 37-67 of human parathyroid hormone-related protein (PTHRP) is described. In spite of the poor immunogenicity of this sub-fragment of PTHRP, a high percentage of specific hybrids were produced by boosting with conjugate and free peptide prior to cell fusion. Seven of the MAbs produced cross-reacted with PTHRP37-67, P T H R P I - 8 6 and native forms of PTHRP. Inhibition studies with peptide sub-fragments of PTHRP37-67 indicated that the majority recognised the 45-59 region, in a R I A for PTHRP1-86, detection limits ranged from 0.17 to 0.9 ng P T H R P I - 8 6 / t u b e , and no cross-reaction was found with PTH1-84. Two MAbs 1DII and 4BI0 were shown to be of potential use in measuring P T H R P 1 - 8 6 in a two-site immunoradiometric assay in combination with either a solid phase consisting of a MAb to PTHRP1-34, or iodinated affinity purified rabbit antibodies to P T H R P I - 3 4 . MAb I D ! 1 coupled to Sepharose was suitable for immunoextraction of PTHRP, and successfully Iocali~d PTHRP on immunoblots. Two additional MAbs were produced which recognised an epitope unique to PTHRP37-67 located in the 37-46 region of the peptide. Key words: Monoclonalantibody; Parathyroid hormone-related protein; Two-site immunoradiometricassay; Western blots

Introduction

Parathyroid hormone-related protein (PTHRP), which was first isolated in 1987 (Moseley et al., 1987), shares homology at its NH2-terminus

Correspondence to: Dr. Wendy A. Ratcliffe, Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Birmingham BI5 2TH, U.K. Abbret'iations: PTH, parathyroid hormone; PTHRP, parathyroid hormone-related protein; MAb, monoclonalantibody; BSA, bovine serum albumin; PEG, polyethyleneglycol; IRMA, immunoradiometricassay; RIA, radioimmunoassay.

with that of PTH, allowing it to act via receptors on PTH target tissues such as bone and kidney (Abou-Samra et al., 1989). There is now overwhelming evidence that PTHRP is produced by a wide range of tumours and that it is an important mediator of humoral hypercalcaemia of malignancy (HHM) (Martin and Suva, 1989). PTHRP can reproduce in vivo and in vitro all the main biochemical and pathological features of HHM, including hypercalcaemia (Kukreja et al., 1988), increased bone resorption (Thompson et al., 1988), decreased calcium excretion, increased phosphate excretion and increased nephrogenous cyclic AMP (Ebeling et al., 1989). Aspects of the

structure, function, tissue expression and proposed pathophysiological roles of PTHRP have been covered in recent reviews (Orloff et al., 1989; Martin, 1990). The humoral role of PTHRP has been confirmed by measurement of increased concentrations of PTHRP in the plasma of patients with HHM. Although a direct radioimmunoassay (RIA) (Henderson et ai., 1990) and extraction RIAs for PTHRP1-34 (Budayr et al., 1989; Kao et ai., 1990) suggested increased PTHRP concentrations in a proportion of patients with HHM, plasma PTHRP1-86 concentrations measured by a highly sensitive two-site immunoradiometric assay (IRMA) were increased in more than 90% of patients with HHM (Ratcliffe et al., 1991a). Two IRMAs and an ELISA have been reported to date, and measure plasma 1-74, 1-86 or 1-87 immunoreactivity by combining compatible antibodies of NH2-terminal and mid-region specificity. In the IRMA reported by Burtis et al. (1990), an antiserum to PTHRP1-74 was affinity-purified to yield two subpopulations of antibodies of 1-36 and 37-74 specificity. The only antiserum produced using a mid-region fragment of PTHRP as immunogen, has been used indirectly labelled with iodinated PTHRP37-67 to quantitate the two-site IRMA for PTHRP1-86 (Ratcliffe et al., 1991a). The ELISA reported by Kramer et al. (1991) used a MAb to a mid-region sequence for antigen capture; however, the assay lacked the sensitivity required for assay of plasma. In this paper we describe the production, characterisation and potential immunodiagnostic and research applications of MAbs specific for the 37-67 region of PTHRP.

Material and methods PTHRP37-67 and subfragments of PTHRP for epitope mapping were prepared by Aita Bioscience (University of Birmingham, England) as described previously (Ratcliffe et al., 1990b). PTHRP1-86 was obtained from Bachem (Saffron Walden, England) and PTHRPI-141 (Hammonds et al., 1989) was a gift from Professor A.D. Care (University of Leeds, England). PTHI-84 was obtained from Scientific Marketing Associ-

ates (Barnet, England). Mouse antibody subclasses were determined by inhibition of agglutination (Sera-Lab, Crawley Down, England). Polyethylene glycol (PEG) for cell fusion was from Boehringer Mannheim (Lewes, England). Sources of materials for cell culture (Ratcliffe et al., 1990b), RIA of PTHRP1-34 (Ratcliffe et al., 1990a) and IRMA for PTHRP1-86 (Ratcliffe et al., 1991a) have been described previously. The Elite ABC anti-mouse immunoglobulin kit was from Vector Laboratories, Peterborough, England.

lmmunisation of mice and cell fusion The immunisation schedule used is given in Table I. In the primary immunisation a PTHRP 37-67-bovine serum albumin (BSA) conjugate (Ratcliffe et al., 1991a) was injected s.c. into 10 BALB/c mice in complete Freund's adjuvant: saline (2:1 v/v). Boost immunisations were in the presence of incomplete Freund's adjuvant, and i.v. injections were given in saline. Splenic lymphocytes were fused with NSO myeloma cells (Galfr~ and Milstein, 1981) at a ratio of 5 : 1 using 50% PEG 6000. The selection medium for hybridomas has been described previously (Ratcliffe et al., 1990b). Specific hybrids were detected by RIA, cloned at least twice by limiting dilution and grown as ascitic tumours in BALB/c mice.

Radioimmunoassay To determine the titre of antisera or detect the presence of hybrids secreting PTHRP antibodies, diluted mouse sera or culture supernatants (100 ttl) were incubated for at least 4 h at room temperature with m25I-PTHRP37-67 or t25I-PTHRP1-86 (15000 cpm) in assay diluent consisting of phosphate buffered saline (PBS) pH 7.4 containing 0.25% Polypep, 0.1% Triton X-100, and 0.01% sodium azide. Separation of the antibodybound fraction was by addition of sheep antimouse immunoglobulin and 5% PEG as described previously (Ratcliffe et al., 1990b). Titre was defined as the dilution of serum or culture medium required to bind 20-25% of the tracer in a R1A. In standard curves and cross-reactivity

70C 70C

30C 30C

25C i.v.

i.v. 50C 35P 50C 35P

i,p 25C 15P 25C 15P

25C i.p.

50C 25P 50C 25P

i.p.

3 25c s.c.

3 25c s.c. 25c i.v.

I IOC s.c.

IIOC s.c. 25C i.p.

3

2

Mice were immunised with PTHRP37-67 (P), or PTHRP37-67-BSA conjugate (C).

i.v.

25C i.p.

i.p.

3 25C s.c.

Number of boosts Quantity ofimmunogen (ttg) Route

Pre-fusion immunisations Route Quantity of immunogen (/zg) 4 days prior to fusion 3 days prior to fusion 2 days prior to fusion

110C s.c.

1

Fusion

Quantity of primary immunogen (#g) Route

lmmunisation schedule

IMMUNISATION SCHEDULE

TABLE 1

50C 25P 50C 25P

i.v.

25c i.v.

i.v.

25C i.p.

50C 25P 50C 25P 50C 25P 50C 25P 50C

i.p.

2 25c s.c.

IIOC s.c.

4

36 studies, standard PTHRP37-67 or PTHRPI-86 (0-10000 pg), 125I-PTHRP37-67 or 1251 PTHRP1-86 (15000 cpm) and PTHRP MAbs or antisera at an appropriate titre in a total volume of 500 p.I, were incubated for 16 h at 4°C. Separation was as described above. Cross-reactivity was calculated as the ratio of PTHRP37-67 or 1-86 (pmol/tube) and the cross-reacting substance (pmol/tube) expressed as a percentage, both giving 50% displacement of tracer. Avidity constants were calculated by the method of Miiller (1983).

Preparation of iodinated PTHRP37-67 and PTHRP1-86 PTHRP37-67 and PTHRP1-86 (2 /zg) were iodinated as described previously and purified by CI8 Sep-Pak cartridge (Ratcliffe et al., 1990b). The incorporation of 1.0 mCi of Nal~I was between 60 and 90% yielding tracers of 300-450 mCi/mg.

Affinity purification of PTHRP Affinity gels were prepared by coupling MAbs purified by Protein A to CNBr-activated Sepharose. Affinity chromatography using MAb 9D to PTHRP1-34 was used to purify native PTHRP for cross-reaction studies from conditioned media from the BEN lung cancer cell line (Moseley et al., 1987), human keratinocytes (Merendino et al., 1986) and human milk (Ratcliffe et al., 1990a). In these studies, PTHRP was assayed by RIA using a rabbit antiserum to PTHRP1-34, and the efficiency of extraction of PTHRP1-86 by MAbs 1Dll and 1A6 to PTHRP37-67 coupled to Sepharose, was quantitated by a RIA for PTHRP1-34 (Ratcliffe et al., 1990a).

Preparation of iodinated affinity purified antibodies to PTHRPI-34 Antiserum (code 3) raised in a rabbit to FITIRP1-34 (Ratcliffe et al., 1990a) was purified on a 2 ml affinity column consisting of 2 mg PTHRP1-34 coupled to CNBr-activated Sepharose. Specific antibodies were eluted in 0.2 M glycine HCI pH 2.5, and dialysed extensively against 0.05 M phosphate buffer pH 7.4. The antibodies (25 p.g) were iodinated in the presence of chloramine T (25 /zg) to a specific activity of

11.2 mCi/mg (Dobson et al., 1986) and purified by gel filtration on a column of Sephadex G-25.

lmmunoradiometric assays for PTHRP In the two-site IRMA described for PTHRP186, MAb ID5 to PTHRP1-34 coupled to cellulose is used for immunoextraction of the sample. The bound PTHRP is quantitated by addition of a rabbit antiserum to PTHRP37-67, which is in turn labelled at its second binding site by addition of ~z~I-PTHRP37-67 (Ratcliffe et al., 1991a). To test the compatibility of the MAbs produced to PTHRP37-67 with MAb 1D5, the rabbit antiserum to PTHRP37-67 was replaced by ascitic fluid containing MAb 1Dll or 4B10 diluted 1500-fold. In an alternative two-site IRMA to assess the suitability of MAbs to PTHRP37-67 for immunoextraction, MAb 1Dll was coupled to cellulose, and iodinated affinity purified antibodies to PTHRP1-34 were used as the label. Thus PTHRP1-86 (0-505 pmol/I, 200 /zl) was incubated for 16 h at 4°C with iodinated affinity purified antibodies to PTHRP1-34 (120000 cpm, 50 /zl). A solid phase suspension (100 /zl) was added consisting of MAb 1Dll or 4B10 coupled to particulate cellulose (Chapman and Ratcliffe, 1982), the tubes were shaken for 1 h, and the solid phase was successively washed twice with 2% Tween 20 in 0.9% NaCl (3 ml). Bound radioactivity was counted for 2 min in a model 1261 Multigamma counter (Pharmacia, Milton Keynes, England). The assay diluent consisted of PBS pH 7.4 containing 2% horse serum, 1% mouse serum, 0.1% Triton X-100, and 0.01% sodium azide.

Epitope mapping The binding sites of the MAbs to PTHRP37-67 were determined by RIA from the cross-reaction of synthetic subfragments of PTHRP (20 n g / tube). The peptides examined were PTHRP3241, 36-50, 37-46, 39-53, 42-51, 42-56, 45-54, 45-59, 47-56, 48-62, 50-59, 51-65, 52-61, 54-68 and 57-66.

Sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of PTHRP from milk PTHRP was extracted from bovine milk by an affinity gel consisting of MAb 9D to PTHRP1-34

TABLE II CHARACTERISTICS OF ANT1SERA FROM IMMUNISED MICE Mouse

Antiserum dilution

1251-PTHRP37-67 bound (%)

I 2 3 4

I/300 l/500 1/300 I/400

20 24 10 28

Detection limit in PTHRP37-67 RIA (rig/tube) 0.4 0.7 0.05 0.8

I~SI-PTHRP1-86 bound (%) 0.1 0.8

0.8 |.3

Specific binding of the tracers 12SI-PTHRP37-67and I"-SI-PTHRPI-86(15000 cpm) was determined as described in the text. The detection limit in the PTHRP37-67 R1A was defined as the mass/tube of PTHRP37-67 corresponding to 95% of specificbinding.

coupled to Sepharose as described previously (Ratcliffe et al., 1990a). P T H R P was further purified by Cla Sep-Pak cartridge, and eluted from the cartridge in 60% acetonitrile in 0.1% trifluoroacetic acid. Following S D S - P A G E and immunoblotting onto 0.2/~w nitrocellulose, P T H R P was Iocalised by incubation with ascitic fluid diluted 500-fold c o n t a i n i n g M A b 5 D l l to P T H R P 1 - 3 4 or M A b 1 D l l to P T H R P 3 7 - 6 7 . P T H R P was detected with the Elite A B C biotinylated anti-mouse immunoglobulin kit, and diaminobenzidine-nickel chloride was used as a substrate for peroxidase.

Results

O f ten B A L B / c mice i m m u n i s e d with P T H R P 3 7 - 6 7 - B S A conjugate, four responded by producing low titres of antisera which bound between 10 and 28% of the P T H R P 3 7 - 6 7 tracer. Binding to the P T H R P I - 8 6 tracer was in the

region of 1% (Table II). W h e n these antisera were used in RIAs for P T H R P 3 7 - 6 7 the detection limits of the standard curves ranged from 0.05 to 0.8 n g / t u b e . The cross-reactivities of the antisera with native forms of P T H R P were studied using P T H R P affinity purified from conditioned media from BEN cells and keratinocytes. Partial inhibition of binding, non-parallel to standard PTHRP37-67, was obtained in all cases, suggesting lack of immunological identity between the standard and native PTHRP. Four cell fusions were carried out and all except fusion 2 produced specific hybrids. Fusions 1 and 3 produced one and four specific hybrids respectively. All antibodies were of low titre a n d low avidity, a n d b o u n d e i t h e r P T H R P 3 7 - 6 7 tracer alone or P T H R P I - 8 6 tracer alone. Fusion 4 produced 237 positive wells from 376 wells screened (Table l i d of which 72 bound both P T H R P 1 - 8 6 and 37-67 tracers; the remainder bound P T H R P 3 7 - 6 7 tracer alone. Preliminary cross-reaction studies carried out on these

TABLE !II YIELD OF HYBRIDS FROM FUSION 4 WHICH SECRETED SPECIFIC ANTIBODIES TO PTHRP Tracer

Number of wells screened

Positivewells detected 400-2,000 cpm 2,000-5,000 cpm

125I-PTHRP37-67 1251-PTHRPI-86

376 376

126 48

63 16

> 5,000 cpm

Total

48 8

237 72

Hybrids secreting specific antibodies te PTHRP were detected by binding to tracers 12-Sl-PTHRP37-67 and '~I-PTHRPI-86 (15000 cpm/tube). Non-specific binding was approximately 200 cpm/tube, All antibodies which bound I!~I-PTHRPI-86, also bound 1251-PTHRP37-67.

38 s u p e r n a t a n t s indicated t h a t t h e a n t i b o d i e s w h i c h b o u n d b o t h 1 - 8 6 a n d 3 7 - 6 7 tracers also cross-rea c t e d with P T H R P derived f r o m h u m a n milk a n d c o n d i t i o n e d m e d i u m f r o m B E N cells. In contrast, t h o s e a n t i b o d i e s w h i c h b o u n d only t h e 3 7 - 6 7 tracer, failed to cross-react with P T H R P 1 - 8 6 a n d native f o r m s o f P T H R P . B a s e d o n this i n f o r m a tion s e l e c t e d h y b r i d o m a s w e r e cloned, a n d yielded seven MAbs which bound the PTHRP1-86 tracer a n d two w h i c h b o u n d only t h e PTW-tRP37-67 tracer. T h e characteristics o f t h e M A b s are s h o w n in T a b l e IV. T h e two M A b s o f h i g h e s t avidity ( 1 D l l a n d 4B10) gave d e t e c t i o n limits o f 0.17 a n d 0.2 n g / t u b e o f P T H R P t - 8 6 in a R I A . T h e b i n d i n g sites r e c o g n i s e d by t h e M A b s w e r e d e t e r m i n e d by s t u d y i n g t h e cross-reactivity o f synthetic s u b f r a g m e n t s o f P T H R P 3 7 - 6 7 . In a P T H R P 1 - 8 6 R I A , M A b s 1A6, 1 D l l , 2E12, 3B3, 4 H 9 a n d 4B10 c r o s s - r e a c t e d with P T H R P 4 5 - 5 9 a n d to a lesser e x t e n t P T H R P 4 8 - 6 2 , w h e r e a s M A b 2B3 cross-reacted with PTHRP54-68. In a R I A f o r PTHRP37-67, MAbs 2H7 and 4G7 cross-reacted with P T H R P 3 7 - 4 6 only. T h e cross-reactivity o f s e l e c t e d M A b s with n a tive P T H R P w a s e x a m i n e d (Fig. 1). A n t i b o d i e s 4B10 a n d 1DI1 c r o s s - r e a c t e d 7 0 % a n d 5 8 % respectively with P T H R P 3 7 - 6 7 , a n d b o t h cross-rea c t e d 8 5 % with P T H R P I - 1 4 1 ( n o t shown). B o t h

10

% TRACER BOUND

5

1

10

100

PTHRP 1 - 8 6 nMOLIL

Fig. 1. RIA standard curve for PTHRPI-86 (e

e) using

MAb 4BI0. Cross-reaction is shown with PTHRP37-67 (o o) and PTHRP affinity purified from conditioned medium from the BEN lung cancer cell line ([7 []) and human milk ( A ,x ).

M A b s failed to cross-react with P T H I - 8 4 a n d P T H R P 1 - 3 4 at c o n c e n t r a t i o n s u p to 100 n g / t u b e . Native P T H R P purified f r o m h u m a n milk a n d c o n d i t i o n e d m e d i u m f r o m B E N cells cross-related in t h e R I A , a l t h o u g h P T H R P f r o m B E N cells d i l u t e d n o n - p a r a l l e l to s t a n d a r d P T H R P 1 86. In contast, a n t i b o d i e s 2 H 7 a n d 4 G 7 s h o w e d n o cross-reaction with P T H R P 1 - 8 6 o r P T H R P

TABLE 1V CHARACTERISTICS OF MONOCLONAL ANTIBODIES TO PTHRP37-67 Hybrid

IA6 1DI I 2B3 2E12 3B3 4H9 4B! 0 2H7 4G7

lsotype

IgM lgG1 lgG3 IgG3 IgGl lgGl IgG 1 lgGl lgG1

Titre '~ Culture fluid

Ascitic fluid

Detection limit h in RIA for PTHRP 1 - 8 6 ng/tube

Affinity constant Ka (L/M)

I : 300 1:300 1 : 10 1 : 150 1 : 100 1:40 1:500 1:45 c 1:30 ¢

1 : 25,000 1:600,000 ND 1 : ],200 1 : 125,000 1:3,000 1:50,000 ND ND

0.25 0.17 0.70 0.80 0.90 0.30 0.20 1.0 ¢ 1.4 ¢

1.3 x 109 5.7× 109 8.9 × 10v !.7 × 109 1.2 × 10~ 4.2 × 10s 6.2 x 109 1.6X l0 s 1.8× l0 s

a Titre is defined as the dilution binding 20-25% of tracer in a RIA. b Minimum detection limit is defined as the mass of PTHRPI-86/tube corresponding to 95% of specific binding of tracer. c Titre and detection limit in a RIA for PTHRP37-67. ND, not done.

toss

-56

COUNTS BOUNO

soo¢

.-----

,

lO

los

-

----"--

1sos

~-.-~

PTHRP 1-86 PMOL/L

Fig. 2. Standard curves for PTHRPI-86 by two-site immunoradiometric assay. Either rabbit anti-PTHRP37-67 (e e) or MAb IDll (o o) MAb 4BI0 (ll O) were used indirectly label led with tX~lPTHRP37-67 in combination with MAb 1D5 to PTHRPI-34 coupled to cellulose (see text).

affinity purified from conditioned medium from BEN cells. Antibodies 1 D l l and 1A6 coupled to Sepharose extracted more t h a n 95% of P T H R P 1 - 8 6 which had been added to culture fluid. Ouantitation of the extraction efficiency was by a R I A for PTHRP1-34. MAbs 1 D l l and 4B10 successfully replaced the rabbit antiserum to P T H R P 3 7 - 6 7 in the two-site I R M A for P T H R P I - 8 6 (Fig. 2), indicating t h e i r compatibility with the M A b to

10oo0 COUNTS BOUND

-...-.

~

A.

-29

-19 -15

~

-10

B,

Fig. 4. lmmunoblotting of PTHRP affinity extracted from bovine milk. PTHkP was Iocalised in A with a MAb to PTHRPI-34, and in B with a MAb to PTHRP37-67. The sample in the left hand lane in each case is PTHRPI-86, and in the right hand lane is PTHRP extracted from bovine milk.

P T H R P 1 - 3 4 coupled to the solid phase. In the alternative two-site assay, M A b 1 D l l coupled to cellulose as the solid phase was compatible with iodinated affinity-purified antibodies to F I T I R P 1-34 as the tracer (Fig. 3). Following S D S - P A G E and immunoblotting, P T H R P affinity purified from bovine milk was iocalised using MAbs to P T H R P 1 - 3 4 a n d 37-67 (Fig. 4). In each case major components of 29, and 19 kDa were identified, together with minor components of 56, 15 and 10 kDa. lmmunoblotling of P T H R P 1 - 8 6 and 1-141 (not shown) gave single bands of 10 and 22 kDa respectively.

Discussion o

i l

11o

i lOS

i lOSS

PTHRP 1-116 PMOLll.

Fig. 3. Standard curve for PTHRPl-86 by a two-site immunoradiometric assays. MAb IDII (e o) coupled to cellulose was used in combination with iodinated affinity purified antiserum to PTHRPI-34 (see text).

The MAbs to P T H R P 3 7 - 6 7 were produced using synthetic subfragments corresponding to a mid-region sequence which shows no homology with PTH. In our experience, mid-region fragments 37-67 and 67-86 coupled to carrier pro-

4o teins are both poorly immunogenic, which may in part reflect conservation of the primary structure between species (Yasuda et al., !989). The method of conjugation and the carrier l~totein are also important facters in determining immunogenicity, since studies with PTI-IRP37-67 coupled to BSA using carbodiimide, or coupled to thyroglobulin using glutaraidehyde, failed to produce a response in BALB/c mice. The PTHRP37-67-BSA conjugate prepared using het,~robifunctionai reagents, and used as immunogen in the present study, was immunogenic in rabbits and produced antisera of high titre and high avidity (Ratcliffe et al., 1991a). However, antisera from two out of five rabbits failed to cross-react with native PTHRP, a phenomenon not uncommon when ~ynthetic subfragments are used for the generation of antibodies to a parent molecule. In a recent study only two out of five MAbs produced to PTH1-34 cross-reacted with PTH1-84 (Logue et al., 1991). 30% of antibodies from positive wells from fusion 4 cross-reacted with PTHRP37-67, the larger subfragment PTHRP1-86 and a common epitope on native PTHRP. Epitope mapping located the binding sites of the MAbs in the region of residues 45-59. The remaining antibodies cross-reacted with epitopes unique to fragment 37-67, and in the case of MAbs 2H7 and 4G7 these were mapped to the 37-46 region of the peptide. The primary structure of the 45-59 region is as follows: ASN SER LYS PRO SER PRO ASN THR LYS ASN HIS PRO VAL ARG PHE. It is of interest that the only other MAbs to PTHRP with similar mid-region specificity reported to date were produced after immunisation with synthetic peptides and recombinant PTHRP19-87 and 1-141 (Kramer et al., 1991). Since the majority of these MAbs cross-reacted with PTHRP38-64, this confirms the presence of important immunogenic sequence(s) in this region suitable for the generation of antibodies cross-reacting with native PTHRP. In general the efficiency of production of specific hybrids to PTHRP37-67 was low; indeed, prior to the studies described in this paper, we performed eight similar fusions using mice immunised with the same conjugate (but omitting prefusion boosts with PTHRP37-67) and failed to

produce specific hybrids with usefld characteristics. It is accepted that high doses of antigen are desirable in the final immunisation before fusion (St.~hli et al., 1983). Immunisation with conjugate and free peptide prior to fusion may have been an important factor in generating MAbs to PTHRP1-34 (Ratcliffe et al., 1990b) and the same approach appears to have been successful in the present study. In view of the extremely low titre of the mice antisera to PTHRP37-67 and particularly to PTHRP1-86, the high frequency of specific hybridomas produced in fusion 4 may reflect an increase in the number of antigen specific B-lymphocyte blast cells at the time of fusion (Stiihli et al., 1983). The MAbs produced to PTHRP37-67 can be used in RIAs, although none exceed the avidity of the best rabbit antiserum to PTHRP37-67 which detects approximately 20 pg/tube (Ratcliffe et al., 1991b). The characteristics of the MAbs to PTHRP38-64 produced by Kramer et al. (1991) were not reported. In a RIA using MAb 4B10, PTHRP from human milk diluted approximately in parallel to standard PTHRP186. PTHRP from human milk has been shown by gel filtration and immunoblotting to contain a wide range of molecular forms of PTHRP (Ratcliffe et al., 1990a). In contrast, PTHRP from BEN cells which contains major species of 23 and 17 kDa as shown by immunoblotting (Hammonds et al., 1989), diluted non-parallel to standard. MAb 4BI0 and 1Dll were both satisfactory when they substituted in the IRMA for a rabbit antiserum labelled at its second binding site by 1251PTHRP37-67, thus demonstrating their compatibility with the MAb 1D5 to PTHRP1-34 coupled to solid phase. The lower detection limit of the IRMA standard curve with the rabbit antiserum may reflect the superior avidity of the antiserum to that of the MAbs. MAb 1Dll coupled to a solid phase, quantitatively extracted PTHRP1-86, and when paired with iodinated affinity purified antibodies to PTHRP1-34 provided a sensitive two-site IRMA. Both IRMAs are at least 30-fold more sensitive than the ELISA using a MAb to PTHRP38-64 for antigen capture (Kramer et al., 1991). On immunoblotting of PTHRP from bovine milk, MAb 1Dll to PTHRP37-67 and MAb 5 D l l

to P T H R P 1 - 3 4 both identified c o m p o n e n t s with a p p a r e n t molecular weights similar to those rep o r t e d previously (Ratcliffe et al., 1990a). This confirms the p r e s e n c e o f mid-region binding sites in all P T H R P species purified from bovine milk using a M A b of NH2-terminal specificity. In conclusion, these M A b s to P T H R P 3 7 - 6 7 are likely to provide valuable reagents, b o t h for i m m u n o e x t r a c t i o n and immunolocalisation of P T H R P , and for d e v e l o p m e n t of highly sensitive two-site I R M A s for those molecular forms of P T H R P in p l a s m a which provide good diagnostic discrimination in malignancy-associated hypercalcaemia.

Acknowledgements We thank the D e p a r t m e n t of Health for financial s u p p o r t , Ms. G. Ubhi for secretarial assistance, and Dr. J. Fox for p r e p a r a t i o n o f synthetic peptides.

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Production and characterisation of monoclonal antibodies to the mid-region 37-67 sequence of parathyroid hormone-related protein.

The production and characterisation of monoclonal antibodies (MAb) to the mid-region sequence 37-67 of human parathyroid hormone-related protein (PTHR...
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