A radioimmunoassay for bradykinin based on monoclonal antibodies

Journal of Neuroimmunology, 23 (1989) 179-185

179

Elsevier JNI 00794

A radioimmunoassay for bradykinin based on monoclonal antibodies Elsa Phillips a n d M i c h a e l W e b b Sandoz Institute for Medical Research, London WC1E 6BN, U.K.

(Received25 July 1988) (Revised, received12 December1988) (Accepted 12 December1988)

Key words: BradykinJn; Radioimmunoassay; Monoclonalantibody

Summary We describe two monoclonal antibodies which recognise the peptides bradykinin and kallidin. These antibodies were used in a study to assess the feasibility of using monoclonal antibodies in place of conventional sera for radioimmunoassay. Antibody SBK1 recognises bradykinin with a K d of 0.67 _ 0.17 nM. It fails to recognise bradykinin from which any carboxyl terminal amino acid has been removed. Antibody SBK2 recognises bradykinin with a K d of 58.11_ 7.55 nM; it recognises 1-8 and 1-7 bradykinin about 30% as effectively as the full-length sequence, and shorter carboxyl terminal truncated sequences with a progressively declining efficiency. In this model system, bradykinin concentrations of 0.2 nM could be reliably measured, and carboxyl truncated versions of the peptide could be distinguished from the parent molecule. The feasibility of using monoclonal antibodies to assay bradykinin and other small peptides is discussed in the light of these results.

Introduction

Kinins are peptide mediators whose involvement in a large number of physiological processes and pathological conditions is known or suspected (general references in Erdos, 1979). Bradykinin (NH 2-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-ArgCOOH) and kallidin (N-lysyl-bradykinin) are produced from high molecular weight precursors by the action of proteases known as soluble or glan-

Address for correspondence: Michael Webb, Sandoz Institute for Medical Research, 5 Gower Place, London WC1E 6BN, U.K.

dular kallikreins respectively. These enzymes are activated at sites of tissue damage and inflammation, and the kinins released by their action mediate a variety of effects including vasodilatation, an increase in vascular permeability, and a direct stimulation of primary nociceptive afferent neurons (Baccaglini and Hogan, 1983). Bradykinin is rapidly metabolised by a variety of peptidase enzymes. Kininase 1, which is present in most tissues, cleaves the carboxy terminal arginine to yield des-Argg-bradykinin; this ligand is inactive at 'B2'-receptors, which are responsible for mediating the best understood actions of the kinins. It has been claimed that this peptide is active at a distinct class of bradykinin receptors,

0165-5728/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (BiomedicalDivision)

180

referred to by Regoli and co-workers as 'Bl'-receptors (Regoli et al., 1977). Kininase 2 (angiotensin converting enzyme), which is richest in the lung and only present in relatively lower quantities in blood, plasma and the membranes of vascular epithelial cells, removes the terminal dipeptide from the carboxyl terminus to leave 1 - 7 bradykinin which is biologically inactive. These initial products are rapidly metabolised further by a variety of different enzymes, and the pattern and sequence of appearance of the various products vary in different organs. In addition to these enzymes which degrade bradykinin from its carboxyl terminus, aminopeptidase activity present predominantly in lung rapidly degrades the peptide from its amino terminus. Because of the importance of this system in inflammatory and other pathological conditions, it is frequently necessary to measure the concentrations of kinins and their metabolites in fluids such as blood and inflammatory exudate. Radioimmunoassay has been employed for this purpose in many studies. These include the investigation of the kallikrein/kinin system in hypertension (Odya et al, 1983; Ishida et al., 1986), in the allergic response to airborne allergen (Proud et al., 1983), and in models of allergic encephalomyelitis (Germain et al., 1986, 1988). Radioimmunoassay has also been employed to demonstrate and quantify the presence of kinins in the central nervous system (Kariya et al., 1985; Yamauchi et al., 1985). Recently, Bonner et al. (1987) drew attention to the very wide range of normal kinin blood concentrations reported. These authors used radioimmunoassay employing four different polyclonal antisera to measure the kinin concentration in aliquots of the same blood sample. They found values of 7.4, 5.6, 127.5 and 693.0 ng/1 with these different antisera, and concluded that the antibodies used could be a major factor in determining the range of the measured kinin concentration. The advantages of a standardised and well-characterised antibody for use in such studies are clear. Monoclonal antibodies offer the possibility of obtaining unlimited quantities of an unvarying reagent which would allow the standardisation of radioimmunoassay between laboratories and over time. These possibilities have not yet been exploited in spite of the clear technical improve-

ment offered by these reagents. We report here a study designed to assess the feasibility of bradykinin radioimmunoassay based on monoclonal antibodies.

Materials and methods

Preparation of immunogen and immunisation Bradykinin and des-Arg9-bradykinin were obtained from Cambridge Research Biochemicals, Harston, Cambridge, U.K. [3H]Bradykinin was obtained from New England Nuclear, Stevenage, Herts, U.K., and 12SIodine was from Amersham International, Amersham, Bucks, U.K. All other reagents were from the Sigma Chemical Co., Poole, Dorset, U.K. 20 mg of bradykinin and 100 mg of bovine thyroglobulin were dissolved in 7.5 ml of phosphate-buffered saline (PBS) (0.15 M sodium chloride, 20 m M sodium phosphate) and 5 /,1 of [3H]bradykinin were added as tracer. Glutaraldehyde was added to a final concentration of 0.002% ( v / v ) and the mixture was agitated at room temperature for 30 min. After the removal of samples for scintillation counting, the mixture was dialysed against PBS (three changes of 21) at 4 ° C for 18 h. Determination of the amount of [3H]bradykinin incorporated into non-dialysable material showed that approximately 46 mol of bradykinin were conjugated per mol of thyroglobulin. This preparation was aliquoted and stored at - 3 0 ° C until use.

A group of B A L B / c mice was immunised with 100 t*1 (about 500 t'g) of a 1 : 1 emulsion of the peptide conjugate and Freund's complete adjuvant. This subcutaneous injection was repeated 2 weeks later, and was followed by a series of subcutaneous injections of peptide emulsified in Freund's incomplete adjuvant. Mice with high titres of serum anti-bradykinin antibodies were selected as spleen cell donors, and received an intravenous injection of 400 ~g of peptide conjugate 3 days prior to fusion.

ELISA A solid-phase enzyme-linked immunosorbent assay (ELISA) was used to determine the titre of anti-bradykinin antibodies in the serum of

181 immunised mice. The same assay was used to screen for hybridomas producing anti-bradykinin antibodies. 100/~1 of 1 mM bradykinin in 0.1 M NaHCO3, pH 9.0, were added per well of 96-well flexible microtitre plates (Falcon 3912). The plates were washed with PBS after incubation at 4 ° C for 2-4 h, and residual binding sites on the plastic were blocked by incubation at room temperature for 60 rain with 3% bovine serum albumin (BSA) in PBS. Samples were diluted in 3% BSA in PBS containing bacitracin (0.1 mM) and enalapril (0.5 mM) and 50/~1 were incubated for 1-2 h at 4 ° C. After washing in PBS to remove unbound antibody, bound mouse immunoglobulin was detected by a second incubation for 60 rain in 1/1000 diluted peroxidase-coupled sheep anti-mouse F(ab') 2 (Amersham). Bound antibody was revealed by the addition of 0.4% (w/v) o-phenylene diamine, 0.02% (v/v) hydrogen peroxide in 24 mM citric acid, 51 mM sodium phosphate buffer pH 5.0. Colour development was quantitated with a Titertek plate scanner (Flow Laboratories). As a control on non-specific binding of mouse antibodies, some assays were carried out in parallel with plates in which the samples were made 2 mM with respect to bradykinin.

Production of monoclonal antibodies. 2 × 108 spleen cells from mice with high titres of serum anti-bradykinin antibody were fused with 2 x 107 NS0 myeloma cells using 50% (v/v) polyethylene glycol by standard methods (Kohler and Milstein, 1975). The products of one fusion were resuspended in Dulbecco's modified Eagle's medium containing hypoxanthine, aminopterin and thymidine, and plated in five 96-weU tissue culture plates with the addition of 108 supplementary mouse splenocytes as feeders. The medium was changed at 4-day intervals, and the wells were screened for the presence of hybridomas secreting anti-bradykinin antibody on the 12th day after fusion. Antibody-secreting hybridomas were cloned twice by limiting dilution. Ascitic fluid was produced by the injection of 5 × 106 cells into the peritoneal cavity of BALB/c mice which had been previously primed by two 0.25 ml injections of pristane (2,4,6,10-tetramethylpentadecane). Hybridomas were stored frozen in liquid nitrogen. Antibodies from clones 2-3-3/3 and 2-42-3/1 were

selected for further study, and were named SBK1 and SBK2 respectively.

Bradykinin radioimmunoassay procedure Radioimmunoassay for bradykinin was carried out in PBS. Monoclonal antibodies and tracer were diluted in PBS containing 0.15% (w/v) BSA. Each tube received 100 #1 of buffer, bradykinin standard or unknown, 100 ~1 of appropriately diluted monoclonal antibody (1/120000 for SBK1; 1/2000 for SBK2; see Results), and 100/xl [3H]bradykinin, about 10000 cpm (radioactive concentration approximately 100 Ci/mmol). In some experiments, [125I]Tyr-kallidin (iodinated by the method of Hunter and Greenwood (1962) and purified on high-performance liquid chromatography (HPLC) by Dr. C.R. Snell, Sandoz Institute) was used as ligand in place of [3H]bradykinin. In this case, 10 000 cpm, about 2.5 fmol, were added per tube (radioactive concentrations were between 1500 and 2000 Ci/mmol). After vortex mixing the components in LP3 (Luckham) tubes, the tubes were left at 4 °C overnight. Bound ligand was separated from free by the addition of 100 /~l per tube of a commercial anti-mouse antibody attached to cellulose (SacCel, obtained from Immunodiagnostics, Washington, U.K., supplied as a slurry and used without further treatment). The tubes were vortexed briefly, and allowed to stand at 4°C for 30 min. 2 ml of ice-cold PBS were added per tube, and the tubes were centrifuged at 2700 × g for 30 rain. The supernatants were removed and the pellets were resuspended in 500 /tl of PBS. 400 #l were removed for scintillation counting in a Beckman fl-counter equipped with on-line correction for counting efficiency. All samples were assayed in duplicate, and all experiments included blank controls lacking first antibody or bradykinin respectively.

Data analysis All data were analysed on a VAX mainframe computer. The K d of both anti-bradykinin antibodies was determined by the use of the curve-fitting program in RS1. The relative reactivities of bradykinin-related peptides were calculated by assigning 100% reactivity t o bradykinin and apply-

182 ing the formula: moles of reference antigen to give 50% inhibition x 100% moles of test antigen to give 50% inhibition

Results

Time course for antibody association P r e l i m i n a r y t i t r a t i o n e x p e r i m e n t s with b o t h antibodies showed t h a t SBK1 (ascitic fluid) b o u n d a p p r o x i m a t e l y 25% of the tracer at a d i l u t i o n of 1 / 1 2 0 0 0 0 in the a b s e n c e of c o m p e t i n g u n l a b e l l e d b r a d y k i n i n . S B K 2 b o u n d a b o u t 25% of the a d d e d tracer at a d i l u t i o n of 1 / 2 0 0 0 ascitic fluid. T h e s e c o n c e n t r a t i o n s were then used r o u t i n e l y in subseq u e n t experiments. W e have, however, o b s e r v e d t h a t the o p t i m a l d i l u t i o n of b o t h a n t i b o d i e s m a y v a r y with different b a t c h e s of c o m m e r c i a l [3H]b r a d y k i n i n . W e w o u l d therefore expect to c a r r y o u t a p r e l i m i n a r y t i t r a t i o n e x p e r i m e n t on each fresh b a t c h of [3H]bradykinin. I n practice, this is u n l i k e l y to cause inconvenience, as we have f o u n d [ 3 H ] b r a d y k i n i n as s u p p l i e d b y N e w E n g l a n d N u c l e a r to b e stable for m o n t h s at - 30 ° C. A t the s t a t e d c o n c e n t r a t i o n of each a n t i b o d y , the a s s o c i a t i o n b e t w e e n l i g a n d a n d a n t i b o d y was c o m p l e t e b y 6 h at 4 ° C (Fig. 1). T h e s e c o n d r e a g e n t c o u l d b e a d d e d at this stage, b u t for

A

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CONCENTRATION

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15

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I I 1 O0 10 Bk CONCENTRATION

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Fig. 2. Quantitative displacement of [3H]bradykinin (Bk) by unlabelled Bk from SBK1 (top panel) and SBK2 (lower panel). The curves were fitted by the use of the curve-fitting program in RS1 to experimental data obtained as described. The data yielded mean K d values of 0.67+0.17 nM for SBK1 and 58.11 + 7.55 nM for SBK2. Note different scale of X-axis in the two figures.

~30

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r e a s o n s of c o n v e n i e n c e we r o u t i n e l y i n c u b a t e d the tubes o v e r n i g h t p r i o r to the a d d i t i o n of the SacCel. 6 TIME

2i4

(HOURS)

Fig. 1. Time course of [3H]bradykinin binding by SBK1 (squares, 1/120000) and SBK2 (circles, 1/2000). Mean and standard deviation of three determinations. The X-axis shows the amount of [3H]bradykinin (Bk) bound at different time points as a percentage of total [3H]Bk present.

Affinity of SBK1 and SBK2 and bradykinin The affinity of both anti-bradykinin antibodies for b r a d y k i n i n was e s t i m a t e d b y a n a l y s i n g q u a n t i tative d i s p l a c e m e n t d a t a using the curve-fitting p r o g r a m in R S 1 . T h e s e e x p e r i m e n t s y i e l d e d K d

183

° ~ 3(: O Z

24

~

SBK1

SBK2

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o

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2

o-1

1.o lo Bk CONCENTRATION

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7

1

2

3

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(nM)

Fig. 3. Displacement of [3H]bradykinin (Bk) from SBK1 by 1-7 Bk (squares, curve 1), 1-8 Bk (circles, curve 2) and Bk (triangles, curve 3).

values of 0.67 + 0.17 nM for SBK1, and 58.11 + 7.55 nM for SBK2 (Fig. 2).

Specificity of anti-bradykinin antibodies We examined the ability of both monoclonal antibodies to recognise derivatives of bradykinin in which the amino acid sequence was shortened from the carboxyl terminus (Fig. 3). SBK1 failed to recognise 1-8 bradykinin (des-Argg-bradykinin) even at concentrations of 1/~M; this concentration is over 1000-fold greater than that required to inhibit 50% of the tracer binding with bradykinin and at least 10-fold greater than the concentration of bradykinin at which 100% displacement of the tracer is seen. In contrast, SBK2 recognised both 1-7 and 1-8 bradykinin at about 30% of the efficiency with which it recognised the full-length

5--

4

Fig. 5. Effect of dynorphin A (columns 2), dynorphin B (3), enkephalin (4), somatostatin (5), or substance P (6), all at 1 /~M, on the binding of [3H]bradykinin (Bk) by SBK1 and SBK2. Columns 1: control; colunms 7: arginine, 1 raM.

3

sequence (Fig. 4). Removal of further amino acids from the carboxyl terminus resulted in a progressive decrease in the capacity of SBK2 to recognise the peptide, although even the 1-5 peptide was recognised to some degree. We tested the ability of both antibodies to recognise several other peptides at a concentration of 1 ~M (Fig. 5). Dynorphin A, dynorphin B, enkephalin, somatostatin and substance P were not significantly recognised by either antibody. In view of the absolute requirement for the C-terminal arginine for recognition by SBK1, we considered the possibility that the antibody was specific for this amino acid, in which case it would presumably have acted as a hapten in the initial immunisation. Free arginine at 1 mM was without

25

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~IO 5

o'.,

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i

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o.,

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CONCENTRATION ( n M )

Fig. 4. Displacement of [3H]bradykinin (Bk) from SBK2 by Bk (triangles, curve 1), 1-8 Bk (diamonds, curve 2) and 1-7 Bk (squares, curve 3).

,~,

,'o

,~oo

CONCENTRATION ( rim )

Fig. 6. Displacement of [3H]bradykinin (Bk) from SBK1 and SBK2 by Bk or kallidin. Curves 1 and 2, displacement from SBK1 by Bk (circles, curve 1) and kallidin (triangles, curve 2). Curves 3 and 4, displacement from SBK2 by Bk (diamonds, curve 3) and kallidin (squares, curve 4).

184

effect on the recognition of bradykinin by either antibody (Fig. 5). Thus the antigenic determinant recognised by SBK1 must consist of the amino terminal arginine in the context of the carboxyl terminal region of the bradykinin sequence. In the absence of this context, arginine is unrecognised by the antibody. Both antibodies were tested for their ability to recognise kallidin (N-lysyl-bradykinin). SBK1 recognised this peptide as effectively as bradykinin, while SBK2 recognised kallidin about 46% as effectively as bradykinin (Fig. 6).

Discussion

We have presented data on two monoclonal antibodies which recognise bradykinin. SBK1 has a K d for bradykinin of less than 1 nM, and in addition is able to recognise only the pharmacologically active peptides bradykinin and kallidin. It is unable to recognise any of the breakdown products commonly produced from bradykinin by peptidase action at the carboxyl terminus. In contrast, SBK2 is able to recognise 1-8 bradykinin with about 30% of the efficiency with which it recognises the full-length sequence. This shortened version of bradykinin is produced from the parent peptide by the action of kininase 1, and has been claimed to be active at a distinct class of bradykinin receptors (Regoli and Barabe, 1980). 1-8 bradykinin is, however, inactive at the 'B2'-receptors about which most is known, and which are probably responsible for most of the commonly observed effects of bradykinin including the stimulation of pain sensory neurons. The sensitivity of a binding assay such as a radioimmunoassay is determined by the affinity of the antibodies employed. In the majority of published radioimmunoassays, conventional polyclonal sera have been used; as these are mixtures of many different components, it is not possible to measure their affinity as strictly defined. In the case of monoclonal antibodies, where there is a single class of homogeneous binding sites, it is possible to measure the antibody affinity. SBK1 was found to have a K d value in the subnanomolar range, and the sensitivity of radioimmunoassays employing this antibody was corre-

spondingly high. We were able to measure reliably bradykinin concentrations down to 0.2 nM in the final reaction mixture. This sensitivity is high enough to be useful in many situations, although it is lower than that reported for some radioimmunoassays based on conventional sera (Odya et al., 1983; Bonner et al., 1987). SBK2 has an affinity for bradykinin about 100-fold lower than SBK1, and a correspondingly lower sensitivity when used in radioimmunoassay. However, unlike SBK1, the antibody recognises the immediate breakdown fragments produced by the action of peptidases kininase 1 and kininase 2 (1-8 and 1-7 bradykinin respectively); these enzymes represent the major bradykinin breakdown system in blood, and the primary means of terminating the vascular actions of bradykinin in vivo (Erdos, 1979). Other enzymes further degrade the initial products, but the relevant point is that SBK2 can be used to assess the total level of bradykinin and its early breakdown fragments, while SBK1 will only recognise the pharmacologically active species kallidin and bradykinin. In many physiological situations, the rapid breakdown of bradykinin is likely to result in a higher level of inactive fragments than active peptide, and the relatively lower sensitivity of SBK2 than SBK1 is thus unlikely to affect its utility. We would conclude from this study that it should be possible to design monoclonal antibody-based radioimmunoassay procedures for bradykinin and other small peptides. The major requirement for such assays is the availability of antibodies of appropriate specificity and high affinity. This study demonstrates that it is possible to produce antibodies of very high specificity which can readily distinguish between the parent peptide and its breakdown products. As this was a pilot study to assess the practicality of using monoclonal antibodies in place of conventional sera, the antibodies described in this paper were not originally selected by a method which would reveal only high affinity antibodies. The plate binding assay is relatively insensitive, and the conformation of bradykinin bound to the plate may be different from the range of conformations found in solution. However, screening methods based on the binding of tritiated ligand in solution in the presence and absence of low concentrations

185 of unlabeUed c o m p e t i t o r w o u l d r e a d i l y reveal those a n t i b o d i e s of sufficiently high affinity even in the p r i m a r y screening. This assay c a n be carried o u t b y c o a t i n g the screening wells with an a n t i - m o u s e a n t i b o d y , a p p l y i n g the test s u p e r n a t a n t s to these wells, a n d then a p p l y i n g a s o l u t i o n of 3H - l i g a n d to the wells. C o n t r a r y to p o p u l a r m i s c o n c e p t i o n s , it is certainly p o s s i b l e to p r o d u c e m o n o c l o n a l antib o d i e s with affinities of at least 10 -1° M ( L o et al., 1984). Such m o n o c l o n a l a n t i b o d i e s w o u l d significantly increase the sensitivity of the assay a n d b r i n g it into the range c o m m o n l y r e p o r t e d for conventional radioimmunoassays. Single m o n o c l o n a l a n t i b o d i e s are u n a b l e to m a n i f e s t the ' b o n u s effect' on affinity p r o d u c e d b y m u l t i v a l e n t b i n d i n g of the l i g a n d b y the different c o m p o n e n t s of p o l y c l o n a l sera. F o r small antigens the size of which p r e c l u d e s the b i n d i n g of m o r e t h a n one a n t i b o d y m o l e c u l e p e r m o l e c u l e of antigen, there is n o a d v a n t a g e in the presence of mixtures o f a n t i b o d i e s recognising m u l t i p l e epitopes. However, in the case of larger antigens, the b o n u s effect can be m i m i c k e d b y suitable d e f i n e d mixtures o f m o n o c l o n a l a n t i b o d i e s if required. M i x t u r e s of m o n o c l o n a l a n t i b o d i e s r e t a i n the p r o p e r t i e s of r e p r o d u c i b i l i t y shown b y their indiv i d u a l c o m p o n e n t s . M o n o c l o n a l a n t i b o d i e s represent a n u n v a r y i n g source of r e a g e n t in essentially u n l i m i t e d quantity, thus allowing s t a n d a r d i s a t i o n o f assays over time a n d b e t w e e n l a b o r a t o r i e s . T h e a d o p t i o n of high affinity m o n o c l o n a l a n t i b o d i e s as the basis for r a d i o i m m u n o a s s a y will e l i m i n a t e m a n y of the p r o b l e m s caused b y the use o f irrep r o d u c i b l e a n d sometimes p o o r l y c h a r a c t e r i s e d antisera.

Acknowledgements W e t h a n k Mr. P. C o o t e for m u c h helpful advice a n d discussion d u r i n g the course of this work. W e are grateful to Dr. C.R. Snell for a critical discussion of the m a n u s c r i p t a n d for p r e p a r i n g [125I]Tyrkallidin.

References Baccaglini, P.I. and Hogan, P.G. (1983) Some rat sensory neurons in culture express characteristics of differentiated pain sensory cells. Prec. Natl. Acad. Sci. U.S.A. 80, 594-598. Bonnet, G., Iwerson, D. and Schimamoto, K. (1987) The analytical value for kinin concentration in blood depends on the antiserum used in the bradykinin radioimmunoassay. J. Clin. Chem. Clin. Biechem. 25, 39-43. Erdos, E.G. (1979) Handbook of Experimental Pharmacology, Vol. 25 (Suppl.): Bradykinin, KaMdin and Kallikrein. Springer-Verlag, Berlin. Germaln, L., Barabe, J. and Galeano, C. (1986) Blood levels of kinins in experimental allergic encephalomyelitis. J. Neuroimmunol. 13, 135-142. Germain, L., Barabe, J. and Galeano, C. (1988) Increased blood concentrations of des-Arg9-bradykinin in experimental allergic encephalomyelitis. J. Neurol. Sci. 83, 211-217. Hunter, W.M. and Greenwood, F.C. (1962) Preparation of 125I-labelledhuman growth hormone of high specific activity. Nature 194, 495-496. Ishida, H., Shimamoto, K., Nishitani, T., Hosoda, S., Yokoyama, T., Nakahashi, Y., Ando, T., Tanaka, S. and Limura, O. (1986) Interrelation between the renin-angiotensin system and kallikrein-kinin system in patients with essential hypertension. Adv. Exp. Med. Biol. 198, 329-336. Kariya, K., Yamauchi, A. and Sasaki, T. (1985) Regional distribution and characterisation of kinin in the CNS of the rat. J. Neurochem. 44, 1892-1897. Lo, M.S., Tsong, T.Y., Conrad, M., Strittmatter, S., Hester, L.D. and Snyder, S.H. (1984) Moneclonal antibody production by receptor mediated electrically induced cell fusion. Nature 310, 792-794. Odya, C.E., Wilgis, F.P., Walker, J.F. and Oparil, S. (1983) Immunoreactive bradykinin and des-Arg9-bradykinin in low renin essential hypertension before and after treatment with enalapril (MK 421). J. Lab. Clin. Med. 102, 714-721. Proud, D., Togias, A., Naclerio, R.M., Crush, S.A., Norman, P.S. and Lichtenstein, L.M. (1983) Kinins are generated in vivo following nasal airway challenge of allergic individuals with allergen. J. Clin. Invest. 72, 1678-1685. Regoli, D. and Barabe, J. (1980) Pharmacology of bradykinin and related kinins. Pharmacol. Rev. 32, 1-46. Regoli, D., Barabe, J. and Park, W.K. (1977) Receptors for bradykinin on the rabbit aorta. Can. J. Physiol. Pharmacol. 55, 855-867. Yamauchi, A., Nakayama, A. and Kariya, K. (1985) Determination of kinin in the rat brain by a sensitive radioimmunoassay. J. Pharmacobiodyn. 8, 606-613.