a sensitive and specific two-site, sandwich-amplified enzyme immunoassay for neuropeptide y

Peptides, Vol. 13, pp. 1049-1054, 1992 0196-9781/92 $5.00 + .00 Printed in the USA. Copyright © 1992 Pergamon Press Ltd.

A Sensitive and Specific Two-Site, Sandwich- Amplified Enzyme Immunoassay

for Neuropeptide Y

E. G R O U Z M A N N , L J. F. AUBERT, B. W A E B E R A N D H. R. B R U N N E R

Hypertension Division and Cardiovascular Research Group, University Hospital, Lausanne, Switzerland

Received 17 January 1992

GROUZMANN, E., J. F. AUBERT, B. WAEBER AND H. R. BRUNNER. A sensitive and specific two-site, sandwich-amplified enzyme immunoassayfor neuropeptide Y. PEPTIDES 13(6) 1049-1054, 1992.--The development of a new enzyme immunoassay for neuropeptide Y (NPY) is reported. Two monoctonal antibodies directed against distinct epitopes of NPY are used, one as a capture antibody (NPY02) and the other one as an indicator antibody (NPY05), this latter antibody being labeled with alkaline phosphatase. The assay calibration curve was performed over concentrations of I to 250 pM in a NPY-free plasma. The intra- assay coefficient of variation (CV) ranged from 0.025 to I 1.9%, whereas the interassay CV was comprised between 5 and 12%. The limit of detection of this assay was l pM (100 amol/well). Neuropeptide Y levels are related to sampling conditions; basal concentrations of NPY with low SEM are found when less than 1.2 ml of blood is taken in EDTA tubes, the sample is centrifuged at 4°C, and immediately frozen. Unanesthetized spontaneously hypertensive rats exhibited higher NPY plasma concentrations than normotensive Wistar-Kyoto controls (53 _+ 7 pM and 25 + 2 pM, respectively, mean + SEM, p < 0.01 ). Plasma NPY levels are similar in 16- and 36-week-old animals. In conclusion, this technique makes it possible to assay a large number of samples within 24 h without requiring radioactivity.

Amplified enzyme immunoassay Neuropeptide Y Monoclonal antibody

NEUROPEPTIDE Y (NPY) is a 36 amino acid peptide isolated for the first time from porcine brain (22). It is colocalized with catecholamines in nerve endings (16) and is thought to be in- volved in cardiovascular regulation by modulating the activity of the sympathetic nervous system (10). In humans, the measurement of NPY has been used as a marker of several neuroendocrine tumors. Thus, high plasma levels of NPY have been described in some patients with pheochromocytoma, carcinoid tumors, or neuroblastoma (1,5,8,9). Increased plasma NPY concentrations have also been found during intense sympathetic stimulation (physical exercise, endotoxic shock) as well as in patients with severe congestive heart failure (4,18,25).

A few years ago, we produced hybridomas secreting two monoclonal antibodies directed against two distinct epitopes of NPY. With these antibodies a specific and sensitive immunoradiometric assay (IRMA) for NPY was developed (8). The present work was undertaken to set up a two-site amplified enzyme immunoassay (AEIA) to measure precisely NPY and re- quiting no radiolabeled ligand. We utilized the same monoclonal antibodies as before, one serving as capture antibody and the other, after conjugation to alkaline phosphatase, as indicator antibody.

METHOD

Materials

Microplates (Polysorp immunoplate, ref 4-75094) and min- isow tubes used for the preparation of the standard curve (ref 4-68808) were obtained from Nunc (Roskilde, DK). Alkaline phosphatase (ALP) was purchased from Biozyme (ALPI2G, specific activity 8303 U/mg, San Diego, CA). Murine monoclonal antibodies (mAbs) NPY02 and NPY05 were prepared against human neuropeptide Y as described previously (8). Por- cine neuropeptide Y was synthetized by BioMega (Laval, Que- bec), and peptide YY (PYY), bovine pancreatic polypeptide (bPP), humanPP (hPP), and ratPP (rPP) by Sigma (Buchs, Swit- zerland). Bovine serum albumin of radioimmunoassay grade was also obtained from Sigma. The ELISA amplification kit Immunoselect was purchased from Gibco (NY). SPDP and Tween 20 (Surfact-Amps 20) were from Pierce (Oud Beijerland, The Netherlands). All reagents were of analytical grade.

Preparation of Conjugates

Alkaline phosphatase was conjugated to the mAbs with N- succinimidyl 3-(2-pyridyldithio) proponiate (SPDP) according

Reque~sforreprintsshould be addressed to E. Grouzmann, Division ofHypeaension, Centre HospitalierUnive~itaireVaudois, 1011Lausanne, Swi~edand.

1049

1050 GROUZMANN ET AL.

NPY05 labelled with

Alksnne PhoW~,t~m

~ -C

EI . I~ t ,

FIG. 1. Principle of the NPY amplified enzyme immunoassay.

to Jeanson et al. (13). Briefly, purified mAbs and ALP were diluted in phosphate buffer saline (PBS) 0.1 M, pH 7.5, con- raining 0.1 M NaC1 and then incubated with SPDP 20 m M in absolute ethanol according to the molar ratio SPDP/ALP = 60 and SPDP/mAb = 3. The reactions were allowed to take place for 30 min at room temperature under mixing. Excess SPDP and released N-hydroxysuccinimide were removed by ultrafiltration (Diafio ultrafiltration membranes, YM 30, Amicon, Danvers, MA). Modified mAbs were diluted in PBS and ALP in sodium acetate buffer 0.1 M, pH 4.5, containing 100 mhd NaC1. Dithiothreitol was added to ALP to reach a final concentration of 25 m M a n d incubated for 20 min at room temperature. The mixture was ultrafiltered and diluted in PBS. Alkaline phosphatase and modified mAbs were mixed in a 1/! proportion and left overnight at room temperature. The conjugates were chromatographed on a superose 12 column (Pharmacia, Upp- sala, Sweden) preequilibrated with triethanolamine 100 mM, pH 7.0, containing 100 mMNaC1, 1 mMMgCI2, 0.1 mMZnC12, and 0.02% sodium azide.

Amplified Enzyme Immunoassay

The amplified enzyme immunoassay of NPY was performed as follows. The microplates were coated with 100 #l/well of the monoclonal antibody NPY02 (40 ng) diluted in Tris 50 mM, pH 7.5, for 16 h at 4°C. Plates were washed four times with Tris buffer containing 0.08% Tween 20, 5% lactose, and 0.5% casein. Wells were filled with either 100 #1 of the plasma sample (the blood being collected in EDTA tubes, Vacutainers, Becton Dickinson, N J) or NPY standards diluted in blank plasma. After a 16-h incubation at room temperature, the plates were washed with Tris buffer containing 0.08% Tween 20. One hundred #1 of the alkaline phosphatase conjugate of NPY05 diluted 1/30,000 with Tris buffer containing 0.08% Tween 20 and 5% nonfat dry milk was then added to each well and the plate incubated there- after for 7 h at room temperature. The plates were washed again four times with Tris buffer containing 150 m M NaCI before addition of 50 #1 of the substrate. The amplifier (50 #1) was added 45 min later according to the Immunoselect kit recom- mendations. Optic density was measured kinetic.ally at 492 nm using a Molecular Devices microplate reader (Molecular Devices,

CA) and the data analyzed by a computer (Soft Max, Molecular Devices). Figure l depicts the principle of this amplified enzyme immunoassay.

Determination of Plasma NPY Concentrations in Spontaneously Hypertensive Rats (SHR) and Normotensive Rats (WKY)

Sampling conditions for NPY measurements were investigated with Wistar-Kyoto rats. We first evaluated the effect of the volume of blood drawn from the animals. We then assessed the effect of two anticoagulants, of the temperature during centrifugation of the sample, and of immediate quick-freezing after decanting on the amount measured. Finally, the influence of the presence of platelets on the NPY assay was evaluated.

Plasma NPY concentrations were measured in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto controls (WKY). The animals were purchased from Iffa Credo (Lyon, France). Sixteen- as well as 36-week-old SHR (n = 18) and WKY rats (n = 19) were studied. On the day of the exper- iment, the rats had the right femoral artery cannulated with a PE-50 tube (Portex, Hythe, Kent, UK) filled with a heparinized 0.9% NaCI solution. This was done under a light ether anesthesia. The rats were then placed in Plexiglas tubes for partial restriction of their movements. Intra-arterial pressure and heart rate were measured during 15 min after a 2-h recovery period from anesthesia, i.e., at a time when blood pressure and heart rate were stabilized. These hemodynamic parameters were recorded using a computerized data acquisition system (7). A 0.8 ml sample of blood was then collected through the arterial line for NPY determination. Samples were centrifugated at 2800 rpm for 10 min at 4°C and immediately quick-frozen. Results are expressed as means -+ SEM. Statistical evaluation of the results was made using one-way analysis of variance (ANOVA).

RESULTS

Purification of Conjugates

Purification by gel filtration of NPY05 conjugated to alkaline phosphatase led to three distinct fractions absorbing at 280 nm. The second fraction gave the best immunoreactivity while ex-

TWO-SITE, SANDWICH-AMPLIFIED ENZYME IMMUNOASSAY 1051

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NPY concentration ( pM )

FIG. 2. (A) Relationship between the signal/noise at 492 nm (the noise corresponding to the optic density measured in the absence of NPY) and the concentration of NPY added to a blank plasma. The curves were established at either 4°C or at room temperature (RT). A refers to the temperature of the coating procedure, B that of the incubation for the NPY assay. (B) Relationship between the signal/noise at 492 nm and the concentration of NPY added to a blank plasma in the presence of increasing amounts of capture monoclonal antibody (NPY02). (C) Effect of different dilutions of the ALP-NPY05 conjugate on the relationship between signal/noise at 492 nm and NPY concentrations. The conjugate was diluted in Tris buffer containing 5% nonfat dry milk and 0.5% Tween 20. Dilutions of the conjugate from 1/1000 to 1/35,000 were tested. All these experiments were performed in triplicate.

hibiting a strong ALP activity (data not shown). It was therefore chosen for the development of the NPY assay.

Amplified Enzyme Immunoassay

Establishment of coating conditions. Two types of buffer were tested for the coating of NPY02. In the initial experiments, the plates were coated with 75 ng/well of NPY02 either in carbonate buffer 50 mM, pH 9.6, or Tris 50 mM, pH 7.5. This was done at room temperature for 12 h as well as at 4°C for the same period. Incubation with NPY standards was sub-

sequently performed either at room temperature or 4°C. The results of these studies are displayed in Fig. 2A. The best sensitivity was achieved when the plates were coated at 4°C and the standards incubated at room temperature. The sensitivity was improved by diluting NPY02 in Tris buffer (data not shown). NPY02 was diluted in Tris buffer over a range of 370 to 1500 ng/ml. Figure 2B illustrates the results observed by adding to the wells 12 to 122 ng of NPY02 in Tris buffer when performing all experiments at room temperature. Based on these data, a 40 ng amount of NPY02 was considered suitable for the assay.

Indicator antibody. In preliminary experiments, we added the indicator antibody directly to the plasma sample to be as- sayed. Using this one-step strategy the measurements were poorly reproducible and the enzymatic activity of the ALP was reduced. Stevens et al. observed the same phenomenon when developing an assay of angiotensin converting enzyme (21). They could solve this problem by adding 5% w/v nonfat dry milk to the Tris buffer used to dilute their conjugate. The same procedure was applied to our assay. Serial dilutions of the ALP-NPY05 conjugate ( 1 / 1000 to 1/35,000) were performed. At the two extreme dilutions, blank values were 20.9 and 1.4 milliabsorbance/min. Increasing the concentrations of the conjugate not only raised signal values, but also the background. The influence of the concentration of the conjugate is illustrated in Fig. 2C. The signal/ noise ratio at 492 nm is depicted for different NPY concentrations at the test dilutions of the conjugate. The slope of the 1/ 30,000 dilution curve appeared to be the most suitable and was retained for the final assay. The influence of the Tween 20 concentration on the NPY assay was also investigated. There was no impact with concentrations ranging from 0.06% to 0.09%. The 0.08% Tween 20 concentration was therefore kept for the definitive assay.

Specificity. The two-site amplified enzyme immunoassay was found to be very specific for NPY. No cross-reactivity with the apparented peptides of NPY (PYY and PP) has been observed in our assay even at high concentrations such as 10 nM (data not shown).

Standard curve and recovery experiments. Our goal was to reach sensitivity in the picomolar range with an assay requiring no plasma extraction and that could be performed within no more than 2 days. In the present assay, optimal coating of the capture mAb was obtained within 16 h, equilibrium for the incubation of the samples and the detecting mAb were reached in 16 and 7 h, respectively. Assay sensitivity was not enhanced by increasing the sample volume.

The assay calibration curve was performed by diluting synthetic NPY (over a range of 1 to 250 pM) in a NPY-free plasma (Fig. 3). The intra-assay coefficient of variation (CV) ranged from 0.025 to 11.9%, whereas the interassay CV was comprised between 5 and 12% (n = 6). Using this standard curve, the NPY concentration of a rat plasma pool was found at 28 pM. The intra-assay CV (n = 8) and interassay CV (n = 5) of this plasma were 7% and 6.4%, respectively. The plasma NPY concentration of a blank plasma spiked with 3.2 pM of NPY was also measured in quadruplicate in three separate assays. The intra-assay CV for this plasma was 14% with a recovery of 87%. The corresponding value for the interassay CV was 20%. The limit of detection of this new method, as assessed by the blank absorbance + 3 SD, was 1 pM (100 amol/well). Figure 4 illustrates a typical standard curve established by adding NPY at seven increasing concentrations into the wells. The measurements were done in quadruplicate. Differences in color between the various NPY concentrations can be seen.

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1052 GROUZMANN ET AL

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NPY ( pM ) NPY ( pM ) FIG. 3. Standard curve of NPY. Each point represent the mean _+ 1 SD of three values obtained in six separate assays.

Determination of Plasma NPY Concentration in WKY and SHR Rats

The size of the sample affects the basal level of NPY, as depicted in Fig. 5. Significant differences were observed in animals from which more than 1.2 ml of blood was drawn. Changes of NPY levels were also found to depend on the anticoagulant used and the temperature at which the blood is centrifuged (Fig. 6). Plasma sampled on EDTA tubes, decanted at 4°C, and quick- frozen provide lower levels and lower SEM when compared to those sampled on heparinized tubes (25.1 + 4.05 pM and 37.6 _+ 11.88 pM, respectively) or centrifuged at room temperature (102.1 + 31.58 pM and 79.2 _+ 6.59 pM, respectively). Neuro-

peptide Y levels of the samples on EDTA tubes centrifuged at either 4°C or room temperature but non-quick-frozen gave higher levels (39.9 + 8.7 pMand 148.7 +_ 53.5 pM, respectively). High levels of NPY have been found on plasma rich in platelets (1387 + 186 pM).

The mean arterial blood pressure of young SHR was similar to the one measured in old SHR (197 + 6 mmHg and 185 -+- 4 mmHg, respectively), but significantly higher when compared to both groups of WKY rats (140 _+ 4 mmHg and 145 _+ 2 mmHg, respectively). Plasma NPY concentration was twofold higher in SHR than in WKY rats (53 +_ 7 pM and 25 _+ 2 pM, respectively, p < 0.0 l) but no difference was observed depending on age (Fig. 7).

FIG. 4. Typical standard curve established by adding NPY at seven increasing concentrations (0, 1, 2.5, 5, 7.5, 10, 25, and 50 pM) into the wells. The measurements were done in quadruplicate. Differences in color between the various NPY concentrations can be seen.

TWO-SITE, SANDWICH-AMPLIFIED ENZYME IMMUNOASSAY 1053

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250

200

N P Y pM 150

100

50

0

0.6 ml 1.2 ml 1.8 ml 2.4 ml

n 10 10 10 9

FIG. 5. Effect of increasing the volume of blood drawn from the animals on plasma NPY levels. Sampling and centrifugation were performed at room temperature using EDTA as anticoagulant. Results are expressed as mean _+ SEM.

DISCUSSION

In the present work we describe a new method for the measurement of NPY. As stated above, it involves again the NPY02 and NPY05 antibodies. In contrast, however, it does not require any radiolabeled antibody, the antibody being linked to an enzyme. This amplified enzyme immunoassay provides results very similar to those obtained previously using an IRMA. With both assays, the limit of detection is at 1 pMand there is no problem of cross-reaction with the NPY family of peptides. In addition to the lack of radioactivity, the enzymatic assay has several ad- ditional advantages:

1. the conjugate remains stable for prolonged periods (we now have an experience of six months);

2. the results are not dependent on batch-to-batch differences in the preparation of the indicator antibody;

3. the assay can be more easily automated; 4. the coating of wells is easier than that of beads or tubes; 5. the cost per assay is much lower.

During recent years several radioimmunoassays have been described in the literature (2,6,11,17,19,23,26). Most of them utilized polyclonal antibodies. This is a potential shortcoming in terms of specificity since these antibodies are expected to cross- react more or less with NPY-related peptides. Only one radioimmunoassay using monoclonal antibodies has been pub- lished (8). That assay we developed 2 years ago is based on the same antibodies used in the present study, i.e., NPY02 and NPY05. The epitopes recognized by these antibodies have been well defined. The 11-24 part of NPY is essential for the binding of NPY02, whereas NPY05 reacts with the amidated C-terminal region of the peptide. The amidated peptide represents the bio- logically active form of NPY. With these two monoclonal antibodies, a sandwich assay can be developed that quantificates specifically the intact (1-36) NPY. This is important since frag- ments of NPY have been detected in the circulation (14,20). Also, related peptides of NPY did not interfere with our two- site amplified enzyme immunoassay. It is therefore not surprising that this assay yielded rat plasma levels of NPY five- to two hundredfold lower than those determined using some of the earlier methods. Furthermore, the conditions at blood sampling are crucial for an accurate determination of NPY. Bleeding or any stress increases the release of NPY. Platelets contain huge

250

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150

NPY pM 100

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FIG. 6. Sampling conditions for NPY measurement. Blood was taken in heparinized or EDTA tubes, centrifuged at 4°C or room temperature, and immediately slow- or quick-frozen. Results are expressed as mean _+ SEM, n = 9.

levels of NPY that could affect the measurement of basal levels of the peptide. It is also essential to sample the first ml of blood on EDTA tubes, centrifuge as soon as possible at 4°C, and quick- freeze the plasma.

With this new method significant differences between plasma NPY levels of spontaneously hypertensive rats (SHR) and normotensive controls (WKY) were observed. This was true in 16- as well as 36-week-old animals. These results are in agreement with previous observations made using conventional radioimmunoassays (12). As anticipated, the levels determined with our sandwich assay are markedly lower.

Potentially, NPY could play an important role in cardiovascular regulation. This peptide exhibits not only direct va- soconstrictor properties, but also enhances the contractile response to various stimuli, including norepinephrine and angiotensin II (24). Moreover, NPY, by stimulating presynaptic receptors, also exerts an inhibitory effect on norepinephrine release (24). Finally, this peptide has been shown to suppress

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FIG. 7. Plasma NPY levels measured in awake spontaneously hypertensive rats (SHR) and normotensive controls (WKY), aged 16 and 36 weeks.

1054 G R O U Z M A N N ET AL.

renin release under certain experimental condi t ions (24). Neuropeptide Y can be released into the circulat ion by sympathetic nerve terminals as well as by the adrenal medul la (3). It seems today that the largest a m o u n t of NPY present in the blood is originating from perivascular adrenergic nerve endings (15). The plasma concentrat ion of NPY may therefore reflect to some extent the activity of the sympathetic nervous system. This view is compatible with the finding by several investigators of elevated plasma NPY levels in patients with an enhanced sympathetic drive, for instance in patients with congestive heart failure or endotoxic shock, as well as in people subjected to a strenuous physical exercise (4,18,25). The NPY concent ra t ion may also be abnormal ly increased in the cir-

culation because of an enhanced release from neuroendocrine tumors such as pheochromocytoma or ganglioneuroblastoma (1,8,9). For all these reasons it appears highly desirable to be able to measure accurately plasma levels of NPY.

In summary, two monoclonal antibodies directed against NPY were used to develop a sensitive amplified enzymatic assay allowing accurate measurement of plasma NPY levels. This technique does not need radioactivity and makes it possible to assay a large number of samples within 24 h.

ACKNOWLEDGEMENTS

This work was supported by grants of Ciba-Geigy and of the Swiss National Foundation.

REFERENCES

1. Adrian, T. E.; Terenghi, G.; Brown, M. J.; Allen, J. M.; Balarese- 15. Lundberg, J. M.; Anggard, A.; Theodorsson-Norheim, E.; Pernow Hamilton, A. J.; Polak, J. M.; Bloom, S. R. Neuropeptide Y in J. Guanethidine-sensitive release of neuropeptide Y immunoreac- phaeochromocytomas and ganglioneuroblastomas. Lancet ii:540- 542; 1983.

2. Allen, J. M.; Yeats, J. C.; Adrian, T. E.; Bloom, S. R. Radioim- munoassay ofneuropeptide Y. Regul. Pept. 8:61-70; 1984.

3. Allen, J. M,; Bloom, S. R. Neuropeptide Y: A putative neurotrans- mitter. Neurochem. Int. 8:13-22; 1986.

4. Chalmers, J.; Morris, M.; Kapoor, V.; Cain, M.; Elliott, J.; Russell, A.; Pilowsky, P.; Minson, J.; West, M.; Wing, L. X. Neuropeptide Y in the sympathetic control of blood pressure in hypertensive subjects. Clin. Exp. Hypertens. [A] l l(Suppl. 1):59-66; 1985.

5. Corder, R.; Lowry, P. J.; Emson, P. C.; Galliard, R. C. Chromato- graphic characterization of the circulating neuropeptide Y immunoreactivity from patients with pheochromocytoma. Regul. Pept. 10:91-97; 1985.

6. Corder, R.; Lowry, P. J, An immunoradiometric assay for the measurement of neuropeptide Y in plasma. Peptides 6:1195-1200; 1985.

7. Fluckiger, J. P.; Gremaud, G.; Waeber, B. Measurement of sympathetic nerve activity in the unanesthetized rat. J. Appl. Physiol. 167:250-255; 1989.

8. Grouzmann, E.; Comoy, E.; Bohuon, C. Plasma neuropeptide Y concentrations in patients with neuroendocrine tumors, J. Clin. En- docrinol. Metab. 64:808-813; 1989.

9. Grouzmann, E.; Gicquel, C.; Plouin, P. F.; Schlumberger, M.; Comoy, E.; Bohuon, C. Neuropeptide Y and neuron-specific enolase levels in benign and malignant pheochromocytomas. Cancer 66: 1833-1835; 1990.

10. Hakanson, R.; Wahlestedt, C.; Ekblad, E.; Edvinsson, L.; Sundler, F. Neuropeptide Y: Coexistence with noradrenaline. Functional im- plications. In: Hokfelt, T.; Fuxe, K.; Pernow, B., eds. Progress in brain research. Amsterdam: Elsevier; 1986:279-285.

11. Higuchi, H.; Yang, H. T.; Costa, E. Age-related bidirectional changes in neuropeptide Y peptides in rat adrenal glands, brain and blood. J, Neurochem. 50:1879-1886; 1988.

12. Howe, P. R. C.; Rogers, P. F.; Morris, M. J.; Chalmers, J. P.; Smith, R. M. Plasma catecholamines and neuropeptide Y as indices of sympathetic nerve activity in normotensive and stroke-prone spontaneously hypertensive rats. J. Cardiovasc. Pharmacol. 8 (6): 1113- 1121; 1986.

13. Jeanson, A.; Cloes, J. M.; Bouchet, M.; Rentier, B. Preparation of reproducible alkaline-phosphatase antibody conjugates for enzyme immunoassay using a heterobifunctional linking agent. Anal. Biochem. 172:392-396; 1988.

14. Kitamura, K.; Kangawa, K.; Tanaka, K.; Matsuo, H. Isolation of NPY-25 (neuropeptide Y (12-36)), a potent inhibitor of calmodulin, from porcine brain. Biocbem. Biophys. Res. Commun. 169(3):1164- 1171; 1990.

tivity in the cat spleen by sympathetic nerve stimulation. Neurosci. Lett. 52:175-180; 1984.

16. Lundberg, J. M.; Terenius, L.; Hokfelt, T.; Tatemoto, K. Compar- ative immunohistochemical and biochemical analysis of pancreatic polypeptide-like peptides with special reference to presence of neuropeptide Y in central and peripheral neurons. J. Neurosci. 4:2376- 2386; 1984.

17. Lundberg, J. M.; Martinsson, A.; Hemsen, A.; Theodorsson-Nor- helm, E.; Svedenhag, J.; Ekblom, B.; Hjemdahl P. Co-release of neuropeptide Y and catecholamines during physical exercise in man. Biochem. Biophys. Res. Commun. 133:30-36; 1985.

18. Maisel, A. S.; Scott, N. A.; Motulsky, H. J.; Michel, M. C.; Boublik, J. H.; Rivier, J. E,; Ziegler, M.; Allen, R. S.; Brown, M. R. Elevation of plasma neuropeptide Y levels in congestive heart failure. Am. J. Med. 86:43-48; 1989.

19. Morris, M.; Kapoor, V.; Chalmers, J. Plasma neuropepfide Y concentration is increased after hemorrhage in conscious rats: Relative contributions of sympathetic nerves and the adrenal medulla. J. Cardiovasc. Pharmacol. 9:541-545; 1987.

20. Senanayake, P.; Warner, M. R.; Levy, M. N.; Jacobsen, D. W.; Fer- ratio, C. M. Characterization of neuropeptide Y immunoreactivity in dog plasma by high performance liquid chromatography and radioimmunoassay. In: Allen, J. M.; Koenig, J. I., eds. Central and peripheral significance of neuropeptide Y and its related peptides. New York: New York Academy of Sciences; 1990:340-342. (Ann. NY Acad. Sci., vol. 611 .)

21. Stevens, J.; Danilov, S.; Fanburg, B. L.; Lanzillo, J. J. A sensitive two-site sandwich enzyme immunoassay for human angiotensin converting enzyme utilizing monoclonal antibodies. J. Immunol. Methods 132:263-273; 1990.

22. Tatemoto, K.; Cariquist, M.; Mutt, V. Neuropeptide Y a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature 296:659-660; 1982.

23. Theodorsson-Norbeim, E.; Hemsen, A.; Lundberg, J. M. Radioim- munoassay for neuropeptide Y (NPY): Chromatographic characterization ofimmunoreactivity in plasma and tissue extracts. Scand. J. Clin. Lab. Invest. 45:355-365; 1985.

24. Walker, Ph.; Grouzmann, E.; Burnier, M.; Waeber, B. The role of neuropeptide Y in cardiovascular regulation. Trends Pharmacol. Sci. 12 : l l l - l l5 ; 1991.

25. Watson, J. D.; Sury, M. R. J.; Corder, R.; Carson, R.; Bouioux, P. M.; Lowry, P. J.; Besser, G. M.; Hinds, C. J. Plasma levels of neuropeptide Y (NPY) are increased in human sepsis but are un- changed during canine endotoxin shock despite raised catecholamine concentrations. J. Endocrinol. 116:421--426; 1988.

26. Zukowska-Grojec, Z.; Konarska, M.; Mc Catty, R. Differential plasma catecholamine and neuropeptide Y response to acute stress in rats. Life Sci. 42:1615-1624; 1988.

a sensitive and specific two-site, sandwich-amplified enzyme immunoassay for neuropeptide y

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