Intended use

The intended use of these reagents is for assay of NPY in human serum/plasma by direct assay without extraction.

For professional use within a laboratory.

This product is also available as research use only. Product code: RB 317 RUO

For complete product description, claims and use, please refer to the Instructions For Use. Contact your local representative for availability in your country.

Background

Neuropeptide Y (NPY) is a 36 aminoacid residues peptide which was initially isolated from pig brain. NPY was later extracted from human adrenal-medullary phaeochromocytoma tissue. The human NPY differs from the pig NPY only by the replacement of leucine at position 17 by methione. NPY shares considerable sequence homology with pancreatic polypeptide and peptide YY. NPY-like immunoreactivity has been found in the central nervous system and in peripheral noradrenergic neurons and intestinal neurons. Potent vasoconstrictor activity is exhibited by NPY. NPY also inhibits noradrenaline release via a presynaptic action and has stimulatory effects on the contraction of the heart. Phaeochromocytoma tumours contain NPY and elevated levels of NPY in plasma from phaeochromocytoma patients have been reported. Increased plasma concentrations of NPY have been found in patient with neuroblastoma. Increased plasma concentrations of NPY have also been found in pediatric B-cell precursor leukemia.

Technical information

NPY in the serum/plasma samples is assayed by a competitive radioimmunoassay using an antiserum raised against synthetic NPY conjugated to bovine thyroglobulin. NPY in standards and samples compete with 125I-labelled NPY in binding to the antibodies. 125I-NPY binds in a reverse proportion to the concentration of NPY in standards and samples. Antibody-bound 125I-NPY is separated from the unbound fraction by using double antibody coupled to solid phase. The radioactivity of the antibody-bound 125I-NPY is measured. The antiserum used in this method crossreacts less than 2.0% with human Peptide YY.

Kit components and storage of reagents

The reagents provided in each kit are sufficient for 100 tubes.

Composition of the reagent kit

1. Anti-NPY (Reagent A)

Rabbit antiserum raised against NPY conjugated to bovine thyroglobulin. Lyophilized in

2.0 mL 0.5 M phosphate buffer, pH 7.4, with 2.5% human serum albumin, 2.5% EDTA, 1.0% Triton X-100, 0.5% sodium azide and 5000 KIU Trasylol^a per mL. For 100 tubes. Reconstitution in 22 mL distilled water.

Colour: Yellow.

2. ¹²⁵I-NPY (Reagent B)

Contains 28 KBq or 0.75 μCi at reference date. Synthetic, human NPY is iodinated according to Bolton-Hunter. The monoiodinated form is purified by HPLC. Specific activity: 1700-2100 μCi/nmol (62-77 MBq/nmol). Lyophilized in 2.5 mL 0.5 M phosphate buffer, pH 7.4, with 2.5% human serum albumin, 2.5% EDTA, 1.0% Tween 80,  0.5% sodium azide and 5000 KIU Trasylol^a per mL. Reconstitution in 25 mL distilled water.

Colour: Blue.

3. Double antibody solid phase (Reagent C)

11.0 mL suspension. Anti-rabbit-Ig coupled to cellulose particles.

4. Standard diluent (Reagent D)

10.0 mL NPY-free human serum, lyophilized. Contains 500 KIU Trasylol^a per mL. Reconstitution in 10.0 mL distilled water. For preparation of NPY working standards.

5. NPY standard 3000 pmol/L (Reagent E)

Lyophilized. 2.00 mL standard. Concentration: 3000 pmol/L after reconstitution. The standard is produced from synthetic, human NPY. Lyophilized in 0.05 M phosphate buffer, pH 7.4, with 0.25% human serum albumin, 0.25% EDTA, 0.1% Triton X-100, 0.05% sodium azide and 500 KIU Trasylol^a per mL. For preparation of NPY working standards.

6. Assay buffer (Reagent F)

5.0 mL 0.05 M phosphate buffer, pH 7.4, with 0.25% human serum albumin, 0.25% EDTA, 0.1% Triton X-100, 0.05% sodium azide and 500 KIU Trasylol^a per mL.

To be used instead of antiserum in non-specific binding test tubes.

7. Controls (Reagent G-H)

Lyophilized serum controls with low and high concentration of NPY. 2.00 mL of each control after reconstitution. The NPY concentrations are given on the labels of the vials.

Contains 0.05% sodium azide.

Reagent preparation and storage

Store all reagents at 2-8° C before reconstitution and use. The water used for reconstitution of lyophilized reagents should be distilled in an all-glass apparatus or be of corresponding purity.  Dissolve the contents in a vial by gentle inversion and avoid foaming. The stability of the reagents is found on the labels of the vials. For lyophilized reagents the expiry dates are valid for the unreconstituted reagents. The reconstituted reagents are stable for 10 weeks (no longer than to the expiry date) if stored properly.