Intended use

The intended use of these reagents is for assay of glucagon in human plasma.

For professional use within a laboratory.

This product is also available as research use only. Product code: RB 310 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

Glucagon is a 29 amino acids straight chain peptide produced in the pancreatic -cells. Glucagon is cleaved out from preproglucagon with 159 amino acids. The amino acid sequence of glucagon is found in glicentin, a 69 amino acid peptide. Glicentin has been proposed to be a biosynthetic intermediate for pancreatic and gut glucagon.

Glucagon is involved in carbohydrate, fat and protein metabolism. Basal amounts of glucagon are essential for the maintenance of normoglycemia and a physiological role for glucagon is to prevent hypoglycemia. Increases in the plasma glucagon level affect glucose production first by stimulating a transient phase of glycogenolysis and then a prolonged period of glyconeogenesis.

A sustained increase in the glucagon level continues to modulate hepatic glucose production.

Glucagon also plays a role in the amino acid metabolism. Elevation of glucagon in plasma decreases amino acids whereas glucagon deficiency increases amino acids. The amino acid sequence of human pancreatic glucagon: His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr.

Technical information

Glucagon in plasma is assayed by the competitive radioimmunoassay using a rabbit antiserum raised against a glucagon-albumin conjugate. Glucagon in standards and samples compete with 125I-labelled glucagon in binding to the antibodies in a two steps incubation.

125I-glucagon binds in a reverse proportion to the concentration of glucagon in standards and samples . Antibody-bound 125I-glucagon is separated from the unbound fraction using double antibody solid phase. The radioactivity of the bound fraction is measured in a gamma counter.

The antiserum used in this assay shows less than 0.1% cross reaction with gut-GLI.

Kit components and storage of reagents

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

Composition of the reagent kit

1. Anti-glucagon (Reagent A)

Rabbit antiserum raised against porcine glucagon, conjugated to human serum albumin. Lyophilized in 5.0 mL 2.0 M glycin buffer, pH 8.8, 2.5% human serum albumin, 0.5% sodium azide and Trasylol. Reconstitution in 52 mL distilled water. The reconstituted reagent contains 500 KIU Trasylol/mL. Colour: Yellow.

2. 125I-Glucagon (Reagent B)

Total radioactivity: 0.75 μCi or 28 KBq at reference date. Produced by iodination of synthetic human glucagon. HPLC-purified, monoiodinated.

Specific activity: 1700-2100 μCi/nmol (62-77 MBq/nmol). Lyophilized in 5.0 mL 2.0 M glycin buffer, pH 8.8, 2.5% human serum albumin, 0.5% sodium azide and Trasylol. Reconstitution in 52 mL distilled water. The reconstituted reagent contains 500 KIU Trasylol/mL. Colour: Blue.

3. Double antibody solid phase (Reagent C)

Anti-rabbit-Ig coupled to cellulose particles.

Volume: 11 mL suspension.

4. Assay diluent (Reagent D)

50 mL of 0.2 M glycin buffer, pH 8.8, containing 0.25% human serum albumin,

0.05% sodium azide and 500 KIU Trasylol/mL.

To be used for the preparation of glucagon working standards and instead of antiserum in non-specific binding control tubes.

5. Glucagon standard (Reagent E)

5.00 mL standard. Concentration: 300 pmol/L (1044 pg/mL) of synthetic human glucagon. Lyophilized in 0.2 M glycin buffer, pH 8.8, containing 0.25% human serum albumin, 0.05% sodium azide and 500 KIU Trasylol/mL. Reconstitution in 5.00 mL distilled water.

6. Controls (Reagent F-G)

Lyophilized controls. 2.00 mL of each control after reconstitution. The glucagon concentrations of the controls 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 the lyophilized reagents should be distilled in an all-glass apparatus or be of corresponding purity. Dissolve the contents in the vials by gentle inversion and avoid foaming.

The stability for each reagent is found on the label of the vial. For the lyophilized reagents the expiry date is valid for the unreconstituted reagents. The reconstituted reagents are stable for 10 weeks (or to the expiry date for the labelled glucagon) when stored according to the instructions below.