Canine Insulin ELISA Kit

Transcription

1 Canine Insulin ELISA Kit Cat. No.:DEIA2033 Pkg.Size:96T Intended use This product provides a method for the quantitative determination of insulin in canine serum and plasma. General Description Insulin is a hormone, produced by the pancreas, which is central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen inside these tissues. Insulin stops the use of fat as an energy source by inhibiting the release of glucagon. With the exception of the metabolic disorder diabetes mellitus and metabolic syndrome, insulin is provided within the body in a constant proportion to remove excess glucose from the blood, which otherwise would be toxic. When blood glucose levels fall below a certain level, the body begins to use stored sugar as an energy source through glycogenolysis, which breaks down the glycogen stored in the liver and muscles into glucose, which can then be utilized as an energy source. As a central metabolic control mechanism, its status is also used as a control signal to other body systems ( such as amino acid uptake by body cells ). In addition, it has several other anabolic effects throughout the body. When control of insulin levels fails, diabetes mellitus will result. As a consequence, insulin is used medically to treat some forms of diabetes mellitus. Patients with type 1 diabetes depend on external insulin ( most commonly injected subcutaneously ) for their survival because the hormone is no longer produced internally. Patients with type 2 diabetes are often insulin resistant and, because of such resistance, may suffer from a "relative" insulin deficiency. Some patients with type 2 diabetes may eventually require insulin if other medications fail to control blood glucose levels adequately. Over 40% of those with Type 2 diabetes require insulin as part of their diabetes management plan. Insulin also influences other body functions, such as vascular compliance and cognition. Once insulin enters the human brain, it enhances learning and memory and benefits verbal memory in particular. Enhancing brain insulin signaling by means of intranasal insulin administration also enhances the acute thermoregulatory and glucoregulatory response to food intake, suggesting that central nervous insulin contributes to the control of whole-body energy homeostasis in humans. Human insulin is a peptide hormone composed of 51 amino acids and has a molecular weight of 5808 Da. It is produced in the islets of Langerhans in the pancreas. The name comes from the Latin insula for "island". Insulin's structure varies slightly between species of animals. Insulin from animal sources differs somewhat in "strength" ( in carbohydrate metabolism control effects ) in humans because of those variations. Porcine insulin is especially close to the human version. Principle Of The Test This producct is a solid phase two-site enzyme immunoassay. It is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule. During incubation, insulin in the sample reacts with peroxidase-conjugated anti-insulin antibodies and anti-insulin antibodies bound to the microplate. After a simple washing step that removes unbound enzyme labelled antibody, the bound conjugate is detected by reaction with 3,3-5,5 -tetramethylbenzidine ( TMB ). The reaction is stopped by the addition of acid, giving a colorimetric endpoint that can be read spectrophotometrically. Reagents And Materials Provided 1. Coated Plate: Mouse monoclonal anti-insulin 1 plate, 96 wells. 8-well strips Ready for use. For unused mocroplate, reseal the bag using adhesive tape. 2. Calibrator 1, 2, 3, 4, 5: Porcine insulin. Concentration stated on vial label. Color coded yellow. 5 vials 1000 µl. Ready for use

2 3. Calibrator 0: Color coded yellow 1 vial 5 ml. Ready for use. 4. Enzyme Conjugate 11X: Peroxidase conjugated mouse monoclonal anti-insulin 1 vial 1.3 ml 5. Enzyme Conjugate Buffer: Color coded blue 1 vial 13 ml. Ready for use 6. Wash Buffer 21X: 1 bottle 50 ml. Dilute with 1000 ml redistilled water to make wash buffer. 7. Substrate TMB: TMB, colorless solution. 1 bottle 22 ml. Ready for use 8. Stop Solution: 0.5 M H2SO4 1 vial 7 ml. Ready for use Materials Required But Not Supplied 1. Pipettes for 25, 50, 100, 200 and 1000 µl ( repeat pipettes preferred for addition of enzyme conjugate solution, TMB Substrate and Stop Solution ) 2. Beakers and cylinders for reagent preparation 3. Redistilled water 4. Microplate reader ( 450 nm filter ) 5. Plate shaker ( The recommended velocity is cycles per minute, orbital movement ) 6. Microplate washing device Storage The recommended storage temperature is 2-8. Specimen Collection And Handling 1. Serum: Collect blood by venipuncture, allow to clot and separate the serum by centrifugation. Samples can be stored at 2-8 up to 24 hours. For longer periods, store samples at 20. Avoid repeated freezing and thawing. 2. Plasma: Collect blood by venipuncture into tubes containing heparin, citrate or EDTA as anticoagulant, and separate the plasma fraction. Samples can be stored at 2-8 up to 24 hours. For longer periods store samples at 20. Avoid repeated freezing and thawing. Reagent Preparation 1. Preparation of enzyme conjugate 1X solution: Prepare the needed volume of enzyme conjugate 1X solution by dilution of Enzyme Conjugate 11X, ( 1+10 ) in Enzyme Conjugate Buffer according to the table below. Mix gently. When preparing enzyme conjugate solution for the whole plate or if the reagents are to be used within 2 weeks, pour all of the Enzyme Conjugate Buffer into the Enzyme Conjugate 11X vial. 2. Preparation of samples: No dilution is normally required for serum or plasma. All samples containing canine insulin above the highest standard should be diluted with Calibrator 0 or with Diabetes Sample Buffer. Assay Steps All reagents and samples must be brought to room temperature before use. Perform each determination in duplicate for

3 standards and unknowns. Prepare a standard curve for each assay run. 1. Prepare enzyme conjugate solution ( according to the table on previous page ) and wash buffer. 2. Prepare sufficient microplate wells to accommodate Calibrators and samples in duplicate. 3. Pipette 25 µl each of Calibrators and samples into appropriate wells. 4. Add 100 µl of enzyme conjugate 1X solution into each well. 5. Incubate on a plate shaker ( rpm ) for 2 hours at room temperature ( ). 6. Wash 6 times with 700 µl wash buffer 1X solution per well using an automatic plate washer with overflow-wash function, after final wash, invert and tap the plate firmly against absorbent paper. Do not include soak step in washing procedure. Or manually, Discard the reaction volume by inverting the microplate over a sink. Add 350 µl wash solution to each well. Discard the wash solution, tap firmly several times against absorbent paper to remove excess liquid. Repeat 5 times, Avoid prolonged soaking during washing procedure. 7. Add 200 µl TMB Substrate Solution into each well. 8. Incubate for 15 minutes at room temperature ( ). 9. Add 50 µl Stop Solution to each well. Place the plate on the shaker for approximately 5 seconds to ensure mixing. 10. Read optical density at 450 nm and calculate results. Read within 30 minutes. Note! To prevent contamination between the conjugate and substrate, separate pipettes are recommended. Quality Control Commercial control and/or internal serum pools with low, intermediate and high canine insulin concentrations should routinely be assayed as unknowns, and results charted from day to day. It is good laboratory practice to record the following data for each assay: kit lot number, reconstitution dates of kit components; OD values for the blank, Calibrators and controls. Calculation 1. Computerized calculation: The concentration of canine insulin is obtained by computerized data reduction of the absorbance for the Standards, except Standard 0, versus the concentration using cubic spline regression. 2. Manual calculation: Plot the absorbance values obtained for the Standards, except Standard 0, against the canine insulin concentration on a lin-lin paper and construct a standard curve. Read the concentration of the unknown samples from the standard curve. Detection Range μg/l Sensitivity 0.01 μg/l Specificity

4 The following cross reactions have been found: 1. Porcine Insulin : 100% 2. Porcine C-peptide: < % 3. Porcine Proinsulin: < 0.2 % 4. Human Insulin: 28% 5. Human C-peptide: < 0.01 % 6. Human Proinsulin: < 0.1 % 7. NovoRapid: 0.7 % 8. Levemir: < % 9. Lantus: 5.4 % 10. Humalog: < % Recovery 1. Recovery upon addition is % ( mean 111 % ). 2. Recovery upon dilution is % ( mean 90 % ). Reproducibility Intra-Assay Precision: % Inter-Assay Precision: % Inter-Lot Precision: % Precautions 1. The content of this kit and their residues must not be allowed to come into contact with ruminating animal or swine. 2. The Stop Solution in this kit contains 0.5 M H2SO4. Follow routine precautions for handling hazardous chemicals. 3. All patient samples should be handled as capable of transmitting infections. Limitations Grossly lipemic, icteric or hemolyzed samples do not interfere in the assay. Insulin is, however, degraded over time in hemolyzed samples. The degradation could give falsely low values and contributes to higher inter assay variation. Analyte Gene Information Gene Name INS insulin [ Canis lupus familiaris ] Official Symbol INS GeneID mrna Refseq NM_

5 Protein Refseq UniProt ID Chromosome Location 18 Pathway REFERENCES NP_ P01321 Aldosterone-regulated sodium reabsorption; Amyloids; Diabetes pathways 1. Gupta N, Park E, Sandhu H, Goh T,Tchipashvili V, Giacca A ( 2006 ) Direct and indirect effects of insulin on hepatic glucose production in diabetic depancreatized dogs during euglycemia. J Endocrinol 190: Kim SP, Ellmerer M,Van Citters GW, Bergman RN ( 2003 ) Primacy of Hepatic Insulin Resistance in the Development of the Metabolic Syndrome Induced by an Isocaloric Moderate-Fat Diet in the Dog. Diabetes 52: Catchpole B, Ristic JM, Fleeman LM, Davison LJ ( 2005 ) Canine diabetes mellitus: can old dogs teach us new tricks? Diabetologia 48: