Serotonin high sensitive ELISA Kit

Transcription

1 Serotonin high sensitive ELISA Kit Cat. No.:DEIA6182 Pkg.Size:96T Intended use High Sensitive Enzyme Immunoassay for the quantitative measurement of Serotonin in low concentrated samples and for small sample volumes. General Description Serotonin (5-Hydroxytryptamine), a biogenic amine, is a product of the tryptophan metabolism. It is a well evaluated neurotransmitter of the central nervous system and can be found in high concentrations in the chromaffine cells of the intestinal mucosa, in the platelets and the serotonergic neurones of the brain. Central-serotonergic neurones influence physiological functions such as sleep and the hormonal and cardio- vascular regulation. Increased serum levels can be found with malignant carcinoid, endogenous depression and schizophrenia. Principle Of The Test The assay kit provides materials for the quantitative measurement of derivated Serotonin in low concentrated samples and for small sample volumes. The derivation is performed during the preparation of the samples. By using the acylation reagent the Serotonin is quantitatively derivated into N-acylserotonin. The competitive Serotonin ELISA kit uses the microtitre plate format. Derivated Serotonin compete for a fixed number of antiserum binding sites. When the system is in equilibrium, free antigen and free antigen- antiserum complexes are removed by washing. The antibody bound to the solid phase Serotonin is detected by anti-rabbit/peroxidase. The substrate TMB / peroxidase reaction is monitored at 450 nm. The amount of antibody bound to the solid phase Serotonin is inversely proportional to the Serotonin concentration of the sample. Reagents And Materials Provided 1. 1 x 12x8 - Microtiter Plate: Break apart strips Coated with Serotonin x 4 ml -Standard, Concentrate: Concentration: 500 ng/ml 3. 1 x 2 x 4 ml - Control 1+2, Concentrate (500x): Concentrations / acceptable ranges see QC Certficate x 4 ml - Acylation Buffer, lyophilized 5. 1 x 1 ml - Acylation Buffer, Concentrate 6. 4 x 2.5 ml - Acylation Reagent, lyophilized 7. 1 x 3 ml - Deactivator: Ready to use x 12 ml - Enzyme Conjugate: Ready to use. Contains: Anti-rabbit-IgG-peroxidase x 20 ml - Wash Buffer, Concentrate (25x) x 12 ml - Substrate: Ready to use. Contains: TMB solution x 12 ml - Stop Solution Ready to use. Contains: 0.3 M sulfuric acid x 6 ml - Solvent: Ready to use. Solvent to dissolve the Acylation reagent. Contains: Acetone x 2 ml - Ascorbic acid: Ready to use. Contains: 10% ascorbic acid x 50 ml - Assay Buffer: Before use enrich to 0.1% ascorbic acid. Contains: 10 mm PBS (0.9% NaCl), stabilized x - Reaction Plate: Ready to use x - Adhesive Foil 1

2 Materials Required But Not Supplied 1. Micropipettes (Multipette Eppendorf or similar devices, < 3% CV). Volumes: 10, 20, 25, 50, 100 and 200 µl 2. Orbital shaker (500 rpm) 3. Vortex mixer 4. Polypropylene (PP) tubes or polypropylene (PP) microtubes for Standard dilution 5. 8-Channel Micropipettor with reagent reservoirs 6. Wash bottle, automated or semi-automated microtiter plate washing system 7. Microtiter plate reader capable of reading absorbance at 450 nm (reference wavelength nm) 8. Bidistilled or deionised water 9. Paper towels, pipette tips and timer Storage The kit is shipped at ambient temperature and should be stored at 2-8. Keep away from heat or direct sun light. The storage and stability of specimen and prepared reagents is stated in the corresponding chapters. Specimen Collection And Handling The test is intended for small sample volumes, for low concentrated samples (e.g. tissue homogenates, dialysates) and in general for diluted samples. For the protection of Serotonin against oxidative degradation the samples must contain 0.1% ascorbic acid. The samples can be stored up to 6 hours at 2-8. For longer storage the samples must be frozen at -20. Repeated freezing and thawing should be avoided. Different dilution buffers are suitable but have to be tested beforehand. Evaluation was done with Ringer buffer and PBS (0.9% NaCl). Alternatively the Assay Buffer included in the kit can be used. All applied buffers must contain 0.1% ascorbic acid. For small sample volumes (< 20 µl) a volume correction is necessary. Add dilution buffer (alternatively Assay Buffer) to correct for volume. For Example:Sample volume: 1µL; Volume dilution buffer: 19 µl Reagent Preparation 1.Preparation of lyophilized or concentrated components 1.1. Assay Buffer The Assay Buffer has to be enriched to 0.1% ascorbic acid prior to use: e.g. 50 ml ASSAYBUF ml ASC ACID. The prepared Assay Buffer should be stored frozen at -20 and is stable until the expiration date. Use the same Assaybuffer for the dilution of controls and samples. All applied buffers must contain 0.1% ascorbic acid Standard The concentration of the CAL CONC is 500 ng/ml ( = pg/sample) Serotonin. Dilute Standard for obtaining working concentration as follows: 0 / 0.67 / 2.0 / 6.7 / 20 / 100 pg/sample. Table one. 2

3 Prepare standard dilution always fresh and use only once. Dilution should be done in polyproylene (PP) tubes, Eppendorf Cups or polypropylene (PP) microtubes Control 1/2 The controls have to be diluted 1:500 prior to use: Table two Acylation Buffer Dissolve the content of concentrate. LYO with 4 ml distilled water. Add 200 µl of Acylation Buffer. Mix shortly and leave on a roll mixer for 30 minutes. Handle carefully in order to minimize foam formation. The reconstituted Acylation Buffer should be stored frozen at -20 to stabilize until the expiry date Acylation Reagent Dissolve the content of one bottle ACYL BUF LYO in 2.5 ml SOLVENT and shake for 5 minutes on an orbital shaker. The Acylation Reagent has always to be prepared immediately before use. After use the reagent has to be discarded. The kit contains 4 vials allowing for a maximum of 4 assay runs. If the whole kit is to be used in one run it is recommended to pool the dissolved contents of two vials of Acylation Reagent. Please note that solvent reacts with many plastic materials including plastic trays; solvent does not react with normal pipette tips and with glass devices. Solvent is volatile and the dissolved Acylation Reagent evaporates quickly. Therefore, please do not use a tray with big surface together with a multichannel pipette for pipetting Acylation Reagent. Rather, use an Eppendorf multipette (or similar device), fill the syringe directly from the vial with dissolved Acylation Reagent and well by well Wash Buffer Dilute the content of the WASHBUF CONC with distilled water to a total volume of 500 ml. For further use the diluted wash buffer must be stored at 2 8 for a maximum period of 4 weeks. Assay Steps 1. Pipette 50 µl of each acyladed Standard, acylated Control and acylated Sample into the respective wells of the Microtiter Plate. 2. Cover the plate with adhesive foil and incubate for hours (overnight) at Remove adhesive foil. Discard incubation solution. Wash plate 4 x with 250 µl of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel. 4. Pipette each 100 µl Enzyme Conjugate into all wells. 5. Cover plate with adhesive foil. Incubate 60 min at RT (18-25 ) on an orbital shaker (500 rpm). 3

4 6. Remove adhesive foil. Discard incubation solution. Wash plate 4 x with 250 µl of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel. 7. Pipette 100 µl of TMB Substrate Solution into each well. 8. Incubate min at RT (18-25 ) on an orbital shaker (500 rpm). 9. Stop the substrate reaction by adding 100 µl of TMB Stop Solution into each well. Briefly mix contents by gently shaking the plate. 10. Measure optical density with a photometer at 450 nm (Reference-wavelength: nm) within 15 min after pipetting of the Stop Solution. Quality Control The test results are only valid if the test has been performed following the instructions. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable standards/laws. All kit controls must be found within the acceptable ranges as stated on the labels and the QC certificate. If the criteria are not met, the run is not valid and should be repeated. Each laboratory should use known samples as further controls. It is recommended to participate at appropriate quality assessment trials. In case of any deviation the following technical issues should be proven: Expiration dates of (prepared) reagents, storage conditions, pipettes, devices, incubation conditions and washing methods. Calculation The obtained OD of the standards (y-axis, linear) are plotted against their concentration (x-axis, logarithmic) either on semi logarithmic graph paper or using an automated method. A good fit is provided with cubic spline, 4 Parameter Logisitcs or Logit- Log. For the calculation of the standard curve, apply each signal of the standards (one obvious outlier of duplicates might be omitted and the more plause single value might be used). The concentration of the samples can be read directly from the standard curve. Conversion: Serotonin (ng/ml) x 5.67 = nmol/l Typical Standard Curve Example. Do not use for calculation! 4

5 Sensitivity The lower limit of detection was determined by taking the 2 fold standard deviation of the absorbance of the Zero Reference and reading the corresponding value from the standard curve. Sensitivity: 0.39 pg/sample Specificity Structural related components were tested for posse interference with the antisera against Serotonin used in the ELISA method. Reproducibility The reproducibility of the ELISA method was investigated by determination of the intra-assay-coefficients of variation (cv) by repeated measurements of two samples with the different Serotonin concentrations. Concentrations in pg/ml. Intra-assay: 6.6%-8.7% Precautions 1. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood. 2. In case of severe damage of the kit package please contact your supplier in written form, latest one week after receiving the 5

6 kit. Do not use damaged components in test runs, but keep safe for complaint related issues. 3. Obey lot number and expiry date. Do not mix reagents of different lots. Do not use expired reagents. 4. Follow good laboratory practice and safety guidelines. Wear lab coats, disposable latex gloves and protective glasses where necessary. 5. Reagents of this kit containing hazardous material may cause eye and skin irritations. See MATERIALS SUPPLIED and labels for details. 6. Chemicals and prepared or used reagents have to be treated as hazardous waste according to national biohazard and safety guidelines or regulations. 7. Avoid contact with Stop solution. It may cause skin irritations and burns. Limitations Specimen collection has a significant effect on the test results. REFERENCES 1. Harenberg, J., Huhle, G., Giese, Ch., Wang, L., Feuring, M., Song, X., Hofmann, U. Determination of Serotonin release from platelets by enzyme immunoassay in the diagnosis of heparin-induced thrombocytopenia British Journal of Hematology, 109, (2000) 2. Balaskas, E., Bamihas, G., Karamouzis, M., Voyiatzis, G., Tourkantonis, A. Histamine and Serotonin in uremic pruritus: effect of ondansetron in CAPD-pruritic patients Nephron, 78: (1998) 6