Melatonin ELISA. For the quantitative determination of melatonin in human serum and EDTA and heparin plasma.

Transcription

1 Melatonin ELISA For the quantitative determination of melatonin in human serum and EDTA and heparin plasma. For Research Use Only. Not For Use In Diagnostic Procedures. Catalog Number: Size: 79-MELHU-E01 96 Wells Version: ALPCO April 27, G Keewaydin Drive, Salem, NH P: (800) F: (603) ts@alpco.com

2 INTENDED USE The ALPCO Melatonin ELISA is intended for the quantitative determination of melatonin human serum and EDTA and heparin plasma samples. For Research Use Only. Not for use in diagnostic procedures. PRINCIPLE OF THE ASSAY The assay procedure follows the basic principle of competitive ELISA whereby there is competition between a biotinylated and a non-biotinylated antigen for a fixed number of antibody binding sites. The amount of biotinylated antigen bound to the antibody is inversely proportional to the analyte concentration of the sample. When the system is in equilibrium, the free biotinylated antigen is removed by a washing step and the antibody bound biotinylated antigen is determined by use of streptavidin alkaline phosphatase as a marker and p-nitro phenyl phosphate as the substrate. Quantification of unknowns is achieved by comparing the enzymatic activity of unknowns with a response curve prepared by using known standards. MATERIALS SUPPLIED Component Quantity Preparation Microplate (96 wells) 12 x 8 strips Ready to use Standards (A-F) 1 vial each; 2mL Lyophilized Control Levels 1 and 2 1 vial each; 2 ml Lyophilized Wash Buffer Concentrate 100 ml 10X Melatonin Biotin 3 x 2 ml Lyophilized Melatonin Antiserum 3 x 2 ml Lyophilized Enzyme Conjugate 250 µl 80X PNPP Substrate Solution 2 x 13 ml Ready to use PNPP Stop Solution 15 ml Ready to use Extraction Columns 2 x 10 Ready to use Page 2 of 9

3 MATERIALS REQUIRED Precision pipettes for dispensing up to 1000 µl (with disposable tips) Repeating or multi-channel pipette for dispensing up to 1000 µl Volumetric containers and pipettes for reagent preparation Polystyrene or glass tubes (12 x 75 mm) Distilled/Deionized water for reagent preparation Microplate washer or wash bottle Microplate shaker capable of rpm Microplate reader Centrifuge (100 xg to 200 xg) or vacuum manifold Evaporator centrifuge or sample concentrator by use of nitrogen Vortex for sample preparation Laboratory Balance Foil to cover the microplate Methanol (HPLC grade) PRECAUTIONS 1. The human blood products incorporated into this kit have been tested for the presence of HIV (Human Immunodeficiency Virus), HBV (Hepatitis B Virus), and HCV (Hepatitis C Virus). Test methods for these viruses do not guarantee the absence of a virus; therefore, all reagents should be treated as potentially infectious. Handling and disposal should be in accordance with all appropriate national and local regulations for the handling of potentially biohazardous materials. 2. All materials derived from animal sources are bovine spongiform encephalopathy (BSE) negative. However, all materials should be treated as potentially infectious. 3. Kit reagents contain sodium azide or Proclin as bactericides. Sodium azide and Proclin are toxic. Substrates for the enzymatic color reactions are toxic and carcinogenic. Avoid contact with skin or mucous membranes. 4. The stop solution consists of diluted sulfuric acid, a strong acid. Although diluted, it still must be handled with care. It can cause burns and should be handled with gloves, eye protection, and appropriate protective clothing. Any spill should be wiped out immediately with copious quantities of water. Do not breathe vapor and avoid inhalation. 5. Avoid direct contact with skin. 6. This product is not for internal use. 7. Avoid eating, drinking, or smoking when using this product. 8. Do not pipette any reagents by mouth. 9. Reagents from this kit are lot-specific and must not be substituted. 10. Do not use reagents beyond the expiration date. 11. Variations to the test procedure are not recommended and may influence the test results. STORAGE CONDITIONS The kit should be stored at 2-8 C. The kit is stable until the expiration date on the box label. SAMPLE HANDLING Serum and EDTA and heparin plasma samples are appropriate for use in this assay. Page 3 of 9

4 The usual precautions for venipuncture should be observed. It is important to preserve the chemical integrity of a blood specimen from the moment it is collected until it is assayed. Do not use grossly hemolytic, icteric or grossly lipemic specimens. Samples appearing turbid should be centrifuged before testing to remove any particulate material. When stored at 2-8 C, samples are stable for up to 24 h. When stored at -20 C, samples are stable for up to 3 months. When stored -70 C, samples are stable for up to 12 months. It is recommended to aliquot samples before freezing. Keep samples away from direct heat or direct sunlight and avoid repeated freeze-thaw cycles. Samples suspected to contain concentrations higher than the highest standard have to be diluted with diluted Wash Buffer prior to the extraction step. SAMPLE EXTRACTION The yield of extraction with this procedure is approx %. Filter or centrifuge the samples prior to extraction in order to avoid clogging of the columns. Each sample, standard and control has to be extracted. Extraction may be performed in advance. The dried extracts (after evaporation of methanol) may be stored at 2-8 C or -20 C for up to 24 h. After elution with methanol the Extraction Columns may be used for extraction of the next samples or stored at 2-8 C protected from dust. Extraction Columns may be re-used up to 4 times. In case of re-use, start again by conditioning the column. Standard version: Procedure for Centrifuge and Evaporator Centrifuge Column Conditioning 1. Place the Extraction Columns into polystyrene or glass tubes (12 x 75 mm). 2. Add 1 ml of methanol (undiluted) to the columns. Let the solvent pass through the column by centrifugation for 1 min at 120 xg. Discard eluate. 3. Add 1 ml distilled water to the columns. Let the solvent pass through the column by centrifugation for 1 min at 120 x g. Discard eluate. 4. Proceed with sample application without delay in order to avoid the columns getting dry. Sample Application 5. Place the Extraction Columns into correspondingly marked polystyrene or glass tubes (12 x 75 mm). 6. Add 0.5 ml of Standards, Controls and samples to the columns. 7. Add 0.5 ml distilled water to the columns. Let pass through the column by centrifugation for 5 min at 120 xg. Discard eluate. Washing Page 4 of 9

5 8. Add 2 x 1 ml of 10 % methanol in distilled water (v/v) to the columns. Let the solvent pass through the column by centrifugation for 5 min at 120 xg. Discard eluate. Elution of Extract 9. Place the Extraction Columns into new, correspondingly marked polystyrene or glass tubes (12 x 75 mm). 10. Add 1 ml of methanol (undiluted) to the columns. Let the solvent pass through the column by centrifugation for 5 min at 120 xg. 11. Remove columns from the tubes. Avoid drops to be left at the columns. 12. Use columns for extraction of the next samples or store at 2-8 C protected from dust. Extraction Columns may be re-used up to 4 times. Evaporation and Reconstitution of Extract 13. Evaporate the methanol to dryness by use of evaporator centrifuge. 14. Reconstitute samples with 0.15 ml of distilled water. 15. Vortex at least 1 min and assay immediately. Alternative version: Procedure for Vacuum Manifold instead of a Centrifuge For the extraction scheme follow points 1-5 accordingly. The volumes remain unchanged. Let the solvent pass through the column using vacuum and a flow rate of 5 ml/min. For the samples and extracts use a flow rate of 2 ml/min. The evaporation of the solvent may be performed by using an evaporator centrifuge or by nitrogen. REAGENT PREPARATION All reagents must be equilibrated to room temperature prior to preparation and subsequent use in the assay. Prepare enough reagents for only the number of strips used. The kit can be used up to 3 times within the expiry date stated on the label. Reagents with a volume less than 100 μl should be centrifuged before use to avoid loss of volume. Controls (Levels 1 and 2) and Standards are provided in a lyophilized form. Reconstitute in 2 ml of distilled water. Close the vial with the rubber stopper and cap, gently swirl the vial without foaming, and allow it to stand for 15 minutes prior to use. The contents of the vial should be in solution with no visible particulates. Reconstituted controls and standards can be stored in aliquots at -20 C and are stable until the expiry date on the label. Melatonin Antiserum is provided in a lyophilized form. Reconstitute in 2 ml of distilled water. Close the vial with the rubber stopper and cap, gently swirl the vial without foaming, and allow it to stand for 15 minutes prior to use. The contents of the vial should be in solution with no visible particulates. Reconstituted antiserum is not stable. Melatonin Biotin is provided in a lyophilized form. Reconstitute in 2 ml of diluted wash buffer. Close the vial with the rubber stopper and cap, gently swirl the vial without foaming, and allow it Page 5 of 9

6 to stand for 15 minutes prior to use. The contents of the vial should be in solution with no visible particulates. Reconstituted biotin is not stable. Enzyme Conjugate (80X) must be diluted with diluted wash buffer 1:81 before use. Dilute 70 µl of Enzyme Conjugate with 5.6 ml of diluted wash buffer. The contents of the vial should be in solution with no visible particulates. Reconstituted enzyme conjugate is not stable. Wash Buffer Concentrate (10X) must be diluted with distilled water 1:11 before use (15 ml WASHBUF ml distilled water) and mixed well. Crystals could occur due to high salt concentration in the stock solutions. The crystals must be dissolved at room temperature or in a water bath at 37 C before dilution of the buffer solutions. The WASHBUF is stable at 2 8 C until the expiry date stated on the label. Wash buffer (1:11 diluted WASHBUF) can be stored in a closed flask at 2 8 C for 8 weeks. All other test reagents are ready to use. Test reagents are stable until the expiry date (see label of test package) when stored at 2 8 C. QUALITY CONTROL Control samples should be analyzed with each run. Results generated from the analysis of control samples, should be evaluated for acceptability using appropriate statistical methods. The results for the samples may not be valid if within the same assay one or more values of the quality control sample are outside the acceptable limits. ASSAY PROCEDURE All reagents and microplate strips (while sealed in foil pouch) should be equilibrated to room temperature prior to use. Gently mix all reagents before use. A standard curve must be performed with each assay run and with each microplate if more than one is used at a time. All standards, controls, and samples should be run in duplicate. 1. Pipette 50 µl of each extracted standard, extracted control and extracted sample into the respective wells of the microtiter Plate. 2. Pipette 50 µl of Melatonin Biotin into each well. 3. Pipette 50 µl of Melatonin Antiserum into each well. 4. Cover plate with adhesive foil. Shake plate carefully. Incubate hours at 2-8 C. 5. Remove adhesive foil. Discard incubation solution. Wash plate 3x with 250 µl of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel. 6. Pipette 150 µl of freshly prepared Enzyme Conjugate into each well. 7. Cover plate with new adhesive foil. Incubate 120 min at RT (18-25 C) on an orbital shaker (500 rpm). 8. Remove adhesive foil. Discard incubation solution. Wash plate 3x with 250 µl of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel. 9. For adding of Substrate and Stop Solution use, if available, an 8-channel Micropipettor. Pipetting should be carried out in the same time intervals for Substrate and Stop Solution. Use positive displacement and avoid formation of air bubbles. 10. Pipette 200 µl of PNPP Substrate Solution into each well. 11. Incubate 40 min at RT (18-25 C) on an orbital shaker (500 rpm). 12. Stop the substrate reaction by adding 50 µl of PNPP Stop Solution into each well. Briefly mix contents by gently shaking the plate. Page 6 of 9

7 13. Measure optical density with a photometer at 405 nm (Reference-wavelength: nm) within 60 min after pipetting of the Stop Solution. CALCULATION OF RESULTS 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 Logistics 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 plausible single value might be used). The concentration of the samples can be read directly from the standard curve. In case of diluted samples the values have to be multiplied with the corresponding dilution factor. Samples showing concentrations above the highest standard have to be diluted as described in the sample handling section and reassayed. Conversion Melatonin (pg/ml) x 4.30 = pmol/l Limitations Specimen collection has a significant effect on the test results. See Sample Handling section for details. The following blood components do not have a significant effect (+/-20% of expected) on the test results up to the below stated concentrations: Hemoglobin Bilirubin 8.0 mg/ml 0.36 mg/ml PERFORMANCE CHARACTERISTICS Typical Calibration Curve: (Example. Do not use for calculation.) Page 7 of 9

8 (OD) Melatonin ELISA Standard (pg/ml) Melatonin (pg/ml) ODMean OD/ODmax (%) A B C D E F Analytical Sensitivity = 1.6 pg/ml Precision Range (pg/ml) Intra-assay Inter-assay CV (%) Linearity Dilution Range (pg/ml) Range (%) 1: Page 8 of 9

9 Spiking Recovery Mean (%) Range (%) Cross-reactivity Substance Cross-reactivity (%) 5-Methoxy-Tryptophole 1.2 N-Acetyl-Serotonin Methoxy-Tryptamine 2.5 SUGGESTED PLATE LAYOUT Below is a suggested plate layout for running standards, controls, and up to 40 samples in duplicate A Std A Std A B Std B Std B C Std C Std C D Std D Std D E Std E Std E F Std F Std F G Ctrl 1 Ctrl H Ctrl 2 Ctrl Std = Standard Ctrl = Control Numbered wells = Samples Page 9 of 9