DRG International Inc., USA Fax: (908)

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1 INTENDED USE The DRG Serotonin ELISA kit provides materials for the quantitative determination of derivatized serotonin in serum, plasma, urine, platelets, tissue homogenates, cell culture supernatants and liquor. The derivatization is part of the preparation of the samples. By using the acylation reagent the serotonin is quantitatively derivatized into N- acylserotonin. CLINICAL RELEVANCE 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 cardiovascular regulation. Increased serum levels can be found with malignant carcinoid, endogenous depression and schizophrenia. PRINCIPLE OF THE TEST The competitive Serotonin EIA kit uses the microtiter plate format. Serotonin is bound to the solid phase of the microtiter plate. Acylated serotonin and solid phase bound 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 4 nm. The amount of antibody bound to the solid phase serotonin is inversely proportional to the serotonin concentration of the sample. WARNINGS AND PRECAUTIONS FOR USERS General Remarks Test methods are not available which can offer complete assurance that Hepatitis B virus, Human Immunodeficiency Virus (HIV/HTLV-III/LAV), or other infectious agents are absent from the reagents in this kit. Therefore, all human blood products, including patient samples, should be considered potentially infectious. Handling and disposal should be in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation, where it exists (e.g., USA Center for Disease Control/National Institute of Health Manual, "Biosafety in Microbiological and Biomedical Laboratories," 1984). Avoid contact with Stop Solution - H 2 SO 4. It may cause skin irritation and burns. Replace caps on reagents immediately. Do not switch caps. Solutions containing additives or preservatives, such as sodium azide, should not be used in the enzyme reaction. Do not pipette reagents by mouth. For in vitro diagnostic use only. Do not mix or use components from kits with different lot numbers. Remarks for the Test All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming. Once the test has been started, all steps should be completed without interruption. Use new disposable plastic pipette tips for each reagent, standard or specimen in order to avoid cross contamination. For the dispensing of the Substrate Solution and the Stop Solution avoid pipettes with metal parts. Pipette standards and samples onto the bottom of the well. For pipetting of Conjugate and Stop Solution it is recommended to hold the pipette in a vertical position above the well and dispense the correspondent solution into the centre of the well so that a complete mixing of Conjugate with sample or standard and of the Stop solution with the Substrate Solution is achieved. Vortex-mixing or shaking of wells after the pipetting step is not required. Before starting the assay, it is recommended that all reagents be ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption. The Substrate Solution should be colorless or slightly blue or green. If the solution is dark blue the reagent is unusable and must be discarded. During incubation with Substrate Solution avoid direct sunlight on the microtiter plate. DRG International Inc., USA Fax: (908) corp@drg-international.com 1

2 KIT COMPONENTS Quantity Reagents Storage 6 x 4 ml Standards A - F in the following concentrations: 0; 15; ; 1; 0; 00 ng/ml 2 x 4 ml Control 1 and 2 Range: see q.c. certificate 1 x 30 ml Acylation Buffer 1 x 3 ml Acylation Reagent 12 strips, 8 wells each Serotonin-coated microtiter plate; break apart, precoated with serotonin. 1 x 5. ml Antiserum; blue coloured Rabbit-anti-N-acylserotonin,. 1 x 11 ml Enzyme Conjugate ; green coloured, anti-rabbit-igg conjugated to peroxidase, 1 x ml Washing Buffer, Concentrate Dilute content with dist. water to 0 ml Concentrate; 2 8 C total volume. 1 x 11 ml Substrate TMB solution 1 x 11 ml Stop Solution Contains 0. M sulphuric acid, not corrosive 1 plate Microtiter plate for Derivatization, 96 wells used for the acylation reaction MATERIALS REQUIRED BUT NOT SUPPLIED Pipettes (,,, and 0 µl) Microplate washing device ELISA reader capable of reading absorbance at 4 nm Orbital shaker (400-0 rpm) SAMPLE COLLECTION AND STORAGE Serum and Plasma EDTA-plasma and serum can be used for the assay. Hemolytic and lipemic samples should not be used. Plasma can be stored up to 24 hours at 2-8 C. For a longer storage (up to 6 months) the samples must be frozen at - 20 C. Repeated freezing and thawing should be avoided. Urine The total volume of urine excreted during a 24-hours period should be collected and mixed in a single bottle containing - 15 ml of 6 M hydrochloric acid as preservative. Avoid exposure to direct sun light. Determine the total volume and take an aliquot for the measurement. For patients with suspected kidney disorders the creatinine concentration should be tested, too. Urine samples can be stored at -20 C for at least 6 months. Platelets More than 98 percent of the circulating serotonin is located in the platelets and is released during blood clotting. Blood must be collected by venipuncture into plastic tubes containing EDTA or Citrate. To obtain platelet-rich plasma (PRP) the samples are centrifuged for minutes at room temperature (200 x g). Transfer the supernatant to another tube and count the platelets. DRG International Inc., USA Fax: (908) corp@drg-international.com 2

3 The platelet pellet is obtained by adding 800 µl of physiological saline to 200 µl of PRP (containing between platelets/µl) and centrifugation (40 x g, minutes at 4 C). The supernatant is then discarded. 200 µl of dist. water is added to the pellet and mixed thoroughly on a vortex mixer. This suspension could be stored frozen for several weeks at < -20 C. After thawing of the frozen samples centrifuge at.000 x g for 2 minutes at room temperature. µl of the supernatant are used for the acylation reaction. To measure serotonin in platelet-free plasma (PFP), an aliquot of the PRP is centrifuged at 4.0 x g for min. at 4 C. µl of the supernatant are used in the acylation reaction. Tissue homogenates and cell culture supernatants Centrifuged tissue homogenates and cell culture supernatants may be used without special precautions. Please notice that some cell culture media may contain serotonin! Please note: a protocol for the high-sensitive determination of Serotonin is available upon request! SAMPLE PREPARATION AND ACYLATION Allow the microtiter plate for the derivatization to reach room temperature. Each well should be used only once. 1. Pipet µl of standard A - F, µl of control 1 & 2, µl of serum, (or µl of urine, µl of plasma, µl of platelet-rich plasma, µl of platelet-free plasma, 20 µl of tissue homogenates, µl of CSF) into the respective wells. 2. Pipet each 0 µl of Acylation Buffer into all wells. 3. Pipet each µl of Acylation Reagent into all wells. 4. Incubate for 15 minutes at room temperature on a shaker (400-0 rpm) 5. Take each µl for the EIA. ASSAY PROCEDURE General Remarks Allow all reagents to reach room temperature. Mix all reagents without foaming. Duplicates are recommended. PREPARATION OF REAGENTS Washing Buffer Dilute content of the bottle named Washing Buffer Concentrate with dist. water to 0 ml total volume. The diluted washing buffer must be stored at 2-4 C. All other reagents are ready for use. Assay Procedure 1. Pipet each µl of acylated Standards, Controls and Samples into the respective wells of the serotonin-coated microtiter plate 2. Pipet each µl of Antiserum into all wells. 3. Incubate for 60 minutes at room temperature on an orbital shaker (400-0 rpm). 4. Discard or aspirate the contents of the wells and wash thoroughly with each 0 µl Wash Buffer. Repeat the washing procedure 3 times. Remove residual liquid by tapping the inverted plate on clean absorbent paper. 5. Pipet each µl of Enzyme Conjugate into all wells. 6. Incubate for 30 minutes at room temperature on an orbital shaker (400-0 rpm). 7. Repeat the washing step (4.) 8. Pipet each µl of Substrate into all wells and incubate for 20 to 30 minutes at room temperature on an orbital shaker (400-0 rpm). 9. Pipet each µl of Stop Solution into all wells.. Read the optical density at 4 nm (reference wavelength between 570 and 6 nm) in a microplate photometer. DRG International Inc., USA Fax: (908) corp@drg-international.com 3

4 CALCULATION OF RESULTS On a semilogarithmic graph paper the concentration of the standards (x-axis, logarithmic) are plotted against their corresponding optical density (y-axis, linear). Alternatively, the optical density of each standard and sample can be related to the optical density of the zero standard, expressed as the ratio OD/ODmax, and then plotted on the y-axis. The concentrations of the controls and serum and urine samples can be read directly from this standard curve by using their average optical density. The read concentrations of the plasma samples have to be divided by 2.4, while the read concentrations of the CSF samples have to be divided by 4.4. Typical Calibration Curve Concentration (ng/ml) Mean OD Read Concentration (ng/ml) Standard A 0 2,392 0 Standard B 15 2,187 11,1 Standard C 1,734 53,59 Standard D 1 1, ,18 Standard E 0 0, ,54 Standard F 00 0,409 94,28 Control 1 1,463,49 Control 2 1, ,57 QUALITY CONTROL Good laboratory practice requires that quality control specimens be run with each standard curve to check the assay performance. Low and high level controls should be included in each assay. Kit controls or any other control materials should be assayed repeatedly to establish mean values and acceptable ranges. Each individual laboratory is responsible for defining their system for quality control decisions and is also responsible for making this system a written part of their laboratory manual. Recommended batch analysis decision using two controls: 1. When both controls are within ±2 SD. Decision: Approve batch and release analyte results. 2. When one control is outside ±2 SD and the second control is within ±2 SD. Decision: Hold results, check with supervisor. If no obvious source of error is identified by the below mentioned check of systems, the supervisor may decide to release the results. Technician check of systems: 1. Check for calculation errors 2. Repeat standards and controls 3. Check reagent solutions 4. Check instrument EXPECTED VALUES The reference ranges given below should only be taken as a guideline. It is recommended that each laboratory should establish its own normal values. Serum Female 80-4 ng/ml Male ng/ml Urine: - 0 µg/day DRG International Inc., USA Fax: (908) corp@drg-international.com 4

5 PERFORMANCE CHARACTERISTICS Sensitivity The sensitivity for serum, urine, platelets and tissue homogenates is 7.5 ng/ml, for plasma it is 3 ng/ml and for CSF it is 1,5 ng/ml. LIMITATIONS OF PROCEDURE Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the package insert instruction and with adherence to good laboratory practice. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbances. Complete mixing of Conjugate with standard or sample and of Stop Solution with Substrate Solution is critical. Insufficient mixing will result in poor precision. REFERENCES 1. Attanasio, A. et al., Diurnal rhythms of N-acetylserotonin and serotonin. J. Pineal res. 3 (1986), Kema, I.P. et al., Influence of a Serotonin- and Dopamine-Rich Diet on Platelet Serotonin Content and Urinary Excretion of Biogenic Amines and Their Metabolites Clin. Chem. 38/9 (1992), p Kema, I.P. et al., Improved Diagnosis of Carcinoid Tumors by Measurement of Platelet Serotonin Clin. Chem. 38/4 (1992), p Preparation and Storage of Reagents Store all reagents at 2 8 C Wash Buffer Dilute content of the bottle named Washing Buffer Concentrate with dist. water to 0 ml total volume. The diluted washing buffer must be stored at 2-4 C. All other reagents are ready for use. Sample Collection and Storage Serum and Plasma EDTA-plasma and serum can be used for the assay. Hemolytic and lipemic samples should not be used. Plasma can be stored up to 24 hours at 2-8 C. For a longer storage (up to 6 months) the samples must be frozen at - 20 C. Repeated freezing and thawing should be avoided. Urine The total volume of urine excreted during a 24-hours period should be collected and mixed in a single bottle containing - 15 ml of 6 M hydrochloric acid as preservative. Avoid exposure to direct sun light. Determine the total volume and take an aliquot for the measurement. For patients with suspected kidney disorders the creatinine concentration should be tested, too. Urine samples can be stored at -20 C for at least 6 months. Platelets More than 98 percent of the circulating serotonin is located in the platelets and is released during blood clotting. Blood must be collected by venipuncture into plastic tubes containing EDTA or Citrate. To obtain platelet-rich plasma (PRP) the samples are centrifuged for minutes at room temperature (200 x g). Transfer the supernatant to another tube and count the platelets. The platelet pellet is obtained by adding 800 µl of physiological saline to 200 µl of PRP (containing between platelets/µl) and centrifugation (40 x g, minutes at 4 C). The supernatant is then discarded. 200 µl of dist. water is added to the pellet and mixed thoroughly on a vortex mixer. This suspension could be stored frozen for several weeks at < -20 C. After thawing of the frozen samples centrifuge at.000 x g for 2 minutes at room temperature. µl of the supernatant are used for the acylation reaction. To measure serotonin in platelet-free plasma (PFP), an aliquot of the PRP is centrifuged at 4.0 x g for min. at 4 C. µl of the supernatant are used in the acylation reaction. DRG International Inc., USA Fax: (908) corp@drg-international.com 5

6 Tissue homogenates and cell culture supernatants Centrifuged tissue homogenates and cell culture supernatants may be used without special precautions. Please notice that some cell culture media may contain serotonin! Sample Preparation (Acylation) Standards, Controls Urine, Serum Plasma CSF PRP PFP Standard/Control /Sample Acylation Buffer [µl] Acylation Reagent [µl] Incubate for 15 min. at RT with shaking (400-0 rpm) Take each µl of the acylated standards, controls and samples for the EIA Assay Procedure Standards Controls Samples Antiserum [µl] Incubate 1 hour at RT, shaking (400-0 rpm) Enzyme Conjugate Incubate 30 min. at RT, shaking (400-0 rpm) Substrate Incubate min. at RT, shaking Stop Solution Standards Controls Samples Wash 3x with 0 µl of Washing Buffer Wash 3x with 0 µl of Washing Buffer (400-0 rpm) Read Absorpti on at 4 nm 4. Expected Values Serum Female 80-4 ng/ml Male ng/ml Urine: - 0 µg/day DRG International Inc., USA Fax: (908) corp@drg-international.com 6