ELISA Assay Kit. Package Insert

Transcription

1 Package Insert LH-sLHCGR ELISA Assay Kit Catalog Number: LHR31-K01 (1 x 96 wells) For Research Use Only. Not for use in diagnostic procedures. v. 1.0 Eagle Biosciences, Inc. 20A Northwest Blvd., Suite 112, Nashua, NH Phone: Fax:

2 Intended Use The human LH-sLHCGR ELISA Assay kit is intended to be used for the in vitro quantitative determination of human soluble LH/hCG receptor (slhcgr) in complex with LH in serum or plasma. This LH-sLHCGR ELISA Assay kit is for research use only and not for use in diagnostic or therapeutic procedures. Introduction LHCGR is a G-protein coupled receptor with luteinizing hormone (LH)/hCG-binding sites at the N-terminus extracellular domain (ECD), six transmembrane (TM) domains and short intracellular C-tail. In addition to mature LHCGR protein, multiple truncated natural variants are produced as a result of alternative splicing. slhcgr is the soluble form of LHCGR. Principle of the Assay The LH-sLHCGR ELISA Assay kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on a sandwich principle. The microtiter wells are coated with monoclonal antibody directed towards the slhcgr molecule. An aliquot of patient sample containing endogenous LH-sLHCGR is incubated in the coated well with enzyme conjugate, which is an anti-lh antibody conjugated with horseradish peroxidase. After incubation the unbound conjugate is washed off. The amount of bound peroxidase is proportional to the concentration of LH-sLHCGR in the sample. After adding the substrate solution, the intensity of color developed is proportional to the concentration of LH-sLHCGR in the sample. Materials Provided P1 - slhcgr Antibody Coated Plate One ready to use microtiter plate containing 96 wells coated with monoclonal slhcgr antibody in each well. Store at 2-8 C until expiration date in sealed pouch with desiccant. AC - LH Antibody Conjugate One vial containing 1.2mL horseradish peroxidase labeled LH antibody in buffer. Store at 2-8 C until expiration date. See Reagent Preparation. CD - Antibody Conjugate Diluent One bottle containing 12mL conjugate diluent. Contains preservative. Store at 2-8 C until expiration date. See Reagent Preparation. S - LH-sLHCGR Standard One ready to use vials containing 250μL LH-sLHCGR Standard in buffer. Contains preservative. Store at 2-8 C until expiration date. See Reagent Preparation. SD - Standard Diluent One ready to use vial, containing 1mL of Standard Diluent. Contains preservative. Store at 2-8 C until expiration date. See Reagent Preparation. DIL - Sample Diluent One ready to use bottle, containing 13mL of Sample Diluent. Contains preservative. Store at 2-8 C until expiration date. See Reagent Preparation. TMB - TMB Solution One ready to use bottle, containing 11.5mL of tetramethylbenzidine (TMB). Store at 2-8 C until expiration date. LH-sLHCGR ELISA Assay Kit 2/9

3 W - Wash Buffer (25x Concentrated) One bottle containing 15 ml of 25x concentrated wash solution. Contains preservative. Store at 2-8 C until expiration date. See Reagent Preparation. ST - Stop Solution One ready to use bottle, containing 12mL of Stop Solution. Contains 1M HCl (Hydrochloric acid). Avoid contact with stop solution. It may cause skin irritations and burns. Store at 2-8 C until expiration date. P2 - Non-coated Plate One non-coated microtiter plate. CO - Plate Cover One microtiter plate cover. Materials required but not provided 1. Distilled or deionized water 2. Precision pipettes and disposable tips 3. ELISA reader capable of reading absorbance at 450nm 4. Lint-free absorbance paper or paper towel 5. Graph paper Warnings and Precautions The LH-sLHCGR ELISA Assay kit is FOR RESEARCH USE ONLY. Not for use in diagnostic or therapeutic procedures. Follow good laboratory practice and safety guidelines. Wear lab coats, disposable latex gloves and protective glasses where necessary. Do not use LH-sLHCGR ELISA Assay kit components beyond the expiration date. LH-sLHCGR ELISA Assay kit components of different lots should not be mixed. It is recommended that calibrators and samples be run in duplicate. TMB substrate (TMB) is light sensitive. Keep away from bright light. The solution should be colorless until use. The STOP Solution (ST) contains 3% Hydrochloric acid and can cause irritation or burns to respiratory system, skin and eyes. Direct contact with skin and eyes should be avoided. If contact occurs, rinse immediately with plenty of water and seek medical advice. Do not re-use microtiter plate wells or pour reagents back into their bottles, once dispensed. Optimal results will be obtained by strict adherence to this protocol. Accurate and precise pipetting, as well as following the exact time and temperature requirements prescribed are essential. Any deviation from this may yield invalid data. Handle all biological samples as potentially hazardous and capable of transmitting diseases. Store and dispose of these materials and their containers in accordance with local regulations and guidelines. Hemolyzed, hyperlipemic and contaminated samples may give erroneous results with the LHsLHCGR ELISA Assay kit. Sample Collection and Preparation 1. Serum and EDTA plasma are the recommended samples. 2. Specimens may be stored refrigerated at 2-8 C for 48 hours. When samples are not processed within 48 hours, store frozen at <-20 C. 3. Avoid multiple freeze-thaw cycles. 4. Prior to assay, frozen sera should be completely thawed and mixed well. 5. Do not use lipemic or hemolyzed specimens. LH-sLHCGR ELISA Assay Kit 3/9

4 Reagent Preparation Bring all reagents to room temperature (18-25 C) prior to use. Wash buffer Prepare the working wash buffer by diluting the 15 ml of 25x concentrated Wash Buffer (W) with 360 ml of distilled or de-ionized water. Store at room temperature (18-25 C). Antibody Conjugate Prepare the working conjugate by diluting the 1.2mL of 10x concentrated LH Antibody Conjugate (AC) with 10.8mL of Antibody Conjugate Diluent (CD), sufficient for 96 tests. Calibrators Prepare each LH-sLHCGR calibrator dilution in the first column (wells A1 through H1) of the Non-coated Plate (P2), by serial dilution of the LH-sLHCGR Standard (S) with Standard Diluent (SD) as shown in the figure below. 1. Pipet 250 μl of the LH-sLHCGR Standard (S) in well A1 of the Non-coated Plate (P2). Do not vortex the LH-sLHCGR Standard (S), but mix gently with a pipet. This is Calibrator 8 (C8). 2. Pipet 125 μl of Standard Diluent (SD) in each well (wells B1 through H1) of the Noncoated plate (P2). 3. Using a new pipet tip, transfer 125 μl of well A1 into well B1 of the Non-coated Plate (P2) and mix by pipetting up and down 15 times. This is Calibrator 7 (C7). 4. Using a new pipet tip, transfer 125 μl of well B1 into well C1 of the Non-coated Plate (P2) and mix by pipetting up and down 15 times. This is Calibrator 6 (C6). 5. Using a new pipet tip, transfer 125 μl of well C1 into well D1 of the Non-coated Plate (P2) and mix by pipetting up and down 15 times. This is Calibrator 5 (C5). 6. Using a new pipet tip, transfer 125 μl of well D1 into well E1 of the Non-coated Plate (P2) and mix by pipetting up and down 15 times. This is Calibrator 4 (C4). 7. Using a new pipet tip, transfer 125 μl of well E1 into well F1 of the Non-coated Plate (P2) and mix by pipetting up and down 15 times. This is Calibrator 3 (C3). 8. Using a new pipet tip, transfer 125 μl of well F1 into well G1 of the Non-coated Plate (P2) and mix by pipetting up and down 15 times. This is Calibrator 2 (C2). 9. The Standard Diluent (SD) alone in well H1 of the Non-coated Plate (P2) is Calibrator 1 (C1). 1 Amount of Standard Diluent (SD) Concentration A 250μL C8 Standard (S) 0 μl 9.5 B C7 125 μl C8 125 μl 4.7 C C6 125 μl C7 125 μl 2.4 D C5 125 μl C6 125 μl 1.2 E C4 125 μl C5 125 μl 0.59 F C3 125 μl C4 125 μl 0.30 G C2 125 μl C3 125 μl 0.15 H C1 0 μl 125 μl 0.00 LH-sLHCGR ELISA Assay Kit 4/9

5 Samples 1. Pipet 100 μl of the Sample Diluent (DIL) in the wells of the Non-coated Plate (P2) designated for the samples (wells A2 through H6). 2. Pipet 25 μl of each sample (S1 through S40) into the wells of the non-coated Plate (P2) containing the Sample Diluent (DIL). See the table below for the pipetting scheme in the non-coated plate A C8 S1 S9 S17 S25 S33 B C7 S2 S10 S18 S26 S34 C C6 S3 S11 S19 S27 S35 D C5 S4 S12 S20 S28 S36 E C4 S5 S13 S21 S29 S37 F C3 S6 S14 S22 S30 S38 G C2 S7 S15 S23 S31 S39 H C1 S8 S16 S24 S32 S40 3. Thoroughly mix the sample and Sample Diluent (DIL) in each well of the non-coated plate by pipetting up and down 15 times using a multichannel pipet and fresh tips for each column. It is very important that sample and diluent are thoroughly mixed to ensure good duplicates in the final assay. 4. The completed non-coated plate contains sufficient material for the calibrators and samples to be transferred in duplicate to the final coated plate (see ASSAY PROCEDURE below). LH-sLHCGR ELISA Assay Kit 5/9

6 Assay Procedure 1. Using an 8-Channel multichannel pipet, transfer 55 μl of each prepared calibrator dilution (C1 through C8) and diluted sample (S1 through S40) from the Non-coated Plate (P2) into each of two strips of the slhcgr Antibody Coated Plate (P1) to create an array of duplicates. Exchange pipet tips after each transfer. See the table below for the pipetting scheme in the coated plate A C8 C8 S1 S1 S9 S9 S17 S17 S25 S25 S33 S33 B C C7 C7 S2 S2 S10 S10 S18 S18 S26 S26 S34 S34 C6 C6 S3 S3 S11 S11 S19 S19 S27 S27 S35 S35 D C5 C5 S4 S4 S12 S12 S20 S20 S28 S28 S36 S36 E F C4 C4 S5 S5 S13 S13 S21 S21 S29 S29 S37 S37 C3 C3 S6 S6 S14 S14 S22 S22 S30 S30 S38 S38 G C2 C2 S7 S7 S15 S15 S23 S23 S31 S31 S39 S39 H C1 C1 S8 S8 S16 S16 S24 S24 S32 S32 S40 S40 2. Keeping the plate flat on a hard surface, gently tap the side of the plate against your hand to ensure that all liquid is in contact with the bottom of the well. 3. Cover the plate with the provided Plate Cover (CO) and incubate for 10 minutes at room temperature (18-25 C). 4. Add 100 μl of the prepared LH Antibody Conjugate to each well. Thoroughly mix for 15 seconds, either by gently tapping the side of the plate as in 2 (above), or, preferably, using the shake function in a plate reader. It is important to have a complete mixing in this step. 5. Cover the plate with the provided Plate Cover (CO) and incubate for 90 minutes at room temperature (18-25 C). 6. Remove liquid from all wells. Wash wells four (4) to six (6) times with 300 μl per well of the prepared wash buffer. At the end of the wash cycle thoroughly blot the plate on lint-free paper towels. 7. Add 100 μl of TMB Solution (TMB) to each well. 8. Incubate for 20 minutes at room temperature (18-25 C) protected from light. Do not use the Plate Cover (CO). 9. Add 100 μl of STOP Solution (ST) to each well. Shake the plate gently to mix the solution. Ensure there are no bubbles. 10. Read absorbance on an ELISA Reader at 450nm within 5 minutes after adding the STOP Solution. Perform a second reading of the absorbance at a wavelength of 650nm. LH-sLHCGR ELISA Assay Kit 6/9

7 Calculation of results The calibration curve is constructed as follows: 1. Check the LH-sLHCGR Standard (S) value on the vial label. This value might vary from lot to lot. Make sure that you check the value on every kit. 2. To construct the calibration curve, plot the absorbance for the LH-sLHCGR calibrator dilution (vertical axis) versus the LH-sLHCGR calibrator dilution concentrations (horizontal axis) on linear graph paper. Draw the best curve through the points. 3. Read the absorbance for each unknown sample from the curve. Record the value for each unknown sample. Remember to adjust the value for each unknown sample according to the dilution of the sample. 4. Alternatively, a calibration curve can be produced using Microsoft Excel software. Remove the value of C1 zero concentration calibrator from each value, then plot Absorbance versus concentration of the calibrator to generate a linear curve that passes through zero. Using the formula for the straight line generated, unknown sample values can be calculated. Typical Calibration Curve Following data is for demonstration only and cannot be used to replace the calibration curve at the time of the assay. OD 450 nm Concentration Calibrator Calibrator Calibrator Calibrator Calibrator Calibrator Calibrator Calibrator Analytical Characteristics All analytical characteristics are expressed in pmol/ml. Analytical Sensitivity The lowest detectable level of LH-sLHCGR which can be distinguished from zero is 0.02 pmol/ml. This value was determined by twenty-four replicates of the zero standard. The sensitivity is calculated on two standard deviations from the mean measured zero calibrator. LH-sLHCGR ELISA Assay Kit 7/9

8 Intra assay variation (within assay) The intra-assay precision was determined from the mean of sixteen replicates each of in-house serum-based controls. Sample N Mean Standard Deviation Coefficient of Variation (%) Inter assay variation (between assays) The inter-assay precision was determined from the mean of average duplicates of in-house serum-based controls for 4 assays. Sample N Mean Standard Deviation Coefficient of Variation (%) Sample dilution Three in-house serum-based controls were spiked with different amounts of LH-sLHCGR and assayed with the LH-sLHCGR ELISA Assay. Sample Expected value (pmol//ml) Observed value Recovery (%) Limitations of the Test The LH-sLHCGR ELISA Assay kit is FOR RESEARCH USE ONLY. Not for use in diagnostic or therapeutic procedures. LH-sLHCGR ELISA Assay Kit 8/9

9 Warranty Information Eagle Biosciences, Inc. warrants its Product(s) to operate or perform substantially in conformance with its specifications, as set forth in the accompanying package insert. This warranty is expressly limited to the refund of the price of any defective Product or the replacement of any defective Product with new Product. This warranty applies only when the Buyer gives written notice to the Eagle Biosciences within the expiration period of the Product(s) by the Buyer. In addition, Eagle Biosciences has no obligation to replace Product(s) as result of a) Buyer negligence, fault, or misuse, b) improper use, c) improper storage and handling, d) intentional damage, or e) event of force majeure, acts of God, or accident. Eagle Biosciences makes no warranties, either expressed or implied, except as provided herein, including without limitation thereof, warranties as to marketability, merchantability, fitness for a particular purpose or use, or non-infringement of any intellectual property rights. In no event shall the company be liable for any indirect, incidental, or consequential damages of any nature, or losses or expenses resulting from any defective product or the use of any product. Product(s) may not be resold, modified, or altered for resale without prior written approval from Eagle Biosciences, Inc. For further information about this kit, its application or the procedures in this kit, please contact the Technical Service Team at Eagle Biosciences, Inc. at info@eaglebio.com or at LH-sLHCGR ELISA Assay Kit 9/9