ELISA PRODUCT INFORMATION & MANUAL. FSH ELISA kit NBP

Transcription

1 ELISA PRODUCT INFORMATION & MANUAL FSH ELISA kit NBP Enzyme-linked Immunosorbent Assay for quantitative detection of Rat FSH. For research use only. Not for diagnostic or therapeutic procedures. - P: P: F: technical@novusbio.com Novus kits are guaranteed for 6 months from date of receipt

2 FOR RESEARCH PURPOSES ONLY Product Manual Unless otherwise specified expressly on the packaging, all products sold hereunder are intended for and may be used for research purposes only and may not be used for food, drug, cosmetic or household use or for the diagnosis or treatment of human beings. Purchase does not include any right or license to use, develop or otherwise exploit these products commercially. Any commercial use, development or exploitation of these products or development using these products without the express written authorization of Novus Biologicals is strictly prohibited. Buyer assumes all risk and liability for the use and/or results obtained using the products covered by this invoice whether used singularly or in combination with other products. LIMITED WARRANTY; DISCLAIMER OF WARRANTIES These products are offered under a limited warranty. The products are guaranteed to meet all appropriate specifications described in the package insert at the time of shipment. Novus Biologicals sole obligation is to replace the product to the extent of the purchasing price. All claims must be made to Novus Biologicals, within five (5) days of receipt of order. THIS WARRANTY IS EXPRESSLY IN LIEU OF ANY OTHER WARRANTIES OR LIABILITIES, EXPRESS OR IMPLIED, INCLUDING WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PURPOSE, AND NON- INFRINGEMENT OF THE PATENT OR OTHER INTELLECTUAL PROPERTY RIGHTS OF OTHERS, AND ALL SUCH WARRANTIES (AND ANY OTHER WARRANTIES IMPLIED BY LAW) ARE EXPRESSLY DISCLAIMED. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

3 TABLE OF CONTENTS Please read entire booklet before proceeding with the assay. Carefully note the handling and storage conditions of each kit component. Please contact Novus Biologicals Technical Support if necessary. Introduction... 2 Principle... 3 Materials Supplied... 4 Storage... 5 Other Materials Needed... 5 Sample Handling... 5 Sample Matrix Properties... 6 Reagent Preparation... 8 Assay Procedure... 9 Calculation of Results Typical Results Performance Characteristics References Contact Information

4 INTRODUCTION Follicle-stimulating hormone (FSH) is a 35.5 KD glycoprotein dimer consisting of - and -subunits. The -subunit of FSH contains 92 amino acids and is identical to those of luteinizing hormone (LH), thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hcg). The -subunit of FSH has 111 amino acids and its biological function is to bind to the FSH receptor 1. FSH is produced by the gonadotropic cells in the anterior pituitary gland. It regulates the development, growth, pubertal maturation and reproductive processes in both women and men. FSH and LH acts synergistically in regulating female ovulation and male spermatogenesis 2. In females, FSH levels are normally low during childhood, early follicular phase, luteal phase and pregnancy. Its level increases when approaching ovulation (Figure 1) or after menopause 3-4. The amount of FSH remains relatively constant in males after puberty 5. Figure 1: Ovulation Cycle and the Glycoprotein Hormones involved in Ovulation 4 Disorders affecting the hypothalamus, pituitary gland, ovaries or testicles can cause the elevation or decline of FSH, resulting in a variety of conditions such as subfertility or infertility, abnormal menstrual cycles, and early or delayed sexual maturation 6-7. Measurement of FSH levels in human serum or plasma is often used in diagnosis of these diseases. The FSH ELISA kit provides a sensitive and efficient assay for accurately quantifying FSH in human specimens. 2

5 PRINCIPLE 1. A monoclonal antibody to FSH is immobilized on a microtiter plate to bind the FSH in the standards or samples. 2. After a short incubation period, the excess sample or standard is washed out and a biotinylated detector antibody to FSH is added. This antibody binds to the FSH captured on the plate. 3. After incubation the excess antibody is washed out and Streptavidin conjugated to Horseradish Peroxidase is added to bind the biotinylated FSH detector antibody. 4. Once the incubation is complete, excess conjugate is washed out and TMB substrate solution is added. An HRPcatalyzed reaction generates a blue color in the solution. 5. Stop solution is added to stop the substrate reaction. The resulting yellow color is read at 450nm. The amount of signal is directly proportional to the level of FSH in the sample. 3

6 MATERIALS SUPPLIED 1. FSH Microtiter Plate, One plate of 96 wells. Plate with break-apart strips coated with a mouse monoclonal antibody specific to FSH. 2. FSH Standard, 0.2ml. Vial containing FSH protein, purified from human origin, at 1000mI U/ml in assay buffer. 3. FSH Detector Antibody, 1 vial. Vial containing 275ng lyophilized FSH detector antibody. 4. SA-HRP Conjugate, 10ml. Solution of Streptavidin conjugated to Horseradish peroxidase. 5. Assay Buffer 13, 50ml. Tris buffered saline containing BSA and detergents. 6. TMB Substrate, 10ml. Solution of 3,3,5,5 tetramethylbenzidine (TMB) and hydrogen peroxide. Protect from prolonged exposure to light. 7. Stop Solution 2, 10ml. A 1N solution of hydrochloric acid in water. capped. Caution: Caustic. 8. Wash Buffer Concentrate, 100ml. 20x Tris buffered saline containing detergents. 9. FSH Assay Layout Sheet, 1 each. 10. Plate Sealer, 3 each. Keep tightly 4

7 Reagents require separate storage conditions. STORAGE All components of this kit should be stored at 4 C except the detector antibody. The FSH Detector Antibody must be stored at -20 C until the kit s expiration date. OTHER MATERIALS NEEDED 1. Deionized or distilled water. 2. Precision pipets for volumes between 100µl and 1,000µl. 3. Repeater pipet for dispensing 100µl. 4. Disposable beakers for diluting buffer concentrates. 5. Graduated cylinders. 6. A microplate shaker. 7. Adsorbent paper for blotting. 8. Microplate reader capable of reading at 450nm, preferably with correction between 570nm and 590nm. 9. Graph paper for plotting the standard curve. SAMPLE HANDLING This assay is suitable for the measurement of FSH in human serum and EDTA plasma in addition to tissue culture media. Prior to assay, frozen specimens should be brought to 4 C and centrifuged. If it is necessary, filter to remove residual debris. Neat (undiluted) samples have been validated for use in this assay (please refer to the Spike and Recovery section on page 7 for details). However, due to variation in samples, a dilution may be required. Users must determine the optimal dilutions for their samples. 5

8 SAMPLE MATRIX PROPERTIES Linearity The minimum required dilution for human female and male serum or EDTA plasma was determined by serially diluting specimens into the provided assay buffer and identifying the dilution at which linearity was observed. Per the below table, the undiluted human serum and plasma are all in the linear detection range. Dilutional linearity, % Dilution Famale Serum Follicular Mid-Cycle Luteal Pregnant Postmenopausal Female EDTA Plasma Male Serum Male EDTA Plasma Neat : : : : Spike and Recovery Purified FSH protein was spiked at concentrations of 80, 20 and 5 miu/ml into undiluted human serum or plasma. Matrix background was subtracted from the spiked values and the recovery was compared to the recovery of identical spikes in assay buffer. The average percent recovery for each matrix at minimum required dilution is presented below. These results show human serum, EDTA plasma and tissue culture medium have no obvious interference with the FSH ELISA assay. Sample Serum EDTA Plasma Tissue Culture Media Spike Concentration, miu/ml % Recovery Minimum Recommended Dilution Neat Neat Neat 6

9 FSH [miu/ml] Product Manual Parallelism Parallelism experiments were carried out to determine if the FSH standard accurately mimicked native FSH in biological matrices. Human serum and plasma were serially diluted into assay buffer and run in the assay. The FSH concentration in each sample was assigned using the standard curve. The parallel response indicates the FSH standard effectively mimics the native protein. 1, Standard Follicular Mid-Cycle Luteal Post-Menopausal Female plasma Male serum Male plasma FSH Dilution Factor

10 REAGENT PREPARATION 1. Wash Buffer Prepare the Wash Buffer by diluting 30ml of the supplied concentrate with 570ml of deionized water. The diluted wash buffer can be stored at room temperature for up to 3 months. 2. FSH Standard Curve The FSH standard stock as well as the diluted standards and samples should be kept on ice and used within 60 minutes of preparation for optimal performance. Allow the FSH standards to warm to room temperature before loading to the plate. Label eight disposable 12 x 75mm (or similar) polypropylene tubes #1 through #8. Pipet 450µl Assay Buffer 13 into tube #1 and 250µl Assay Buffer 13 into tubes #2 through #8. Pipet 50µl of 1000mI U/ml FSH standard stock into tube #1 and vortex gently. Serially dilute 250µl of tube #1 standard to tubes #2 through #8 by gently vortexing after each dilution. After loading the plate, discard any unused standard dilutions. 3. FSH Detector Antibody Reconstitute the detector antibody in 11ml of the Assay Buffer 13. Keep it on ice before using. Aliquot and store at - 20 C if it will not be used right away. Avoid repeated freeze thaw cycles. 8

11 ASSAY PROCEDURE Refer to the Assay Layout Sheet to determine the number of wells to be used. Remove unneeded wells and return them, with the desiccant, to the plate bag and seal. Store the unused wells at 4 C. 1. Pipet 100µl of Assay Buffer 13 into NSB and standard 0 wells. 2. Pipet 100µl of standards #1 through #8 into the appropriate wells. 3. Pipet 100µl of the samples into the appropriate wells. 4. Seal the plate and incubate at room temperature on a plate shaker for 30 min at ~500rpm*. 5. Wash 4x400µl with Wash Buffer. 6. Pipet 100µl of reconstituted FSH Detector Antibody into all wells except for the NSB and blank. Pipet 100µl of Assay Buffer 13 to NSB wells. 7. Seal the plate and incubate at room temperature on a plate shaker for 30 min as in #4 above. 8. Wash 4x400µl with Wash Buffer. 9. Add 100µl of SA-HRP into each well including blank. 10. Seal the plate and incubate at room temperature on a plate shaker for 30 minutes as in #4 above. 11. Wash 4x400µl with Wash Buffer. 12. Pipet 100µl TMB substrate into all wells. 13. Seal the plate and incubate at room temperature on a plate shaker for 30 minutes as in #4 above. 14. Pipet 100µl of the Stop Solution 2 into each well. 15. After zeroing the plate reader against the substrate blank, read optical density at 450nm. * The plate shaker speed was based on a BellCo Mini Orbital Shaker (model no ).The actual speed of the plate shaker should be such that the liquid in the plate wells mixes thoroughly, but does not splash out of the well. 9

12 CALCULATION OF RESULTS Several options are available for the calculation of the concentration of FSH in the samples. We recommend that the data be handled by an immunoassay software package utilizing a four parameter logistic curve fitting program. Such software is often supplied by plate reader manufacturers. Alternatively, the concentration of FSH can be calculated as follows: 1. Calculate the average net optical density (OD) bound for each standard and sample by subtracting the average NSB OD from the average OD for each standard and sample. Average Net OD = Average OD - Average NSB OD 2. Plot the average net OD versus concentration of FSH for the standards. Approximate a line through the points. The concentration of FSH in the unknown samples can be determined by interpolation. Samples with concentrations outside of the standard curve range will need to be reanalyzed using a different dilution. 10

13 Optical Density (450nm) Product Manual TYPICAL RESULTS The results shown below are for illustration only and should not be used to calculate results. Sample FSH [miu/ml] Net OD (450nm) NSB Std Std Std Std Std Std Std Std Std *FSH conversion: 3.75 x 10 6 miu/ml = 1 mg/ml FSH [miu/ml] 11

14 PERFORMANCE CHARACTERISTICS Specificity The specificity of this assay was determined by measuring the cross reactants at a concentration of ten times the high standard. The cross-reactivity of each reactant is listed in the below table. Sensitivity Analyte Cross Reactivity FSH 100% LH <0.088% TSH <0.029% hcg Undetected The sensitivity or limit of detection of this assay is 0.5 miu/ml. The assay sensitivity was determined by interpolation at two standard deviations above the net OD of 20 zero standard replicates utilizing a four parameter logistic (4PL) curve fit. Intra-assay precision was determined by analyzing 20 replicates of three matrix controls containing FSH in a single assay. Intra-assay precision [miu/ml] %CV Inter-assay precision was determined by measuring matrix controls containing FSH in multiple assays over several days. Inter-assay precision [miu/ml] %CV

15 REFERENCES 1. Fan QR, Hendrickson WA. Structure of human folliclestimulating hormone in complex with its receptor. Nature Jan 20; 433(7023): Hirano M, Igarashi A, Suzuki M. Dynamic changes of serum LH and FSH during pregnancy and puerperium. Tohoku J Exp Med Mar; 118(3): Howles CM. Role of LH and FSH in ovarian function. Mol Cell Endocrinol Mar 30; 161(1-2): Menstrual Cycle: Pre & Post Ovulation. (September 2014). Retrieved from 5. Petak SM, Nankin HR, et al. American Association of Clinical Endocrinologists Medical Guidelines for clinical practice for the evaluation and treatment of hypogonadism in adult male patients update. Endocr Pract Nov-Dec; 8(6): Daya S. Follicle-stimulating hormone in clinical practice: an update. Treat Endocrinol. 2004; 3(3): Sharma TP, Nett TM, Karsch FJ, Phillips DJ, Lee JS, Herkimer C, Padmanabhan V. Neuroendocrine control of FSH secretion: IV. Hypothalamic control of pituitary FSHregulatory proteins and their relationship to changes in FSH synthesis and secretion. Biol Reprod Jun 7; 86(6):

16 Catalog Number: NBP Copyright Rev. 12/01/2014