KRIBIOLISA Bovine Serum Albumin (BSA) ELISA REF : KBBP10 Ver1.1 RUO Immunoassay for the measurement of Bovine Serum Albumin RUO For Research Use Only REF Catalog Number Store At Manufactured By Expiry Date LOT Batch Code Biological Risk Consult Operating Instructions For Research Purposes Only. Not for use in diagnostic or therapeutic procedures. Purchase does not include or carry the right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of KRISHGEN BioSystems is strictly prohibited. KRISHGEN BioSystems 1537 Ashley Ct, Whitter, CA Tel: (213)291 3096 Fax: (213)402 2025 Email: sales_usa@krishgen.com Cat No#KBBP10, Ver1.1 www.krishgen.com 1
Introduction: The manufacture of products by various biotechnological processes such as cell or tissue culture can result in residual contamination of the desired product by components used in the culture media. The use of so called serum free defined media greatly reduces the number of potential contaminants but it may still be necessary to determine trace contamination levels of the proteins and growth factors used in these media. Most commercial formulations of serum free media contain significant amounts of albumin and transferrin either of bovine or human origin, and insulin from various species. This assay is designed to provide a simple to use, precise, and highly sensitive method to detect BSA contamination. Intended Use: This kit is used for determination of BSA contamination in various antibody products that are purified through immobilized BSA chromatographic techniques. Principle: This assay is based on the Sandwich ELISA procedure. Samples containing BSA are reacted with microtiter wells already coated with affinity purified capture BSA antibody. This immunological reaction results in formation of a complex of solid phase antibody-bsa. The wells are washed to remove any unbound reactants as per the wash procedure (see Assay procedure section mentioned below). Anti-BSA-HRP antibody is added to wells to form a sandwich complex. After a washing step, the substrate 3, 3, 5, 5 Tetramethyl Benzidine is then reacted. The amount of hydrolyzed substrate is read on a microtiter plate reader and it is directly proportional to the concentration of BSA present. Materials Provided: 1. Anti-BSA coated Microtiter plate (96 wells) 1 no 2. BSA Standards, (1ml/vial) 6 vials 3. Anti-BSA HRP Conjugate 1 vial 4. Wash Buffer (10X) 50ml 5. Sample Diluent 70ml 6. TMB Substrate 15ml 7. Stop Solution 15ml 8. Instruction Manual Materials to be provided by the End-User: 1. Microplate Reader able to measure absorbance at 450 nm. 2. Adjustable pipettes to measure volumes ranging from 50ul to 5000µl. 3. Deionized (DI) water. 4. Wash bottle or automated microplate washer. 5. Semi-log graph paper or software for data analysis. 6. Timer. 7. Absorbent paper. Cat No#KBBP10, Ver1.1 www.krishgen.com 2
Handling/Storage: 1. All reagents should be stored at 2 0 C to 8 0 C for stability. 2. All the reagents and wash solution are stable until the expiration date of the kit. Once opened all kit components are stable for up to 2 months at 2 0 C to 8 0 C. 3. Before using, bring all components to room temperature (18-25 ºC). Upon assay completion return all components to appropriate storage conditions. Unused wells should be refrigerated and protected from moisture in the original cover carefully resealed. 4. The diluted wash solution can be used for at least 30 days when stored at 2 0 C to 8 0 C. 5. The Substrate is light-sensitive and should be protected from direct sunlight or UV sources. Health Hazard Warnings: 1. All the reagents provided may be harmful if ingested, inhaled or absorbed through the skin. 2. For Research or Manufacturing use only. Reagent Preparation: Please refer to lot specific instructions for preparation of the reagents. Procedural Notes: 1. For good assay reproducibility and sensitivity, proper washing of the ELISA plate to remove excess/unbound reagents is essential. 2. If the BSA concentration of the undiluted sample is less than the diluted sample, this may be indicative of the Hook Effect. High Dose Hook Effect may be observed in samples with very high concentrations of BSA, usually in samples from the initials stages of purifications. To overcome Hook Effect samples to be assayed should be sufficiently diluted with our recommended diluent. 3. Avoid assay of samples containing sodium azide (NaN 3 ), as it may destroy the HRP activity of the conjugate resulting in the under-estimation of the levels of BSA. 4. All Standards and Samples should be assayed at least in duplicates. 5. Maintain a repetitive timing sequence from well to well for all the steps to ensure that the incubation timings are same for each well. 6. If the Substrate has a distinct blue color prior to use it may have been contaminated and use of such substrate can lead to compromisation of the sensitivity of the assay. 7. The plates should be read within 30 minutes after adding the Stop Solution. 8. Make a work list in order to identify the location of Standards and Samples. Specimen Collection and Handling: This Bovine Serum Albumin kit is intended for the determination of the bovine serum albumin content in diluted sample. The dilution varies in dependence from the material. Samples collected shouldn t be stored longer than 48 hours at 2-8 C. For long term storage, samples should be stored at -20 C. Avoid repeated freezing and thawing of the same sample. Frozen samples should be warmed to room temperature and mixed thoroughly before testing. Cat No#KBBP10, Ver1.1 www.krishgen.com 3
Assay Procedure: 1. Bring all reagents to room temperature prior to use. It is strongly recommended that all Standards and Samples be run in duplicate or triplicate. A standard curve is required for each assay. 2. Pipette out 100µl of each Standards and Samples into the respective wells. 3. Cover the plate and incubate for 1 hour at room temperature. 4. Aspirate and wash plate 5 times with 300µl Wash Buffer (1X) and blot residual buffer by firmly tapping plate upside down on absorbent paper. Wipe off any liquid from the bottom outside of the microtiter wells as any residue can interfere in the reading step. 5. Pipette out 100µl of Anti-BSA-HRP Conjugate into each well. 6. Cover the plate and incubate for 1 hour at room temperature. 7. Wash plate 5 times with 300µl Wash Buffer (1X) as in step 4. 8. Pipette out 100µl of TMB Substrate in each well. 9. Incubate the plate at room temperature for 15 minutes in dark. DO NOT SHAKE or else it may result in higher backgrounds and worse precision. Positive wells should turn bluish in color 10. Pipette out 100µl of Stop Solution. Wells should turn from blue to yellow in color. 11. Read the absorbance at 450nm. Calculation of Results: Calculate the mean absorbance of standards and samples. Create a reference curve from the mean absorbance of the standards (y-axis) and corresponding bovine serum albumin concentrations of these standards (x-axis). Plot the mean absorbance of the calibrator of standards (y-axis) against the corresponding BSA concentrations of these standards (x-axis). Determine the BSA concentration of the unknown samples by referring their mean absorbance to the reference curve. Samples with BSA concentrations exceeding those of 500ng/ml should be retested in higher pre-dilution. In case of sample pre-dilution values have to be multiplied with the dilution factor. Test Validity: A test run is valid if: The mean absorbance of the standard 500ng/ml is 1.50. The mean absorbance of the standard 15ng/ml is 0.50. Repeat the test if the above mentioned quality criteria are not met. Make sure that the test procedure is correct. Incorrect results may be caused by contamination of reagents or working instruments with BSA. Cross contamination of the kit reagents and samples, bacterial or fungal contaminations of reagents and/or samples, incorrect washing and incorrect incubation times may also cause erroneous results. Safety Precautions: This kit is for in vitro use only. Follow the working instructions carefully. The expiration dates stated on the kit are to be observed. The same relates to the stability stated for reagents Do not use or mix reagents from different lots. Do not use reagents from other manufacturers. Avoid time shift during pipetting of reagents. All reagents should be kept in the original shipping container. Some of the reagents contain small amount of sodium azide (< 0.1 % w/w) as preservative. They must not be swallowed or allowed to come into contact with skin or mucosa. Source materials maybe derived from human body fluids or organs used in the preparation of this kit were tested and found negative for HBsAg and HIV as well as for HCV antibodies. However, no known test Cat No#KBBP10, Ver1.1 www.krishgen.com 4
guarantees the absence of such viral agents. Therefore, handle all components and all patient samples as if potentially hazardous. Since the kit contains potentially hazardous materials, the following precautions should be observed - Do not smoke, eat or drink while handling kit material - Always use protective gloves - Never pipette material by mouth - Wipe up spills promptly, washing the affected surface thoroughly with a decontaminant. In any case GLP should be applied with all general and individual regulations to the use of this kit. LIMITED WARRANTY Krishgen Biosystems does not warrant against damages or defects arising in shipping or handling, or out of accident or improper or abnormal use of the Products; against defects in products or components not manufactured by Krishgen Biosystems, or against damages resulting from such non-krishgen Biosystems made products or components. Krishgen Biosystems passes on to customer the warranty it received (if any) from the maker thereof of such non Krishgen made products or components. This warranty also does not apply to Products to which changes or modifications have been made or attempted by persons other than pursuant to written authorization by Krishgen Biosystems. THIS WARRANTY IS EXCLUSIVE. The sole and exclusive obligation of Krishgen Biosystems shall be to repair or replace the defective Products in the manner and for the period provided above. Krishgen Biosystems shall not have any other obligation with respect to the Products or any part thereof, whether based on contract, tort, strict liability or otherwise. Under no circumstances, whether based on this Limited Warranty or otherwise, shall Krishgen Biosystems be liable for incidental, special, or consequential damages. This Limited Warranty states the entire obligation of Krishgen Biosystems with respect to the Products. If any part of this Limited Warranty is determined to be void or illegal, the remainder shall remain in full force and effect. Krishgen Biosystems. 2016 THANK YOU FOR USING KRISHGEN PRODUCT! Cat No#KBBP10, Ver1.1 www.krishgen.com 5