Human Insulin ELISA Kit User Manual Catalog number: GTX15041 GeneTex
Table of Contents A. Product Description... 2 B. Kit Components... 3 C. Additional Required Materials (not included)... 3 D. Reagent Preparation... 4 E. Assay Procedure... 5 F. Calculation of Results... 6 G. Storage... 6 H. Troubleshooting... 7 1
A. Product Description GeneTex s Human Insulin levels in serum and plasma, urine, and cell culture supernatants. A 96-well plate (in a strip format with 12 strips and 8 wells per strip) is coated with an antibody specific for human Insulin. Insulin standards and experimental samples (prepared as described in Reagent Preparation ) are added to the wells where the Insulin is captured by the bound antibody. A series of washes is performed and the immobilized Insulin in the standard and sample wells is then bound by biotinylated anti-human Insulin antibody. The wash sequence is repeated to remove the unbound detection antibody, followed by addition of HRP-conjugated streptavidin that will lock on to the detection antibody. The washes are repeated and the specific color signal is generated with TMB substrate pipetted into the wells. Stop Solution will make the blue color turn to yellow, and the strip is then ready to read at 450 nm. Benefits: Quantitative colorimetric assay Sensitivity to less than 4 IU/ml Strip/well format offers experimental flexibility 2
B. Kit Components Item A. Microplate with immobilized Capture Antibody Component One 96-well (12 strips with 8 well/strip) microplate pre-coated with antibody to human Insulin B. Wash Buffer 25 ml of wash buffer concentrate (20x) C. Standards Two vials of recombinant human Insulin protein D. Assay Diluent A 30 ml of diluent buffer with 0.09% sodium azide as preservative (For standard and sample (serum/plasma) dilution) E. Assay Diluent B 15 ml of 5x concentrated buffer (For standard and sample (urine/cell culture medium) dilution) F. Detection Antibody Two vials of biotinylated human Insulin antibody (each vial is enough to assay one-half of the microplate) G. HRP-Streptavidin 200 µl of 600x concentrated HRP-conjugated streptavidin H. TMB Substrate 12 ml of 3,3,5,5 -tetramethylbenzidine (TMB) in buffer solution I. Stop Solution 8 ml of 0.2 M sulfuric acid Item I (Stop Solution) contains a strong acid that may cause severe skin burns and eye damage and should be handled with protective gloves/ protective clothing/ eye protection/ face protection. If in contact with skin or eye, flush with plenty of water for at least 15 minutes (remove contact lenses if possible). See a physician immediately. C. Additional Required Materials (not included) 1. Microplate reader capable of measuring absorbance at 450 nm 2. Precision pipettes to deliver 2-1000 µl volumes 3. Adjustable 1-25 ml pipettes for reagent preparation 4. 100 ml and 1 liter graduated cylinders 5. Absorbent paper 6. Sterile ddh 2 O 7. Log-log graph paper or computer and software for ELISA data analysis 8. Tubes to prepare standard or sample dilutions 3
D. Reagent Preparation 1. Bring all reagents and samples to room temperature (18 25 C) before use. 2. Sample dilution : If your samples require dilution, Assay Diluent A (Item D) should be used for dilution of serum/plasma samples. Assay Diluent B (Item E) (first diluted 1:5 with sterile ddh 2 O to a 1x final working concentration) should be used for dilution of urine and culture supernatants. A 2-10 fold dilution is suggested for normal serum/plasma samples. Please note that levels of the target protein may vary between different specimens. Optimal dilution factors for each sample must be determined empirically by the investigator. 3. Preparation of standards: Briefly centrifuge a vial of the recombinant protein standard (Item C) and add 400 µl of either Assay Diluent A (for serum/plasma samples) or 1x Assay Diluent B (for urine/culture supernatants) into the vial to prepare a 1,400 IU/ml standard. Dissolve the lyophilized powder thoroughly by gently mixing and centrifuge briefly to collect all liquid. Repeat the mixing and centrifuge steps if necessary until the protein is completely dissolved. 4. Add 150 µl standard from the vial of Item C into a tube with 550 µl Assay Diluent A or 1x Assay Diluent B to prepare a 300 IU/ml stock standard solution. Next, pipette 300 µl Assay Diluent A or 1x Assay Diluent B into the appropriate number of tubes. Then use the 300 IU/ml standard solution to produce a set of two-fold serial dilutions (as shown below) by transferring 300 µl of each tube to the 300 µl of buffer in the next tube. Mix each tube thoroughly before the next transfer. Assay Diluent A or 1x Assay Diluent B alone serves as the zero standard (0 ng/ml). 5. If the Wash Buffer Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until completely dissolved. Dilute 20 ml of Wash Buffer Concentrate (20x) into 380 ml sterile ddh 2 O to yield 400 ml of 1x Wash Buffer. 4
6. Briefly centrifuge the Detection Antibody vial (Item F) before use. Add 100 µl of 1x Assay Diluent B into the vial to prepare a Detection Antibody concentrate. Pipette gently to mix (the concentrate can be stored at 4 C for up to 5 days). An appropriate volume of Detection Antibody concentrate should then be diluted 80-fold with 1x Assay Diluent B for use in Step 4 of Section E. Assay Procedure. 7. Briefly centrifuge the HRP-Streptavidin concentrate vial (Item G) and gently pipette to mix before use. An appropriate volume of the HRP-Streptavidin concentrate should then be diluted 500-fold with 1x Assay Diluent B for use in Step 6 of Section E. Assay Procedure. E. Assay Procedure 1. Bring all reagents and samples to room temperature (18-25 C) before use. It is recommended that all standards and samples be run at least in duplicate. 2. Add 100 µl of each standard and sample (see Section D. Reagent Preparation, Step 3 and 4) into appropriate microplate wells (Item A). Make sure the bottom of each microplate well is completely covered with standard or sample. Cover microplate wells and incubate for 2.5 hours at room temperature or overnight at 4 C with gentle shaking. 3. Discard the solution and wash 4 times with 1x Wash Buffer (see Section D. Reagent Preparation, Step 5). Wash by filling each well with 1x Wash Buffer (300 µl) using a multi-channel pipette or autowasher. Complete removal of liquid at each step is essential for optimal performance. After the last wash, remove any remaining 1x Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. 4. Add 100 µl of the diluted Detection Antibody solution (see Section D. Reagent Preparation, Step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking. 5. Discard the solution. Repeat the washes as in Step 3. 6. Add 100 µl of the diluted HRP-Streptavidin solution (see Section D. Reagent Preparation, Step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking. 7. Discard the solution. Repeat the washes as in Step 3. 5
8. Add 100 µl of TMB Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking. 9. Add 50 µl of Stop Solution (Item I) to each well. Read at 450 nm immediately. Stop Solution (Item I) contains strong acid that causes severe skin burns and eye damage and should be handled with protective gloves/ protective clothing/ eye protection/ face protection. If in contact with skin or eye, flush with plenty of water for at least 15 minutes (remove contact lenses if possible). See a physician immediately. F. Calculation of Results For each set of duplicate standards, controls, and samples, calculate the mean absorbance and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or by using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points. * The antibody pair ( Capture and Detection ) used in this Human Insulin ELISA Kit (GTX15041) is optimized to detect human Insulin. Reactivity to other species has not been determined. G. Storage Kit may be stored for up to six months at 2-8 C from the date of shipment. Standard protein (recombinant Insulin) should be stored at -20 C or -80 C following reconstitution. Opened microplate strips or reagents may be stored for up to one month at 2-8 C. Return unused wells to the pouch containing desiccant pack and reseal completely. The kit can be used within one year if the entire kit is stored at -20 o C. Avoid repeated freeze-thaw cycles. 6
H. Troubleshooting Problem Possible Cause Solution 1. Poor standard curve 1. Pipetting errors. 2. Errors in dilution of standards. 1. Check pipette calibration. 2. Ensure that vial of standard is completely dissolved in buffer. 3. Ensure every dilution is mixed thoroughly before transfer. 2. Weak signal 1. Incubation times too short 2. Errors in reagent preparation 1. Verify correct incubation times. Please note that incubation time in Step 2 of Section E Assay Procedure can be overnight as indicated. 2. Check pipettes and review reagent preparation procedure. 3. High background Inadequate washes or contaminated wash buffer 1. Check wash procedure. If using a plate washer, check that all ports are functioning. 2. Prepare fresh wash buffer. 4. Low sensitivity 1. Improper kit storage 2. Stop Solution 1. Review storage conditions. 2. Remember to add Stop Solution to each well before measurement. 7