|
Principle
|
This assay employs the competitive inhibition enzyme immunoassay technique. A polyclonal antibody specific for human HB has been pre-coated onto a microplate. A competitive inhibition reaction is launched between HRP labeled human HB and unlabeled human HB (Standards or samples) with the pre-coated antibody specific for human HB. The more the amount of human HB in samples, the less the HRP labeled human HB bound by pre-coated antibody. The substrate solution are added to the wells, respectively. And the color develops in opposite to the amount of human HB bound in the initial step. The color development is stopped and the intensity of the color is measured.
|
|
Reagent Preparation
|
1. Bring all kit components and samples to room temperature (18-25 °C ) before use. 2. Standard - Reconstitute the Standard with 0.8 ml of Standard Diluent, kept for 10 minutes at room tempreture, shake gently(not to foam). The concentration of the standard in the stock solution is 100 g/mL. Use the stock standard solution to produce a dilution series (shown below). Mix each tube thoroughly before the next transfer. Set up 5 points of diluted standard such as 100 g/mL, 33.3 g/mL, 11.1 g/mL, 3.7 g/mL,1.23 g/mL, and the last EP tubes with Standard Diluent is the blank as 0 g/mL. 3. Samples - Serum/plasma samples require about a 4,000 fold dilution. Sample should be diluted by 0.02 M PBS(PH=7.0-7.2). 4. Assay Diluent A - Dilute 6mL of Assay Diluent A (2 ) with 6ml of deionized or distilled water to prepare 12 mL of Assay Diluent A . The prepared working dilution can't be frozen. 5. Detection Reagent A - Briefly spin or centrifuge the stock Detection A before use. Dilute to the working concentration with working Assay Diluent A(1:100). 6. Wash Solution - Dilute 20mL of Wash Solution Concentrate(30 ) with 580ml of deionized or distilled water to prepare 600 mL of Wash Solution(1 ). 6. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again. Note: 1. Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37 °C directly. 2. Making serial dilution in the wells directly is not permitted. 3. Please carefully reconstitute Standards or working Detection Reagent A according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10l for once pipetting. 4. The reconstituted Standards, Detection Reagent A can be used only onceonce. 5. If crystals have formed in the Wash Solution concentrate warm to room temperature and mix gently until the crystals have completely dissolved.
|
|
Sample Collection
|
Sample Serum - Use a serum separator tube and allow Samples to clot for two hours at room temperature or overnight at 4 °C before centrifugation for 20 minutes at approximately 1000 g. Assay freshly prepared serum immediately or store Samples in aliquot at - 20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge Samples for 15 minutes at 1000 g at 2-8 °C within 30 minutes of collection. Remove plasma and assay immediately or store Samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles. Other biological fluids: Centrifuge samples for 20 minutes at 1000 g. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.
|
|
Assay Procedure
|
Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments. 1. Determine wells for diluted standard, blank and sample. Prepare 5 wells for standard, 1 well for blank. Add 50 each of dilutions of standard (read Reagent Preparation ), blank and samples into the appropriate wells, respectively. And then add 50 of Detection Reagent A to each tube immediately. Shake the plate gently. Cover with a Plate sealer. Incubate for 1 hour at 37 °C°C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform. 2. Aspirate the solution and wash with 400 of 1X Wash Solution to each well using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1-2 minutes. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Repeat 5 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper. 3. Add 90 of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 25 minutes at 37 °C (Don't exceed 30 minutes). Protect from light. The liquid will turn blue by the addition of Substrate Solution. 4. Add 50 of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 5. Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the surface of the liquid. Then, run the microplate reader and conduct measurement at 450nm immediately. Note: 1. Assay preparation: Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4 until the kits expiry date. 2. Samples or reagents addition Please use the freshly prepared Standard. Please additionPlease carefully add samples to wells and mix gently to avoid foaming. Do not touch the well wall as possible. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and specimens, although not required, is recommended. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. 3. Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation time and temperature must be observed. 4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate. Insufficient washing will result in poor precision and falsely elevated absorbance reading. 5. Controlling of reaction time: Observe the change of colour after adding TMB , Substrate (e.g. observation once every 10 minutes), if the colour is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading. 6. TMB Substrate is easily contaminated. Please protect it from light.
|
|
Calculation of Results
|
This assay employs the competitive inhibition enzyme immunoassay technique, so there is an inverse correlation between HB concentration in the sample and the assay signal intensity. Low levels of HB result in a high luminescence intensity, while a high concentration of HB results in a low signal. Average the duplicate readings for each standard, control, and samples. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the HB concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
|
|
Components
|
Reagents (Quantity): Pre-coated, ready to use 96-well strip plate (1), Plate sealer for 96 wells (4), Standard (liquid) 2 Standard Diluent (1 × 20ml), Detection Reagent A (1 × 120µl), Assay Diluent A (2 x concentrate) (1 × 6ml), TMB Substrate (1 × 9ml), Stop Solution (1 × 6ml), Wash Buffer(30 x concentrate) (1 × 20ml)
|
|
Material not included
|
1. Microplate reader with 450 ± 10 nm filter. 2. Precision single and multi-channel pipettes and pipette tips with disposable tips. 3. Eppendorf Tubes for diluting samples. 4. Deionized or distilled water. 5. Absorbent paper for blotting the microtiter plate. 6. Container for Wash Solution
|
|
Storage
|
All the reagents should be kept according to the labels on vials.The Standard Detection Standard, Reagent A and the 96-well strip plate should be stored at -20 °C upon being received. The unused strips should be kept in a sealed bag with the desiccant provided to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit (six months from the date of manufacture). Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above.
|
|
Restrictions
|
For Research Use only
|
Alternatives 
