Rat Hepatitis A virus cellular receptor 1,HAVcr-1 ELISA Kit
Catalog No: E0785r
FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!
PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!
Hepatitis A virus cellular receptor 1,HAVcr-1
This immunoassay kit allows for the in vitro quantitative determination of rat Hepatitis A virus cellular receptor 1 ,HAVcr-1 concentrations in serum, plasma, tissue homogenates, cell culture supernates, and other biological fluids.
The ELISA is based on the competitive binding enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with an antibody specific to C4a, During the reaction, C4a in the sample or standard competes with a fixed amount of biotin-labeled C4a for sites on a pre-coated Monoclonal antibody specific to C4a. Excess conjugate and unbound sample or standard are washed from the plate. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of C4a in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Materials and components
Assay plate 1
Sample Diluent 1 × 20ml
Assay Diluent A 1 × 10ml
Assay Diluent B 1 × 10ml
Detection Reagent A 1 × 60μl
Detection Reagent B 1 × 120μl
Wash Buffer(25 x concentrate) 1 × 30ml
Substrate 1 × 10ml
Stop Solution 1 × 10ml
Plate sealer for 96 wells 5
Other supplies required
Pipettes and pipette tips.
Deionized or distilled water.
Storage of the kits
The Assay Plate, Standard, Detection Reagent A and Detection Reagent B should be stored at -20℃ upon being received. After receiving the kit , Substrate should be always stored at 4℃.Other reagents are kept according to the labels on vials. But for long term storage, please keep the whole kit at -20℃. 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.
Sample collection and storage
Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 × g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2 - 8℃ within 30 minutes of collection. Store samples at -20℃ or -80℃. Avoid repeated freeze-thaw cycles.
Urine - Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particulate matter, assay immediately or aliquot and store at ≤ -20℃. Avoid repeated freeze-thaw cycles.
Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissue was rinsed with 1X PBS to remove excess blood, homogenized in 20 mL of 1X PBS and stored overnight at ≤ -20℃. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 x g. Remove the supernate and assay immediately or aliquot and store at ≤ -20℃.
Cell culture supernates and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃ or -80℃. Avoid repeated freeze-thaw cycles.
Limitations of the procedure
Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer.
Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 10.0 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard (10.0 ng/mL ). The Sample Diluent serves as the zero standard (0 ng/mL).
ng/mL 10.0 5.00 2.50 1.25 0.62 0.31 0.15 0
Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A and B (1:100), respectively.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37℃ directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. 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. Prepare all reagents, working standards and samples as directed in the previous sections. 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. Add 50 μl of Standard, Blank, or Sample per well.
2. Immediately add 50 μl of Detection A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C.
3. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (approximately 400 μl) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
4. Add 100 μl of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 45 minutes at 37℃.
5. Repeat the aspiration/wash process for five times as conducted in step 3.
6. Add 90 μl of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 15-30 minutes at 37°C. Protect from light.
7. Add 50 μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8. Determine the optical density of each well at once, using a microplate reader set to 450 nm.
1. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents should be freshly prepared prior to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.
2. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards Detection Reagent A and B can be used only once. This assay requires pipetting of small volumes. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10μl for once pipetting.
3. 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.
4. For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 10 minutes.
5. 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.
6. The wash procedure is critical. insufficient washing will result in poor precision and falsely elevated absorbance readings.
7. Duplication of all standards and specimens, although not required, is recommended.
8. Substrate Solution is easily contaminated. Please protect it from light.
This assay recognizes recombinant and natural Rat C4a. No significant cross-reactivity or interference was observed.
Limited by current skills and knowledge, it is impossible for us to complete the cross- reactivity detection between rat C4a and all the analogues, therefore, cross reaction may still exist.
The minimum detectable dose of Rat C4a is typically less than 0.39 ng/mL.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.
Calculation of results
Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. 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 C4a concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 1.3. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
This graph data is shown as an example. A standard curve must be generated each time the assay is run.