Guinea-pig IL1a (Interleukin 1 Alpha) Elisa Kit

Awaiting product image

Guinea-pig IL1a (Interleukin 1 Alpha) Elisa Kit

Sku Size Price Quantity
EK245363 96 Wells $380
MSDS
SKU:EK245363
Categories: ,
Uniprot ID:Q60480
Species:Guinea-pig
Assay Principle:Quantitative
Assay Type:Sandwich
Target Name:IL1a (Interleukin 1 Alpha)
Sample Type:Serum, Plasma, other Biological Fluids
Sensitivity:6.6 pg/ml
Detection Range Low:15.63 pg/ml
Detection Range High:1000 pg/ml
Standard:1000 pg/ml
Assay Time:3.5 h
Sample Volume:50 - 100 μL
Detection Wavelength:450 nm
Research Area:Cytokine - Apoptosis

Components

Components Quantity
1 x 96 Tests		 Storage Condition
Pre-Coated Microplate8 wells ×12 strips4°C/-20°C
Standard2 vials4°C/-20°C
Biotinylated-Conjugate (100×)1x120μL4°C/-20°C
Streptavidin-HRP (100×)1x120μL4°C/-20°C
Standard/Sample Diluent Buffer1x20mL4°C/-20°C
Biotinylated Antibody Diluent1x12mL4°C/-20°C
HRP Diluent1x12mL4°C/-20°C
Wash Buffer (25×)1x20mL4°C/-20°C
TMB Substrate Solution1x10mL4°C/-20°C (Store in Dark)
Stop Reagent1x6mL4°C/-20°C
Plate Covers2 piecesRT

Test Principle

This assay employs the sandwich enzyme immunoassay technique. The microtiter plate included in this kit has been pre-coated with an antigen specific to IL1a (Interleukin 1 Alpha). Standards or samples are added to the appropriate microtiter plate wells, followed by a biotin-conjugated antibody specific to IL1a (Interleukin 1 Alpha). Next, avidin conjugated to horseradish peroxidase (HRP) is added to each microplate well and incubated. After incubation, the TMB substrate solution is added. The enzyme-substrate reaction is then terminated by the addition of the stop solution, and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 10 nm. The concentration of IL1a (Interleukin 1 Alpha) in the samples is determined by comparing the OD of the samples to the standard curve.

Sample Collection

Serum: Allow samples to clot for 1 hour at room temperature or overnight at 2–8°C before centrifugation at 1,000×g for 20 minutes at 2–8°C. Collect the supernatant to carry out the assay.

Plasma: Collect plasma using EDTA-Na₂ as an anticoagulant. Centrifuge samples at 1,000×g for 15 minutes at 2–8°C within 30 minutes of collection. Collect the supernatant to carry out the assay.

Tissue Homogenates: It is recommended to consult specific literature for different tissue types before processing. Note that hemolyzed blood may interfere with results; therefore, tissues should be minced into small pieces and rinsed thoroughly in ice-cold PBS (0.01M, pH 7.4) to remove excess blood. Weigh the tissue pieces and homogenize them in PBS at a ratio of 1:9 (e.g., 1g tissue to 9mL PBS) using a glass homogenizer on ice. To further disrupt the cells, sonicate the suspension with an ultrasonic cell disrupter or subject it to repeated freeze-thaw cycles. Centrifuge the homogenates at 5,000×g for 5–10 minutes at 2–8°C and collect the supernatant.

Cell Lysates: For adherent cells, gently wash the cells with a moderate amount of pre-cooled PBS and dissociate them using trypsin. Collect the cell suspension into a centrifuge tube and centrifuge at 1,000×g for 5 minutes. Discard the medium and wash the cells three times with pre-cooled PBS. Resuspend the cells in pre-cooled PBS (approximately 150–250 μL of PBS per 1×10⁶ cells). Lyse the cells via repeated freeze-thaw cycles or ultrasonic cell disruption until the cells are fully lysed. Centrifuge at 1,500×g for 10 minutes at 2–8°C. Remove cell debris and collect the supernatant for the assay.

Urine: Collect the first midstream urine of the day directly into a sterile container. Centrifuge to remove particulate matter. Assay immediately or store in aliquots at ≤ −20°C. Avoid repeated freeze-thaw cycles.

Saliva: Collect saliva using a specialized collection device. Centrifuge the samples at 1,000×g for 15 minutes at 2–8°C. Remove any remaining particulates and assay immediately, or store in aliquots at ≤ −20°C. Avoid repeated freeze-thaw cycles.

Feces: Collect dry feces, ideally weighing more than 50 mg. Dilute the sample with PBS or lysis buffer at a ratio of 1:9 (w:v) (e.g., add 900 µL of buffer to 100 mg of feces). Homogenize the sample by mashing or sonication. Centrifuge the mixture at 5,000×g for 10 minutes and collect the supernatant for testing.

Cell Culture Supernatant and Other Biological Fluids: Centrifuge samples at 1,000×g for 20 minutes at 2–8°C. Collect the supernatant to carry out the assay.

Recommended Reagents for Sample Preparation: 10× EDTA Anticoagulant, PMSF Protease Inhibitor, 0.25% Trypsin Solution.

Notes

  1. Samples intended for use within 5 days may be stored at 4°C; otherwise, samples must be stored at −20°C (for up to 1 month) or −80°C (for up to 2 months) to avoid loss of bioactivity and contamination.
  2. Sample hemolysis can influence the results, so hemolytic specimens should not be used.
  3. When performing the assay, bring the samples to room temperature.

Reagent Preparation

Bring all reagents to room temperature (18–25°C) before use. If the kit will not be fully utilized in one assay, please take out only the necessary strips and reagents for the current experiment and store the remaining strips and reagents under the required conditions. If the kit will not be used in one session, only take out the strips and reagents needed for the current experiment, and save the remaining strips and reagents as specified.

Dilute the 25× Wash Buffer to 1× Wash Buffer with double-distilled water.

Centrifuge the standard at the specified speed for 1 minute. Reconstitute the standard with the designated volume of Standard Diluent Buffer, allowing it to sit at room temperature for approximately 10 minutes. Gently shake the solution to avoid foaming.

Prepare seven tubes, each containing a specific volume of Standard Diluent Buffer, and use the diluted standard to create a double dilution series. Mix each tube thoroughly before proceeding to the next transfer by pipetting the solution up and down several times. Establish a series of diluted standards across the tubes, ending with a final tube that contains only Standard Diluent, which will serve as the blank. To ensure the validity of your experimental results, always use a new standard solution for each experiment. When performing the dilutions, change the pipette tip for each new dilution to prevent cross-contamination. Remember that the last tube is designated as the blank; do not transfer any solution into it from the preceding tube.

Standard dilution tubes
Tube6 (Stock)543210 (Blank)
pg/ml100050025012562.531.250

Preparation of 1× Biotinylated-Conjugate and 1× Streptavidin-HRP Working Solution:

  1. Briefly spin or centrifuge the stock solutions of Biotinylated-Conjugate and Streptavidin-HRP to ensure they are well mixed.
  2. Dilute each solution to a working concentration of 100-fold:
    • For the Biotinylated-Conjugate, use the Biotinylated-Conjugate Diluent.
    • For the Streptavidin-HRP, use the HRP Diluent.
  3. For example, to prepare the Streptavidin-HRP solution, mix 10 µL of Streptavidin-HRP with 990 µL of HRP Diluent.

TMB Substrate Solution — Aspirate the required dosage of the solution with sterile tips, and do not pour the residual solution back into the vial.

Assay Procedure

  1. Determine the wells for the Diluted Standard, Blank, and Sample. Prepare 7 wells for the Standard and 1 well for the Blank. Add 100 μL of the Standard Working Solution or 100 μL of samples into the appropriate wells. Cover with the Plate Cover and incubate for 80 minutes at 37°C. Note: Solutions should be added to the bottom of the micro ELISA plate wells. Avoid touching the inside wall to minimize foaming.
  2. Pour out the liquid from each well. Aspirate the solution and wash each well with 200 μL of 1× Wash Solution, letting it sit for 1–2 minutes. Completely remove the remaining liquid from all wells by snapping the plate onto absorbent paper. Perform a total of 3 washes. After the last wash, invert the plate and blot it against absorbent paper. Notes: (a) When adding Wash Solution, the pipette tip should not touch the walls. (b) Ensure that the washing liquid is poured directly into the wells to prevent contaminating other wells.
  3. Add 100 μL of Biotinylated Antibody Working Solution to each well, cover with the Plate Cover, and incubate for 50 minutes at 37°C.
  4. Repeat the aspiration and wash process a total of 3 times as described in step 2.
  5. Add 100 μL of Streptavidin-HRP Working Solution to each well, cover with the plate sealer, and incubate for 50 minutes at 37°C.
  6. Repeat the aspiration and wash process a total of 5 times as conducted in step 2.
  7. Add 90 μL of TMB Substrate Solution to each well. Cover with a new Plate Cover and incubate for 20 minutes at 37°C (do not exceed 30 minutes) in the dark. The liquid will turn blue. Preheat the microplate reader for about 15 minutes before measuring OD.
  8. Add 50 μL of Stop Reagent to each well. The liquid will turn yellow. Mix by tapping the side of the plate. If the color change does not appear uniform, gently tap the plate. The order of adding the Stop Reagent should be the same as that of the TMB Substrate Solution.
  9. Wipe off any drops of water and fingerprints from the bottom of the plate, and confirm that there are no bubbles on the surface of the liquid. Run the microplate reader and conduct measurements at 450 nm immediately.

Calculation of Results

Average the duplicate readings for each standard, control, and sample, and then subtract the average optical density of the zero standard. Construct a standard curve with concentration on the y-axis and absorbance on the x-axis, and draw a best-fit curve. If samples have been diluted, multiply the concentration by the dilution factor.

Troubleshoot Common Elisa Issues

Pipetting and washing techniques for ELISA
Pipetting technique
  1. Use the correct pipette that is within the range suggested by manufacturer
  2. Confirm tip is firmly seated on the pipette
  3. Confirm there are no air bubbles while pipetting
  4. Change tips between each standard, sample, or reagent
  5. Use different reservoirs for each reagent
  6. Pipette sample into the side of wells to avoid splashing
  7. Always run samples/standards in replicate
Washing Procedure
  1. Completely aspirate liquid from all wells by gently lowering an aspiration tip into the bottom of each well.
    Note: Take care not to scratch the inside of the well.
  2. Fill the wells with at least 500 µL of diluted wash buffer
  3. Let soak for 25 to 30 seconds
  4. Aspirate wash buffer from wells
  5. Repeat as directed in protocol (usually 4-5 times)
  6. After washing is complete, invert plate and tap (forcefully, if necessary) dry on absorbent tissue. Be sure to remove any residual liquid.
  7. Alternatively, a squirt bottle or automated plate washer may be used. Be sure to follow the above.
Problem: No color plate
Possible Cause Solution
Mixed use of component reagents Please read labels clearly when preparing or using
In the process of plate washing and sample addition, the enzyme marker is contaminated and inactivated, and loses its ability to catalyze the color developing agent Confirm that the container holding the ELISA plate does not contain enzyme inhibitors (such as NaN3, etc.), and confirm that the container for preparing the Wash Solution has been washed.
Missing a reagent or a step Review the manual in detail and strictly follow the operating steps.
Problem: Light Color
Possible Cause Solution
The sample uses NaN3 preservative, which inhibits the reaction of the enzyme Samples cannot use NaN3
The sample to be tested may not contain strong positive samples, so the result may be normal In case of doubt, please test again.
Wrong filter used for absorbance reading When TMB is used as the substrate, the absorbance should be read at 450 nm.
Insufficient incubation time Make sure to follow recommended incubation times.
Insufficient color reaction Usually 15 - 30 minutes
The number of washings increases, and the dilution ratio of the concentrated lotion does not meet the requirements Reduce the impact of washing, dilute the concentrated lotion and washing time according to the manual, and accurately record the washing times and dosage.
Distilled water quality problem The prepared lotion must be tested to see if the pH value is neutral.
In the process of plate washing and sample addition, the enzyme marker is contaminated and inactivated, and loses its ability to catalyze the color developing agent. Confirm that the container holding the ELISA plate does not contain enzyme inhibitors (such as NaN3, etc.), confirm that the container for preparing the Washing Solution has been washed, and confirm that the purified water for preparing the Washing Solution meets the requirements and is not contaminated.
The kit has expired or been improperly stored Please use it within the expiration and store it in accordance with the storage conditions recommended in the manual to avoid contamination.
Reagents and samples are not equilibrated before use All reagents and samples should be equilibrated at room temperature for about 30 minutes.
Insufficient suction volume of the pipette, too fast discharge of pipetting suction, too much liquid hanging on the inner wall of the tip or the inner wall is not clean. To calibrate the pipette, the tips should be matched, each time the tips should fit tightly, the pipetting should not be too fast, and the discharge should be complete. The inner wall of the tips should be clean, and it is best to use it once.
Incubation temperature constant temperature effect is not good Keep the temperature constant to avoid the local temperature being too high or too low
When adding liquid, too much remains on the medial wall of wells When adding liquid, the tip should try to add liquid along the bottom of the medial wall of wells without touching the bottom of the hole.
Reuse of consumables The tips should be replaced when different reagents are drawn, and different storage vessels should be used when configuring different reagent components.
The bottom of the microwell is scratched or there is dirt During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other.
Cross-contamination during sample addition Try to avoid cross-contamination when adding samples
Cross-contamination from manual plate washing When washing the plates by hand, the first 3 injections of the lotion should be discarded immediately, and the soaking time should be set for the next few times to reduce cross-contamination.
Cross-contamination when clapping Use a suitable absorbent paper towel when clapping the plate, do not pat irrelevant substances into the well of the plate, and try not to pat in the same position to avoid cross-contamination.
The liquid filling head of the plate washer is blocked, resulting in unsatisfactory liquid addition or large residual amount of liquid suction, resulting in the color of plate is chaotic and irregular. Unblock the liquid addition head, so that each well is filled with washing liquid when washing the plate and the residual amount should be small when aspirating liquid.
Incomplete centrifugation of the sample, resulting in coagulation in the reaction well or interference of sediment or residual cellular components Serum plasma should be fully centrifuged at 3000 rpm for more than 6 minutes
The sample is stored for too long time, resulting in contamination. Samples should be kept fresh or stored at low temperature to prevent contamination
Incorrect preparation of Washing Solution or direct misuse of concentrated Washing Solution Please configure according to the manual
Weak or no signal in ELISA
Possible Cause Solution
Reagents not at room temperature at start of assay It is recommended that all reagents be at room temperature before starting the assay. Allow reagents to sit on bench for 15–20 minutes to reach room temperature.
Incorrect storage of components Double check storage conditions on kit label. Most kits need to be stored at 2–8oC.
Expired reagents Confirm expiration dates on all reagents. Do not use reagents that are past the expiration date.
Reagents added/prepared incorrectly Check protocol, ensure reagents were added in the proper order and prepared to correct dilution.
Incorrect dilutions prepared Check pipetting technique—see below—and double check calculations.
Capture antibody didn’t bind to plate If using a ready-to-use kit: Manufactured kits come with plates pre-coated with capture antibody.
If coating your own plate with an Antibody Pair Kit: Ensure that you are using an ELISA plate, not a tissue culture plate. Dilute antibody in PBS. Ensure correct preparation and incubation time for both coating and blocking steps.
Wells scratched with pipette or washing tips Use caution when dispensing and aspirating into and out of wells. Automated plate washers may need to be calibrated so tips don’t touch bottom of wells.
Plate read at incorrect wavelength Make sure to use recommended wavelength/filter.
Too much signal in ELISA
Possible Cause Solution
Insufficient washing Use appropriate washing procedure—see below. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
Plate sealers not used or reused During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other.
Incorrect dilutions prepared Check pipetting technique—see below—and double-check calculations.
Longer incubation times thanrecommended Make sure to follow recommended incubation times.
Problem: High background in ELISA
Possible Cause Solution
Insufficient washing Use appropriate washing procedure—see below. Increasing duration of soak steps may also help. Add 30 seconds each time you let wash buffer soak. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
Substrateexposed to light prior to use Ensure substrate isnot exposed to light—store in a dark place. Limit exposure to light whilerunning assay.
The yellowingof the whole plate may be caused by wrong addition of other reagents Check the componentsand lot numbers of the reagents before the experiment, and confirm that allcomponents belong to the corresponding kit. Reagents from differentkits or different lot numbers cannot be mixed.
Streptavidin-HRP contaminates the tip and TMB container orpositive control contaminates the Pre-coated Microplate When absorbingdifferent reagents, the tips should be replaced. When configuring differentreagent components, different storage vessels should be used. Please use apipette during operation.
BiotinylatedAntibody or Streptavidin-HRP concentration too high Checkwhether the concentration calculation is correct or use after furtherdilution.
Color development time is too long Pleasestrictly follow the steps of the manual.
Longerincubation times than recommended Make sure to followrecommended incubation times.
Incorrectstandard curve dilutions prepared Check pipettingtechnique—see below—and double-check calculations.
The wrongfilter was used when the absorbance value was read When TMB is used asthe substrate, the absorbance should be read at 450 nm.
Problem: High background in ELISA
Possible Cause Solution
Incorrect standard curve dilutions prepared Check pipetting technique—see below—and double-check calculations.
Capture antibody didn’t bind to plate Ensure that you are using an ELISA plate, not a tissue culture plate. Dilute antibody in PBS. Ensure correct preparation and incubation time for both coating and blocking steps.
Problem: Poor replicate data in ELISA
Possible Cause Solution
Insufficient washing Use appropriate washing procedure—see below. Increasing duration of soak steps may also help. Add 30 seconds each time you let wash buffer soak. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
Capture antibody didn’t bind to plate Ensure that you are using an ELISA plate, not a tissue culture plate. Dilute antibody in PBS. Ensure correct preparation and incubation time for both coating and blocking steps.
Plate sealers not used or reused During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other.
Problem: Inconsistent results assay-to-assay in ELISA
Possible Cause Solution
Insufficient washing Use appropriate washing procedure—see below. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
Inconsistent incubation temperature Manufactured kits have optimized protocols. Make sure to follow recommended incubation temperatures. Be aware of fluctuations in temperature due to environmental conditions.
Plate sealers not used or reused During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other.
Incorrect dilutions prepared Check pipetting technique—see below—and double-check calculations.

Product Information

SKU:EK245363
Categories: ,

Related products