In order to help you better solve the various problems you may encounter in the experiment, we designed these problem guides, equipped with reagents
The instructions in the box are provided for your reference. Many factors will lead to the failure of immunoassay experiments, and most technologies
Mistakes can be avoided by a thorough reading and accurate understanding of the instructions and experimental procedures. If you have any instructions on the kit
Any comments and suggestions are welcome. We look forward to hearing from you to improve our products and better
To serve you.
â—† Read the manual carefully
â—† Check the expiration date on the kit label. If it expires, do not use it.
â—† According to the instructions to confirm that all reagents are complete (quantity, volume)
â—† Specimen preparation should be standardized, each sample volume should be prepared (storage) according to the amount of more than 2-3 complex holes, and packed as a backup as much as possible.
Those who are unable to experiment in the short term, pay attention to cryopreservation
â—† Prepared all additional items required for the experiment, (eg pipette, test tube, washer, microplate reader)
â—† Balance the reagents to room temperature according to the instructions, and determine the amount of reagents required according to the number of test specimens
â—† Check the storage conditions of each reagent in the kit to ensure that all reagents are stored according to the conditions recommended in the instructions in the kit.
â—† Check the unstable or deteriorated reagent solution (such as precipitation or discoloration). Some reagents, such as standard dilutions or
The measured diluent may contain sediment during design. All reagents must be thoroughly mixed before dropping into the microplate. And by
Due to the characteristics of the precipitate, it is necessary to keep stirring before adding the enzyme plate.
â—† Ensure sufficient incubation time and temperature.
â—† Do not use reagents with different production batches to replace the existing reagents or mix the existing reagents.
â—† When mixing or dissolving protein solution, avoid foam.
â—† Before the experiment starts, arrange the experiment flow.
â—† Before the experiment, clean the workbench.
â—† If there is any problem, you should contact the technical support of our company or agent
Washing Techniques
For any successful ELISA test, the correct plate washing method is a very critical and important aspect. Coherent plate washing is also
It is necessary. When diluting the concentrated washing liquid, please use deionized water or distilled water.
â—† Washing bottle or multi-channel pipette
Ensure that the pressure in the wash bottle is good or that the pipette tip is properly adjusted and free of debris. Check whether the slats in the board are installed
it is good. Number the slats to prevent loosening during dumping for easy adjustment. First, dump the microplate to empty the contents. According to the test
Add the lotion dropwise to each well in the volume recommended in the kit. If soaking is required in the experimental procedure, soak each hole regularly. will
The liquid in the plate is poured completely and wiped with a clean paper towel. Repeat the above steps as shown in the manual. After the last wash,
Pour the liquid in the plate completely, pat the surface with a clean paper towel and wipe dry. Do not let the holes dry. Follow the instructions in the kit
Quickly proceed to the next experimental operation.
â—† Automatic plate washer
Connect the automatic plate washer to an appropriate vacuum (according to the manufacturer's instructions). Make sure that each tube is properly aspirated. First, by pumping
Suction or dump to empty the plate. Add wash solution to each well according to the recommended volume in the kit. If soaking is required in the experimental procedure,
Then regularly soak each hole completely. Aspirate each hole completely to ensure that no lotion remains. After the liquid in the hole is completely pumped away
The device should be pumped too far into the hole. Repeat the above steps as shown in the manual. After washing the plate for the last time,
Pour the liquid completely, pat the surface with a clean paper towel and wipe dry. Do not let the holes dry. Follow the instructions in the kit quickly
Go to the next experimental operation.
Pipetting considerations
â—† The key to repeated and accurate pipetting is coherent operation. Smooth and fast pipetting operation is very important. Aspirate and release fluid
The body must avoid violent movements.
â—† Check whether the size of the gun head is appropriate and whether it is fixed intact.
â—† When dropping samples or reagents, replace the tip each time.
â—† Do not transfer less than the minimum volume of liquid recommended by the pipette manufacturer. Otherwise it will reduce the accuracy and repeat of the experiment
Sex.
â—† Some liquids tend to adhere to the inner and outer surfaces of the gun head, so it is best to wash the gun head in advance to avoid this surface tension
And the liquid volume change caused. This will increase the accuracy of pipetting.
â—† If the liquid to be moved is viscous, when absorbing the liquid, please stay in the liquid to be taken for a while until the volume reaches
Move out after balancing.
â—† After aspirating the liquid with a pipette, dry the outer surface of the pipette tip with a cotton-free tissue paper.
â—† If bubbles appear in the pipette tip when aspirating liquid, drain the aspirated liquid and re-absorb slowly from the container. If the gun head
If bubbles still appear inside, replace the gun head.
â—† If there is foam in the pipette tip when aspirating liquid, tilt the pipette slightly and suck the liquid slowly.
â—† Each reagent is separately prepared and stored
Sample Information
Can I collect samples the day before the experiment?
â—† Unless the instruction manual in the kit specifically says that it is not allowed, it is generally acceptable. If the collected samples cannot be tested in time,
Please save to -20 ℃ or save according to the instructions. Avoid repeated freezing and thawing.
◆ -20 ℃ is the recommended storage temperature. The thawing equilibrium process may cause degradation of the sample.
Do I have to prepare samples according to the method in the instructions in the kit?
â—† The recommended sample preparation method is the best method verified by experiment, so it is recommended to prepare samples according to the method in the manual
Goods.
Can I collect plasma without recommended collection methods?
â—† Every measurement according to the recommended steps is verified and feasible. Some anticoagulants are not recommended in certain assays
of. For details, please refer to the instruction manual.
â—† Some analytes to be detected are also present in platelets, so it is recommended to use low platelet plasma. Correct collection operator
Please refer to the instructions for the method.
Can I use my own diluent or buffer?
â—† Each kit contains a dilution solution prepared according to the formula to match the specific sample species to ensure the sample to be tested
Was properly diluted. For details, please refer to the instruction manual.
Suggest
â—† Make sure that the samples to be tested ensure aseptic operation during collection and preparation.
â—† If multiple samples are to be tested, isolate the samples and mark them clearly to prevent mutual contamination.
â—† Before testing, centrifuge the sample to remove particles. Transfer the sample to a new centrifuge tube and mix well.
â—† When mixing samples, use centrifuge tubes with a volume at least 50% larger than the sample volume to ensure full mixing.
I don't have enough diluent to dilute all samples, what should I do?
â—† A sufficient amount of diluent specified in the manual is provided in the kit. It can ensure that all standards and samples are tested twice.
â—† If there is no recommended dilution, or a large amount of dilution is required, please calculate the total amount of dilution required before the experiment, and then
Obtain additional diluent from the technical service department.
Can I use non-recommended sample types in the kit?
â—† The sample type specified in the instruction manual in the kit is verified and feasible. Other unrecommended sample types are not passed
Any detected.
The test results of my control are out of range, why?
â—† If a control is used, its concentration should be within a certain range. If the control value is outside the range, it is invalid.
â—† Make sure the control is repeatable.
Can I use the sample data in the instructions in the kit as a reference?
â—† The sample data in the manual is derived from a limited number of individuals, which can only be used as a general guide.
Can I omit the standard value of the standard curve in the manual?
â—† No. The sample value must be determined by the standard curve for each test.
If it is a hand-drawn standard curve, how do I determine the sample concentration?
â—† To determine the concentration of each sample, first find the photometric value or relative light unit of the Y axis, and then make a horizontal line to the standard curve.
At the intersection, make a vertical line to the X axis and read the corresponding concentration.
How can I compensate for the dilution or concentration of the sample?
â—† If the sample is diluted, the value read by the standard curve must be multiplied by the dilution.
â—† If the sample is concentrated, the value read by the standard curve must be divided by the concentration ratio.
How do I convert units of sample values ​​to Units?
â—† If you can use the NIBSC / WHO international standard, you can use the corresponding conversion equation described in the manual to convert. Insert
Enter your sample value into the equation to convert pg / mL or ng / mL to Units.
The sample values ​​I detected are outside the dynamic range of the determination experiment. Can I use these values?
◆ Only the sample value that decreases with the standard curve is recommended. Values ​​outside the range of the standard curve are non-linear, leading to
Wrong extrapolated value.
â—† If the measured sample value is higher than the value of the highest standard, the sample needs to be further diluted and re-checked
Measurement.
â—† If the measured sample value is far below the measurement range, the sample is considered to be undetectable.
My standard curve looks pretty good, but it does not achieve the results I expected. Why?
â—† The sample may not contain analytes.
â—† The influence of diluent may obscure the detection. Make sure to use the recommended dilution.
â—† If the recommended dilution is indeed used, check whether the dilution operation is correct. Excessive dilution will cause the sample value to fall below the standard
Quasi-curve range.
Precision / Reproducibility
The accuracy of the immunoassay can be determined by the repeatability between the wells of the microtiter plate and the repeatability between each test. Caused by high CV values
Low accuracy is generally caused by two factors: pipetting and washing operations.
Do I need to mix reagents in the wells?
â—† When multiple reagents are dropped into a well (such as diluent and sample), they need to be mixed in the well. Dropping reagents and samples
To the center of each well, tap the microplate to mix thoroughly.
My reagents / samples are sticky and not easy to absorb and move, what should I do?
â—† When sucking viscous liquid, please stay in the liquid to be taken for a period of time, and then remove it after the volume reaches equilibrium.
â—† Pre-wash the pipette tip with the corresponding reagent to compensate for the surface tension of the liquid.
Micropipette
â—† Insufficient or uneven liquid volume in the pipette tip indicates that the pipette needs to be calibrated. Check the function of the pipette and repeat if necessary
New calibration.
â—† When dropping reagents, standard products and samples, the tips should be replaced in time to avoid mutual contamination.
â—† Check whether the gun head is installed well. If installed improperly, it may result in inaccurate volume of liquid suction and discharge.
â—† When removing the pipette tip from the container, lightly lean the pipette tip against the wall of the container to remove the liquid remaining on the outer surface of the pipette tip
The correct way to wash the plate
â—† If using automatic plate washer, suction device or multi-channel pipette. Make sure that each tube is properly aspirated. The liquid is finished in the hole
Do not over-pump the device into the hole after it has been fully drawn. Repeat the above steps as shown in the manual. Last plate wash
After that, pour the liquid in the plate completely, pat the surface with a clean paper towel and wipe dry.
â—† Wash the plate too fast or too slow, do not wash the plate completely or suck.
â—† Various factors such as drying in the hole will affect the accuracy of the test results. Follow the instructions in the kit to quickly proceed to the next experiment
operating.
Can I use the same container for all reagents?
â—† No. Use different containers to hold different reagents to avoid contamination.
â—† Some analytes are extremely sensitive to contamination (such as saliva, oxidizing reagents, etc.). According to the instructions, pay attention to the prevention kit
Internal reagent contamination.
Can I reuse the cover plate?
â—† The reused cover plate may contain residues from the previous step, which may contaminate the existing components in the hole, resulting in reduced accuracy
low. So use a new cover plate every time you incubate. Many factors can affect the results of the standard curve, including
Preparation and operation.
Standard Curve
Many factors can affect the results of the standard curve, including preparation and operation.
Can I change the standard preparation?
â—† No. The recombinant standard appropriately diluted with the designated diluent is shown in the instruction manual.
â—† When mixing and dissolving standard products, avoid foaming.
â—† The dissolved standard should be allowed to stand for at least 15 minutes (according to the instructions). Mix gently before use to ensure all
The solid has dissolved.
â—† When making a standard curve, make sure that all dilution steps have been completed accurately. When diluting, pay attention to replace the tip in time. Pipetting
Mix the diluent thoroughly.
How does the washing method affect my standard curve?
â—† Incomplete washing will reduce the accuracy of the measurement, resulting in unsatisfactory standard curve results. Ensure the normal operation of the washer
The wells of the microtiter plate were washed thoroughly but not dried.
How does pipetting affect my standard curve?
â—† Unreasonable pipetting operation will lead to high CV value and unsatisfactory curve.
â—† In the process of making the standard curve, incorrect pipetting method will lead to wrong dilution, so that the wrong value will enter the standard
In the quasi-curve, or produce a non-linear curve. Make sure the pipette is working properly. The unequal volume of liquid in each hole may be shifting
Caused by malfunction of the liquid container or improper use of the pipette tip.
My computer software does not support the recommended data induction method. Can I use other methods?
â—† Yes, however, it is best to use the data induction method recommended in the manual for each measurement. Using different data induction methods will
Floating results.
â—† For different computer software, set the absorbance of the standard product to the Y axis and the concentration to the X axis. Made with logarithmic paper
The data should be linear, and regression analysis should be applied to logarithmic transformation. If logarithmic paper cannot be used, the logarithm of the density and
The logarithm of the OD value is plotted linearly. The optimal curve is determined by regression analysis.
Can I use the same standard curve when measuring multiple microplates?
â—† When measuring samples in different plates, each plate corresponds to a standard curve. Technical errors or different incubation situations,
Environmental conditions can cause different results between microplates. The sample value determined by a standard curve must be in the same environment
Generated below, otherwise it is invalid value.
My standard curve is flat or non-linear, what could be the cause?
There are many reasons for the unsatisfactory standard curve. The most common reasons are as follows:
â—† Determine whether the dilution is proper.
â—† Determine whether the wavelength is correct.
â—† Standards and samples must be added to the plate within 15-20 minutes. (Unless specifically stated in the instructions in the kit)
â—† Check whether the background is too high.
â—† Determine whether the microplate is washed correctly. Improper washing may cause flat curves with high background.
â—† Repeat the plate during the measurement process. Reading the board after the recommended time will cause erroneous results.
â—† If the reading of the highest standard exceeds the range of the microplate reader, determine whether the standard is dissolved correctly, incubation time and temperature
Is it accurate.
â—† Please repeat the measurement to ensure the accuracy of the experiment.
â—† If a control is used, the concentration of the control must be within the measurement range.
â—† Make sure that the reagents are not contaminated during testing and incubation.
â—† The flat standard curve without any signal may be due to the enzyme conjugate or substrate not reacting. Check each operation process
â—† Because of the limitations of many microplate readers, it is recommended that standard samples be tested from high to low.
Edge Effect
The definition of the edge effect is that there is a significant signal difference between the center part of the well of the microplate and the part far away from the center of the well of the microplate
The difference is generally attributed to two main factors.
Does the incubation environment really affect the experiment?
â—† Yes. Uneven temperature during incubation can cause edge effects. Please strictly follow the recommended temperature for incubation. If conditions permit,
Incubation can be carried out in a temperature-controlled incubator.
â—† Balance all reagents to room temperature before use. (Unless specifically stated)
â—† Check the working ambient temperature before the experiment starts. If the curve is very straight, it may be because the working environment temperature is lower than the air temperature
Caused by.
At the end of the incubation period, I noticed that the side of the cover plate protruded. Will this affect my experimental results?
â—† If the cover is not used properly, it will cause edge effects.
Drift
The definition of hole-hopping is that there is a significant inversion of the signal in the well from the left side to the right side or from the top to bottom
I just stopped when I configured the reagents. Will this affect my experimental results?
â—† This may cause a wavy curve. Determine whether all standards or samples are well prepared before pipetting. please at
Add the reagent to the plate within 15-20 minutes after preparation. (Unless specifically stated)
Do I need a multichannel pipette?
â—† When preparing such as substrate, enzyme conjugate, or diluent, it is best to choose continuous or multi-channel pipette.
What if I don't have equilibration reagents?
â—† Unbalanced reagents will cause uneven temperature between wells, which will variate the signal of the whole enzyme plate. Unless otherwise specified, try
The agent is generally equilibrated to room temperature before use.
â—† Each test will have recommended incubation time and temperature. Frozen reagents generally require longer incubation times. So it's better to make
Use the incubation temperature and time recommended in the instructions.
Do I need a cover?
â—† Refer to the instructions in the kit. If there is a recommendation to use a cover plate in the experimental steps in the instruction manual, you should use the enzyme
The cover plate matched with the target plate makes the edge fit tightly with the enzyme standard plate. If one side of the cover plate protrudes, there will be a difference in signal.
How does the setting of the microplate reader affect my results?
â—† For substrate measurement, if the reading time is equal to or more than 2 seconds / well, the time from the first well to the last well will exist when reading
Delays in this will lead to variability in results. During the substrate reaction phase, horseradish peroxidase will continue to act until the substrate reacts
complete. Due to the delay of time, the substrate is getting less and less, and the substrate reaction is also affected. Set the reading time of the microplate reader
It is 1.0 second / hole.
Can I adjust the incubation time according to my plan?
â—† Changing the incubation time or temperature will cause the test to fail to reach equilibrium or overreact. Please follow the recommended incubation time and
Temperature operation.
â—† If multiple tests are performed at the same time, each well must be timed separately to avoid insufficient or excessive incubation.
Signal Development
The change of signal is influenced by the following factors: substrate, incubation time, failure of enzyme conjugate and substrate reaction, wavelength and instrument.
Can I change the method of substrate processing?
â—† If the reactant solution needs to be mixed, ensure that the volume of the reactant reagent added is correct and fully mixed. The mixed solution must be
Use within 15 minutes (chemiluminescence detection up to 4 hours). If the reactants are mixed too early, the color in the container
Subject to change.
â—† If the substrate is a lyophilized product or a tablet, use the appropriate volume of diluent to completely dissolve it according to the method in the instructions. mixing
Use after completion and within the time recommended in the manual.
Do I have to follow a certain sequence when pipetting?
â—† Perform the pipetting operation according to the order in the manual.
â—† High-sensitivity detection utilizes an amplification system. After the substrate incubation is completed, the amplification reagents are added sequentially in the order of substrate addition.
â—† After the substrate is added, pat the enzyme plate gently to mix thoroughly.
Is the substrate likely to be contaminated?
â—† Yes. If you are using alkaline phosphatase or horseradish peroxidase-labeled conjugates, you should pay attention to the measurement
Avoid contamination. Contamination may result in values ​​that exceed the range of the microplate reader. Avoid contact with metals or oxidizing reagents. Use new
container.
Does the substrate need to be incubated in a dark environment?
â—† No need. However, direct sunlight should be avoided during the incubation process.
How does temperature affect results?
â—† Alkaline phosphatase or horseradish peroxidase are light-sensitive enzymes. Optical density unit or relative light unit will vary with temperature
Changes.
â—† Avoid incubation in places where environmental conditions change drastically. (Such as vents and windows where the temperature and light change dramatically
square)
To ensure that the enzyme conjugate and substrate react completely:
â—† For colorimetric determination:
Mix the same volume of conjugate and substrate solution thoroughly (for high sensitivity kits, add the same volume of conjugate and
After one minute of substrate, an equal volume of amplification reagent is added). If the color does not appear immediately, check if the added component is in
Stored at the correct storage temperature, whether it is contaminated, and whether it is within the validity period.
Do I need to correct the wavelength?
â—† For colorimetric measurement, the wavelength needs to be corrected to reduce the error.
What should I do if I have not corrected the wavelength?
â—† Subtract the original corrected wavelength from the read wavelength. This method can correct optical errors.
My microplate reader does not have the filter recommended to correct the wavelength, can I use another one?
â—† Yes. As long as the selected wavelength is higher than the recommended calibration wavelength.
My microplate reader does not have the wavelength recommended in the manual, can I use another one?
â—† The recommended wavelength is determined by the substrate used and the peak absorbance of the color after reaction. There are corresponding data in the manual, if not
If yes, choose the closest wavelength. However, this data change is relatively large.
Why is my relative light unit inconsistent with the instructions?
â—† Because there is no uniform standard for the measurement of light units in industry, the relative light units vary greatly between microplate readers
Big. The values ​​of relative light units should be comparable.
â—† Use the recommended microplate reader settings, if you use a different microplate reader, set the corresponding equivalent. Different settings will produce
Different signals.
â—† Accurate reading in the reading window.
I measured according to the experimental procedure, but there is no signal, why?
â—† For colorimetric determination using HRP substrate, the addition of the stop solution will change the color of the substrate. The wavelength recommended by the manual is the most
Suitable wavelength.
â—† Mix the reagents in the well by tapping the edge of the microplate. When the stop solution is added, the color changes from blue to yellow (if it is HRP
Substrate). If the reagents in the wells are not mixed before the stop solution is added, the substrate color turns green.
â—† The order of adding reagents may be wrong.
â—† Do not wash the enzyme plate after adding the substrate solution.
â—† Make sure that the standard product has been added during serial dilution.
â—† If there are two components in the substrate system, they must be mixed evenly.
â—† Make sure that the microplate reader is used and operated correctly.
â—† Make sure that the reagents, standards and samples are correctly prepared and added according to the instructions.
â—† For high-sensitivity kits, make sure that the substrate and amplifier used are properly diluted.
What are the reasons for the high background?
â—† Change of incubation time and temperature.
â—† Inappropriate washing.
â—† Reagent is contaminated.
â—† Enzyme plate is contaminated during incubation.
â—† Reuse of cover plate, container or gun head.
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