When using the ischemia modified albumin (IMA) assay kit, the following key points should be noted to ensure the accuracy and experimental safety of the test results:
1. Sample collection and processing
Avoid hemolysis
Hemolytic samples can release hemoglobin, interfere with IMA detection, and lead to higher results. If hemolysis occurs in the sample, it should be collected again.
Sample type and preservation
Serum/plasma: After collection, it should be separated as soon as possible (such as centrifugation at 1000 × g for 10-20 minutes) to avoid repeated freezing and thawing.
. If storage is required, it is recommended to pack and freeze at -20 ℃ or -80 ℃. Other samples, such as cell culture supernatant and tissue homogenate, need to be centrifuged according to the instructions to remove particles and polymers. Sample dilutionIf the concentration of IMA in the sample exceeds the standard curve range, it is necessary to dilute the sample diluent appropriately and retest.
.II. Reagent Preparation and Storage
Reagent Recuperation
All reagents must be restored to room temperature (20-25 ℃) before use to avoid temperature differences affecting reaction efficiency.
.Standard dilution
Standard should be used and prepared according to the instructions, and diluted standard should not be stored.
. During the dilution process, it is necessary to mix thoroughly to avoid uneven local concentrations.Preparation of detergent
Concentrated detergent needs to be diluted in proportion (such as 1:20 or 1:50). If crystals precipitate after dilution, they can be dissolved by heating in a water bath.
.The washing solution should be thoroughly mixed before use to avoid residual sediment blocking the washing machine pipeline.
.Reagent storage
Unopened reagent kits should be stored according to the label instructions (such as 2-8 ℃ or -20 ℃).
. After opening, the remaining reagents should be sealed and stored to avoid contamination and volatilization.III. Experimental Operation Norms
Sample addition sequence and time
Strictly follow the order specified in the instructions to add samples, standards, antibodies, and substrates, ensuring consistent incubation time for all reaction wells.
.When adding samples, avoid generating bubbles and use disposable suction tips to prevent cross contamination.
.Incubation conditions
The incubation temperature (such as 37 ℃) and time should be strictly controlled to avoid temperature fluctuations affecting the reaction results.
. During the incubation process, it is necessary to cover the enzyme-linked immunosorbent assay (ELISA) plate to prevent concentration changes caused by liquid evaporation.Washing steps
The number of washes and soaking time should be carried out according to the instructions (if hand washing needs to be repeated 3-5 times, soaking for 30 seconds each time).
. After washing the plate, the enzyme-linked immunosorbent assay (ELISA) plate should be thoroughly dried to avoid residual liquid interfering with the color reaction.Color development and termination
Color developers (such as TMB) need to be stored away from light and prepared for immediate use.
.After adding the termination solution, the OD value should be measured immediately to avoid color fading and result deviation.
IV. Requirements for Instruments and Equipment
Calibration of ELISA reader
Before use, the ELISA reader should be preheated and calibrated to a wavelength of 450nm to ensure accurate readings.
.Avoid operating the enzyme-linked immunosorbent assay (ELISA) reader under direct sunlight to prevent signal interference.
. Washing Machine SettingsIf using an automatic washing machine, it is necessary to set the washing program in advance (such as washing frequency, soaking time) and verify the washing effect.
.Fifth, Safety and Protection
Personal Protection
During the experiment, gloves, masks, and goggles should be worn to avoid direct contact with reagents and samples.
. Eating, drinking, or smoking are prohibited in the laboratory to prevent the ingestion of toxic substances.Waste disposal
All used reagents, samples, and tips must be treated as biohazard waste to avoid polluting the environment.
. Reagents containing NaN ∝ need to be treated separately as they may inhibit enzyme activity and produce harmful gases.VI. Interpretation of Results and Quality Control
Drawing of Standard Curve
Use the OD value of the standard sample to draw the standard curve, ensuring that the correlation coefficient (R ²) is ≥ 0.990 to ensure the accuracy of linear regression.
. If the standard curve is abnormal (such as non-linear), it is necessary to check the experimental operation or reagent quality.Multiple well testing
It is recommended to set up multiple wells for each sample and standard to reduce accidental errors and improve the reliability of the results.
.Inter batch difference control
Different batches of reagent kits should not be mixed to avoid result deviation caused by reagent differences.
. Regularly verify the coefficient of variation (CV) between batches of the reagent kit to ensure that it meets the instructions (e.g. CV ≤ 15%).source:http://en.zjikon.com/news1081856.html