We then proceed to the point of attention to the various operational steps of the plate-type ELISA.
Color and colorimetric
color
The color of the ELISA is the last step of the temperature-producing reaction, when the enzyme catalyzes the colorless substrate to produce colored products.
The temperature and time of the reaction are still the factors that influence the color rendering.
In a certain time, the negative pore can remain colorless, while the positive hole is enhanced with the lengthening of time.
Proper raising of temperature will help to accelerate the color rendering.
In the quantitative determination, the reaction temperature and time after adding the substrate should be accurate according to the regulation.
Qualitative determination of color can be at room temperature, and generally do not need to strictly control the time, sometimes according to bore positive control and negative control of color appropriate to shorten or extend the reaction time, judgment in a timely manner.
OPD chromogenic substrates in outdoor temperature is 37 ℃ or reaction after 20-30 min deepen no longer, longer reaction time, can make the background value is higher.
The OPD substrate is affected by the light of the light, the color reaction should be avoided by the light, and the end of the color reaction will be added to the termination reaction.
The OPD product was turned from orange to brown when the product was terminated with sulfuric acid.
TMB is not affected by illumination, but it can be placed on the operating table at room temperature.
But in order to ensure the stability of experimental results, appropriate time reading results should be given.
After the function of HRP, the TMB reached its peak of about 40min, and then gradually decreased to no color after 2 hours.
TMB termination fluid has a variety of enzyme inhibitors, such as sodium azide and sodium dodecyl sulfate (SDS), can terminate the reaction.
This kind of termination agent can keep the blue for a long time (12-24 hours). It is a good termination agent for visual judgment.
In addition, all kinds of acid terminates will turn blue into yellow, which can be used at specific wavelengths (450nm) to measure the absorbance.
colorimetric
The liquid that is attached to the bottom of the dry plate should be cleaned before color, but should be avoided as far as possible, and then put the plate correctly in the color frame of the enzyme label colorimeter.
For the test of the soft plate as the carrier, it is necessary to put the plate in the frame of the standard 96 hole to make the color.
It is best to cut the edges of the soft plate before the addition of the substrate, so that the plate can be fully seated.
Color should be first to distilled water zero, sensing substrate hole (without any response only add the substrate liquid hole) and blank hole (replace the specimens with normal saline or diluent for the whole process of hole), to record the test reagent condition.
Then the blank hole can be used for the zero point of distilled water. The absorbance of each hole should be deducted from the blank hole and then calculated.
Colorimetric results the expression of previous general optical density (oplical density, OD), is now with absorbance (absorbence, A) according to regulations, the same meaning.
The usual method is to write the absorption wavelength at the lower right corner of A letter, such as the absorption wavelength of the OPD at 492nm, which means "A492nm" or "OD492nm".
Enzyme comparator
The enzyme bichromator, or enzyme standard, usually refers to a photometer which is used to measure the absorbance of ELISA results.
In view of the different forms of solid - phase vectors, each has special design for plate, bead and small test tube.
Many reagent companies supply the enzyme standard equipment.
The main performance indicators of the enzyme are: reading speed, accuracy of reading, repeatability, accuracy and measurable range, linearity and so on.
Excellent enzyme readings can be accurate to 0.001, accuracy is plus or minus 1%, and repeatability is 0.5%.
For example, if the value of A hole is 1.083, the real value of the hole relative to air should be 1.083 + 0.01 (1.073 ~ 1.093), and the number of repeated tests should be between 1.083 and + 0.05 (1.078 ~ 1.088).
The measurable range of the enzyme is different from the performance of the enzyme markers.
The average enzyme marker is at 0.000 to 2.000, and the upper limit of the new type of enzyme marker is up to 2.900, even higher.
A value exceeding the measurable limit is often expressed in "*" or "over" or other symbols.
Should pay attention to the different measurable range and linear range, the linear range is often less than the measurable range, such as an enzyme standard meter can be measured in the range of 0.000 ~ 2.900, and the linear range is only 0.000 ~ 0.000, the production of standard curve in the quantitative ELISA should be pay attention to.
Enzyme mark should not be placed in the sun or glare, operating room temperature should be at 15 ~ 30 ℃, using preheating equipment before 15 to 30 min, sensing results more stable (there are also some enzyme mark clearly marked on the manual does not need to preheat).
When reading A value, choose the sensitive absorption peak of the product, such as OPD with 492nm wavelength.
Some enzyme mark sensing instrument available double wavelength type, namely each hole has read twice, the first time in optimal wavelength (W1), the second time in less sensitive wavelength (W2), between two measurement does not move the location of the ELISA plate.
For example, the OPD USES 492nm for W1, 630nm for W 2, and the final A value is the difference between the two (w1-w2).
Dual wavelength reading can reduce the optical interference caused by scratches or fingerprints on the container.
The performance of various enzyme markers is different, and the instructions should be read in detail in use.
Results the judgment
Qualitative determination of
The result of qualitative determination is to make a simple answer to whether there are "some" or "no" or "no" or "no" or "no" or "no" in the specimen.
"Positive" indicates that the specimen has a reaction in the measurement system."
Negative is not reactive.
Semi - quantitative results can be obtained by using qualitative method, which can be used to express the intensity of reaction, and its essence is still a qualitative test.
In this semi-quantitative determination, the specimen was tested in a series of dilutions, and the highest dilution of positive reaction was the titer.
According to the height of droplet, the strength of the specimen reactivity can be determined, which is more quantitative than the deep shallow judgment of the color of the non-dilutive specimen.
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