Brief description of Bunsen: ELISA kit testing precautions

Bunsen Brief: ELISA Kit Detection Considerations The ELISA method is widely used in various antigen and antibody assays
.
However, there are many influencing factors in the determination of ELISA, and there are certain technical requirements in its operation, and in addition to normal reactions in the detection process, some erroneous results
are sometimes common.
The main causes of the wrong results of ELISA measurement are: (1) specimen factors; (2) Reagent factors; (3) Operating factors
.
The following is an analysis
of some common problems in the operation of ELISA.
1.
Sample dilution Enzyme-linked immune reaction is a highly sensitive reaction, if the serum is not diluted, it is inevitable to produce a strong non-specific reaction, false positives
.
Therefore, foreign kits for detecting serum antibodies are prescribed to dilute the serum to an appropriate multiple to reduce the non-specific reaction and fully reflect the specific antigen antibody response
.
The sample dilution multiples of the general product are determined by a large number of tests to determine the sensitivity and specificity of
the test.
However, some users fail to operate strictly according to the instructions for use, such as some products should take 10 μl of sample for dilution, individual users take 5 μl or even 1 μl of sample for dilution, because the nozzle is inevitably stained with samples and the accuracy of the micropipette is not enough, so the sample dilution multiple is inaccurate, and the test results are problematic
.
2.
Kit balance All reagents and slats in the kit should be balanced to room temperature (about 25 ° C), generally need to be placed at room temperature for more than
20 to 30 minutes.
Equilibrium time is too short to cause reagent mixing to be insufficient, sample incubation time to be relatively shortened, and insufficient ELISA reaction
.
In winter, the room temperature is low, and the kit can be placed in a 37 °C incubator for 20 minutes
.
3.
Mixing of samples and reagents Dilution, after the sample must be fully mixed, all reagents in the sample must also be shaken evenly, in order to test uniformity
.
4.
Sample In the current ELISA commodity kit, there must be a step
to add samples using a microsampler.
The key point of attention is: the sample should not be added too quickly, and it should be avoided to be added to the upper part of the hole wall, and not to splash out and produce bubbles
.
Sampling is too fast to trace the accuracy and uniformity of
dosing.
The non-coated area added to the upper part of the pore wall can easily lead to non-specific adsorption
.
Spilling can contaminate
adjacent holes.
If bubbles appear, the reaction fluid interface is different
.
Therefore, sometimes a specimen with the same kit this time is positive, the next test is negative, often caused by
the above dosing and reagent errors.
5.
Incubation Incubation is a key factor
in the ELISA assay that affects the success or failure of the assay.
ELISA as a solid-phase immunoassay, the binding reaction of the antigen antibody is carried out in the solid phase, and to bind the antigen or antibody in the liquid phase to the specific antibody or antigen on the solid phase, it must be reacted for a certain period of time
under certain temperature conditions.
The time required for incubation is inversely proportional to the temperature, i.
e.
the higher the temperature, the shorter
the time required.
The commonly used incubation temperatures are 37 °C and room temperature, followed by 43 °C and 2 to 8 °C
.
Some operators, unauthorized change of instruction manual operation, the use of their favorite incubation time and incubation temperature, which causes some unnecessary trouble
.
Because different kits have different incubation times and incubation temperature choices, changing the incubation time or incubation temperature at will will cause deviations
in the test results.
6.
Plate washing Solid phase immunoassay technology is a heterogeneous immunoassay technology, which needs to be used to separate the antigen or antibody specifically bound to the solid phase from the non-specific components adsorbed during the reaction incubation process by washing operation, and the specificity
of ELISA determination is used.
Plate washing is also a critical step
in the ELISA assay.
Try not to spill the wash outside the hole; After adding the lotion, let it stand for 1 minute, and after throwing off the lotion in the plate well, be sure to pat it dry; Replace the absorbent paper in time, especially the absorbent paper that has been photographed with enzyme markers, must be discarded, otherwise it may affect the test results
.
7.
Edge effect In the ELISA assay using a 96-well plate, it is often found that there is a "edge effect", that is, the peripheral pores are darker
than the central pores.
Studies have shown that thermodynamic gradients in incubation may be the root cause
.
Polystyrene itself is a poor thermal conductor, and in the routine ELISA assay in the laboratory, the plate is placed from room temperature (usually around 25 ° C) in a 37 ° C temperature chamber, and when the plate is also warmed, there may be a thermodynamic gradient between the peripheral well and the central well
.
Therefore, using a water bath or when adding the reaction solution to the plate well, heating both the plate and the solution to the incubation temperature (e.
g.
, 37 ° C) can easily exclude the "edge effect" and improve the repeatability
of the assay.
8.
Color rendering Color development must control the time, according to the instructions of the kit can be operated
.
In general, the color development time is too short and the result is low; Excessive color rendering time, increased white space, or increased
non-specific color rendering.
9.
Colorimetric Colorimetric should pay attention to the choice of
wavelength.
Kits with TMB as substrate and OPP as substrate are used, and the colorimetric wavelength is 450nm, the latter is 492nm, and the filter needs to be replaced
at any time upon request.
Therefore, it is easy to have the problem
of filter misuse.
Second, the problem
of single or dual wavelength colorimetric selection.
The so-called single wavelength colorimetric is usually performed on the wavelength of the color development with absorption such as 450nm or 492nm; The dual-wavelength bicolor microplate reader is determined once each at a sensitive wavelength such as 450nm and a non-sensitive wavelength such as 630nm, and the absorbance measurement value above and below the sensitive wave is the sum of the absorbance of the specific color of the sample to determine the enzyme reaction and the absorbance caused by fingerprints, scratches, dust and other dirt on the plate well; The measurement at non-sensitive wavelengths is to change the wavelength to a certain value, so that the absorbance value of the specific color of the sample assay reaction is zero, and the measured absorbance is the absorbance value
of the dirt.
The value given by the microplate reader is the difference between the absorbance value at sensitive wavelengths and the absorbance value at very sensitive wavelengths
.
Therefore, the dual-wavelength colorimetric assay has the advantage
of excluding the influence of non-specific absorption, fingerprints, scratches, dust, etc.
on the specific color-rendering determination of absorbance by the droplet plate itself, the specimen in the plate well.
Since there is a certain degree of uncertainty in the non-specific absorption of a single blank well in the ELISA assay, that is, different absorbance values may be obtained for each assay or the same assay for the different positions of the blank wells, so when determining the colorimetry of ELISA, a dual-wavelength colorimetry
is used.
Bunsen Brief Description: ELISA Kit Testing Precautions BUNSEN can not only meet the special requirements of R&D customers for product types and packaging, but also meet the comprehensive needs
of production-oriented enterprises at all stages from small trial, pilot test to large-scale production 。 Products include PCR consumables (octagrams, single tubes, 96-well plates, 384-well plates, sealing membranes, auxiliary devices, etc.
), ELISA kits, pipette nozzles, centrifuge tubes, cryopreservation tubes, Petri dishes, culture plates, culture bottles, tips, instruments and gloves, culture media, antibodies, serum, chromatography consumables, needle filters and commonly used consumables
in the laboratory.