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How to Operate ELISA Correctly

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#1 Lulucy



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Posted 12 March 2010 - 12:51 AM

The determination of clinical ELISA is usually used with manual solid-phase microplate mode. And it is very simple, typically involving the collection of preserved specimens, reagent preparation, plus sample, incubation, plate washer, color, colorimetric determination, the results of judgments and report on the results and explain the terms of any one of these steps. Every improper step will affect the determination of the results, in particularly to the plus samples, the incubation and the plate washer. The described are as below.

The collection and preservation of clinical samples

Serum (plasma) is the most commonly used clinical sample in ELISA determination. Saliva, cerebrospinal fluid, urine, feces and other samples are also used for some special detection. At present, the markers used for serum determination are generally infectious pathogen antigens and antibodies, tumor markers, hormones, special proteins, cytokines, therapeutic drugs and so on. When collecting the serum sample used to hormones and therapeutic drugs, much more time and the posture may affect the result. For examples, in the morning between 4 to 6 o'clock, there will be a peak occur of cortisone. Growth hormone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are in the paroxysmal form of release. So it is necessary to get several interval samples in a time when this kind of hormones is determined. The renin in serum may apparently higher when standing up from lying. When testing treatment drugs, you should choose the optimum time to test the blood according to pharmacokinetics. The time and the posture will not influence the collection of the serum used to the detections of fectious pathogens, antigens and antibodies, tumor markers and other special proteins. However, please pay attention to the following aspects when processing and preservation.

(1) Prevent serious hemolysis. Hemoglobin contains heme groups, which are similar to peroxidase activity. If the hemoglobin in the serum sample is too high, it is very easy to adsorb on the solid during the incubation phase, and then join with the HRP chromogenic substrate reaction in the ELISA determination with HRP as the maker enzyme.

(2)Prevent bacterial contamination in the sample collection and serum separation. Firstly, the enzyme from the growth of bacteria may result the decomposition of antigens and antibodies. Secondly, endogenous enzymes of some bacteria, such as E. coli β-galactosidase enzyme, itself will be marked with the corresponding determination method to produce non-specific interference.

(3)Serum samples in aseptic separation can save 2 ~ 8 ℃ in a week. Or please cryopreservation. Samples stored for long periods should be below -70 ℃.

(4)Stored frozen serum samples should avoid repeated freezing and thawing caused by power failures or others. Repeated freezing and thawing of specimens produces the mechanical shearing force which will damage the molecules, leading to false negative results. In addition, do not volatility when mix the freeze-thaw specimens, only need to mixed repeatedly reversed.

(5)If there is some turbidity and floc caused by the bacterial contamination in the preservation, please take the supernatant after centrifugation test.


If the reagent are directly take out from the fridge without any other treatment, the result may be detecting false negatives to some weak positive specimen if the Incubation time is not enough. So the preparation of the reagent is the most important step. The correct step is taking the reagent out, and then put it at room temperature in more than 20 minutes to balance the temperature before the testing. The main purpose is to make the temperature inside the porous reaction can rapidly reach the required height to meet the measurement requirements in the following Incubation reaction. Secondly, for the plate washer used now is the dilution of concentrated liquid preparation, so when diluting with distilled or deionized water please ensure the quality. In addition, when the substrate in the kit is OPD, the solution substrate should be compounded provisionally before it color.

The Adding of the serum samples and the regants

Now, in the ELISA Kit, adding the serum sample is the only step which needs to use the micro-plus device. When using this device, following are some advice: adding the sample can not be too fast, to avoid spilling to the hole or out, and some bubbles. If adding too fast, the accuracy and uniformity can not be sure. It is very easy lead to non-specific adsorption for the non-coated area on the pore wall. And it will influence the near wells if spilling out. There will be some differences if there are some bubbles. Sometimes a sample kit with the same determination as the positive, the next determination is negative, is often caused by the fault methods of adding the samples or reagents.


Edited by Lulucy, 12 March 2010 - 12:52 AM.

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