Elisa frequently asked question and analysis ELISA experiment is well accepted as its high sensitivity and specificity in biology research area. However,for beginners,if experiment operation steps are neglected,this will cause a big effect to the experiment result,such as no signal,weak coloration and low sensitivity, non gradient,etc. Now,let’s explain these experiment results one by one.

No signal:After adding TMB substrate, all holes of ELISA plates are colorless. This is no signal. Possible reasons are listed below:

a.Reagent shelf life has expired or different batches of dilution cross used.
b. Added by mistake or forget to add detection A,detection B or TMB substrate.
c. Washing or adding sample,labeled enzyme is contaminated and inactivate;dilution solution containing an enzyme inhibitor such as Sodium azide, etc.
d. The distilled water used to configure dilution solution may be contaminated.
e. Wash buffer is concentrated which is not diluted in proportion.
f. The activity and titer labeled enzyme is low.
g. PH value of solution is incorrect. Normally it should maintained at 7.2-7.4.

Weak coloration and low sensitivity: After adding TMB substrate, all holes of ELISA plates are weak colored which shows weak signal. Possible reasons are listed below:

a. Reagent shelf life has expired or reagent are not proper stored according to our manual.
b. Reagent,Standard or sample didn’t place to room temperature.
c. Less adding amount of reagent or wrong reagent dilution rate.
d. Washing or adding sample, labeled enzyme is contaminated and inactivate.
e. Incubation time and temperature didn’t reach experiment request.
f.Wash buffer dilution times didn’t meet the request;Washing times too much;Washing impact too strong;long time washing.
g. TMB substrate chromogenic time is not enough.

non gradient:After adding TMB substrate, all holes of ELISA plates has color but no gradient and the same time has strong signal.

a. TMB substrate is not placed in a cool dark place which has been blue before test.
b. Incubation time is too high or too long which leads strong nonspecific adsorption.
c. Don’t obey the washing requirement,specially adding less wash buffer.
d. Forget to change pipette tip and cause cross contamination.
e. Low concentration or low activate coated antibody or detection antibody,enclosed concentration temperature,time don’t meet the requirement which lead the coated antibody unstable,washing ineffective and coated plate issues.

In conclusion,we should pay attention to the following problems:

a. Before experiment,reagent should be placed to room temperature according to manual.
b. Coated antibody and detection antibody should have high activity and are against the same antigen.
c. Reagent storage:Protein antibody reagent should be store at -20℃ and coloring liquid and wash buffer should be stored at 2-8℃.
d. To Ensure the uniformity and accuracy of reagent,each reagent should be mixed thoroughly.
e. Strictly control the reaction temperature and reagent.
f. Washing times,volume of wash buffer,time in wash buffer and washing strength should be noted.
g. Related concentrate should be diluted to the appropriate proportion to according to manual before use, while PH value should be maintained at 7.2-7.4.
h.Try to avoid bubbles in micropore during termination reaction.
i. Complete reading enzyme microtiter plate reader in 2 min after finishing termination reaction.

materials required but not supplied.

1. Microplate reader with 450 ± 10nm filter.
2. Precision single or multi-channel pipettes and disposable tips.
3. Eppendorf Tubes for diluting samples.
4. Deionized or distilled water.
5. Absorbent paper for blotting the microtiter plate.
6. Container for Wash Solution.

sample collection and storage .

Serum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximately 1000×g. Assay freshly prepared serum immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8℃ within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.
Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed in ice-cold PBS(0.02mol/L,pH 7.0-7.2) to remove excess blood thoroughly and weighed before homogenization. Minced the tissues to small pieces and homogenized them in 5-10 mL of PBS with a glass homogenizer on ice(Micro Tissue Grinders woks, too). The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifugated for 5 minutes at 5000×g. Remove the supernate and assay immediately or aliquot and store at ≤-20 ℃.
Cell Lysates - Cells must be lysed before assaying according to the following directions.
a. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly).
b. Wash cells three times in cold PBS.
c. Resuspend cells in PBS (1×) and the cells was subject to ultrasonication for 4 times (or Freeze cells at≤-20℃. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.)
d. Centrifuge at 1500×g for 10 minutes at 2 - 8℃ to remove cellular debris. Cell culture supernates and Other biological fluids - Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquot at -20℃ or -80℃. Avoid repeated freeze/thaw cycles.

sample preparation.

a.DL Sci&Tech Development Co.Ltd is only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.
b. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
c. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
d.Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals.
e.Due to the possibility of mismatching between antigen from other origin and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products.
f. Influenced by the factors including cell viability, cell number or sampling time, samples from cell culture supernatant may not be detected by the kit.
g. Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.

precision.

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level the index were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level the index were tested on 3 different plates, 8 replicates in each plate.CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

stability.

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.