Dear Customer:
Thank you for choosing our company's ELISA kits! Since assay steps and detection methods are varied among different manufacturers and kits, to help you conduct your experiments more efficiently, and save your precious samples and valuable time, please carefully read the following ELISA technical guide before starting your experiments.
1. Read the printed manual carefully, learn assay steps of this ELISA kit.
Detection steps and data are based on the printed manual.
2. Sample Preparation
For sample preparation documents, please visit the following link.
3. Confirm the approximate content of the tested sample, estimate the dilution ratio required by each group
Due to limited sample types and quantity, dilution ratio suggested in the printed manual is the detection results of limited samples(e.g. normal serum/plasma, cell lysis buffer or cell culture supernatant), and can’t cover the expression situation of all samples. Affected by different diseases or models, the best dilution ratio for each sample group may be different from the dilution ratio recommended in this manual (10X, 100X, or even 1,000X is possible). To avoid potential waste of kits and samples, you're suggested to check relevant citations firstly to know the change tendency of target in the related disease or model. Then conduct the pilot assay to get the best dilution ratio of control and experimental group.
4. Importance of Pilot Assay
The pilot assay appears to be a waste of time, sample and reagents. However, pilot assay saves a lot via understanding its aim and effect. First, we have a rudimentary grasp of the unknown ELISA kit. It's also a detection to avoid a waste of your valuable samples caused by assay steps or the kit's quality. Second, the detection of less sample in pilot assay can determine the best dilution ratio for each sample group.
5. Sample Dilution Solution in Pilot Assay
5.1. First, calculate the number of wells
The pre-coated ELISA microplate consists of dismountable strips (12 strips * 8 wells). During pilot assay, it's required to measure a standard curve and less samples of different groups at the same time. If the well budget is very limited, the highest, medium, lowest and blank well of the standard should be measured at least. Choose 1-2 samples from different groups to test different dilution ratios. For 4 different dilution ratios, the required number of wells is standard wells + sample quantity*4.
5.2. How to better dilute sample during Pilot Assay?
Is the gradient 1/2,1/4,1/8,1/16 suitable? When to stop dilution? Here, you’re suggested to dilute in 10X. Please refer to recommended dilution ratio range to set 3-4 10X dilution conditions. Take h.CRP (C-reactive protein) for example: If you have 4 groups of serum samples, the normal serum dilution ratio recommended in the manual is 1/5000-1/10000. Through checking citations, C-reactive protein rises rapidly when the organism is infected or the tissue is damaged. Thus, the dilution ratio for your sample group is 1/10000 at least. Then, we design the dilution ratio in 10X (e.g. 1/10,000, 1/100,000, 1/1000,000, 1/10,000,000). If the detection target of other normal serum kit is 1/2, the sick level increases as well. It's suggest to set 1/2, 1/10, 1/100, 1/1000.
5.3. Why to dilute in 10X?
Usually, the standard curve is diluted in gradient (7 points + zero well) and appears to be a ruler across 64X. The increasing or decreasing level of the sample is unknown. Lower dilution range is a waste of more wells or still higher than the highest well of the standard curve at the end of the assay. Higher dilution range may not find the best dilution ratio. Dilution in 10X always contains a dilution ratio just falling in the 64X ruler.
6. Notes for Pilot Assay
6.1. Check ELISA kit components. Check the leakage and availability of all components listed in the manual. Please contact us to solve If abnormal situation happens.
6.2. Less reagents may be adhered to the cap of the reagent tube due to transportation. Please centrifuge and then open to avoid to affect the assay.
6.3. Try to not open many different kinds of ELISA kits to avoid to mix kits' components. Otherwise, the component cap should be labelled with the marking pen to avoid the assay failure caused by mixing components.
6.4. The sealed kit can be stored at 2-8℃. After opening, the microplate and lyophilized standard can be stored at -20℃ to extend the shelf life. Dry environment is more suitable for the storage. The microplate and standard may lose partial activity by getting damp. Other liquid reagents can be stored at 2-8℃. Cryopreservation is not allowed and will greatly decrease the performance.
6.5. The highest dissolved concentration of lyophilized standard offered in this kit can be stored at 2-8℃ and used within 12h.(Cryopreservation is not recommended.) Arrange the assay time properly. You can perform two rounds of assay within 12h. Two vials of standard can perform the assay four times at least.
7. Sample Dilution Skills
If the detected substance requires for higher dilution ratio (e.g. 100,000 times) and there are many samples, the sample dilution job will be a challenge. Here, some skills are offered to help you improve efficiency and accuracy.
7.1. Preparations
96 well 2ml deep-well plate (Sterilization for repeated use)
96 well cell culture plate or ELISA plate (disposable use)
Loading slot (disposable or sterile)
10-100ul 8 or 12 channel pipette
20-200ul or 30-300ul 8 or 12 channel pipette
7.2. Detailed Assay Steps
Select Deep-well Plate
Step 1: Dilute in 500X: arrange the pilot sample according to the order of deep-well plate. Add 3ul sample into the bottom of the empty well with single channel pipette. Add 1.497ml normal saline into each well and slowly shake for 3min with the shaker to mix sample well. Then, the current sample dilution ratio is 1/500 and labelled as S-500X.
Step 2: Dilute in 100X: Following the method in Step 1, continue to suck 10ul sample diluted in Step 1 with multichannel pipette, and add into another new deep-well plate. Then, add 990ul normal saline into the well and shake for 3min to mix well. The sample has a total dilution of 50,000 times, which is labelled as S-50,000X.
Step 3: Finally dilute in 1:2 with sample dilution buffer: add 50ul sample dilution buffer offered by the kit into the well of pre-coated ELISA plate with 50ul multichannel pipette. Then add 50ul sample diluted in 50,000 times(S-50,000X) into ELISA sample well and shake for 3min to mix well. Thus, the dilution of 100,000 times is completed. Seal the ELISA plate and statically incubate in 37℃ incubator.
7.3. Notes
For 1,000,000 times dilution or other situations, please refer to Step 2 to complete.
If you perform large scale dilution for various samples with 96 well ELISA plate or cell culture plate (max volume: 350ul/well), dilute in 100X with normal saline first (dilute 3ul sample into 300ul). Simulate the method above to dilute. Before sample loading, dilute in 1/2 to complete sample dilution with the buffer offered by the kit. (When the minimum percentage of sample dilution buffer in loading amount is 50%, PH value can be effectively stabilized. The accuracy of sample detection result is assured accordingly.)