Bovine Ig(Total Immunoglobulin) ELISA Kit使用说明（二）

Note: Samples to be used within 5 days can be stored at 4°C, besides that, samples must be stored at -20°C (assay ≤1 month) or -80°C(assay≤2 months) to avoid loss of bioactivity and contamination. Hemolyzed samples are not suitable for this assay.
Sample Dilution Guideline
End user should estimate the concentration of target protein in the test sample, and select a proper dilution factor to make the diluted target protein concentration fall in the optimal detection range of the kit. Dilute the sample with the provided dilution buffer, and several trials may be necessary. The test sample must be well mixed with the dilution buffer. And also  standard curves and sample should be making in pre-experiment. The following dilution  solutions are for reference only:

• High concentration (1000-10000ug/ml): Dilution: 1:100. (i.e. Add 1μl of sample into 99μl of Sample/Standard Dilution Buffer.)

• Medium concentration (100-1000ug/ml): Dilution: 1:10.( i.e. Add 10μl of sample into 90μl of Sample/Standard Dilution Buffer.)

• Low concentration (1.563-100ug/ml): Dilution: 1:2. (i.e. Add 50μl of sample into 50μl of Sample/Standard Dilution Buffer.)

• Very low concentration (≤1.563ug/ml): Unnecessary to dilute, or dilute at 1:2.

Reagent Preparation and Storage
Put the kit at room temperature for 20 minutes before use
1, Wash Buffer:
Dilute 30mL Concentrated Wash Buffer into 750 mL Wash Buffer with deionized or distilled water. Put unused solution back at 4°C. If crystals have formed in the concentrate, you can warm it with 40°C water bath (Heating temperature should not exceed 50°C) and mix it gently until the crystals have completely been dissolved. The solution should be cooled to room temperature before use.
2, Standard:
1). 100ug/ml of standard solution: Add 1 ml Sample / Standard dilution buffer into one Standard tube, keep the tube at room temperature for 10 minutes and mix them thoroughly.
2).50ug/ml→1.563ug/ml of standard solutions: Label 6 Eppendorf tubes with 50ug/ml, 25ug/ml, 12.5ug/ml, 6.25ug/ml, 3.125ug/ml, 1.563ug/ml, respectively. Add 0.3 ml  of  the Sample/Standard dilution buffer into each tube. Add 0.3 ml of the above 100ug/ml standard solution into 1st tube and mix them thoroughly. Transfer 0.3 ml from 1st tube to 2nd tube and mix them thoroughly. Transfer 0.3 ml from 2nd tube to 3rd tube and mix them thoroughly, and  so on.

Note: It is best to use Standard Solutions within 2 hours. The Standard Solution shall be at 4°C up to 12 hours. Or store it at -20 °C up to 48 hours. Avoid repeated freeze-thaw cycles.
3, Preparation of Biotin-labeled Antibody Working Solution
Prepare it within 1 hour before experiment.

1. Calculate required total volume of the working solution: 0.1 ml / well × quantity of wells. (Allow 0.1-0.2 ml more than the total volume)

2. Dilute the Biotin-detection antibody with Antibody Dilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1μl Biotin-labeled antibody into 99μl Antibody Dilution Buffer.)

4, Preparation of HRP-Streptavidin Conjugate (SABC) Working Solution:
Prepare it within 30 minutes before experiment.

1. Calculate required total volume of the working solution: 0.1 ml / well × quantity of wells. (Allow 0.1-0.2 ml more than the total volume)

2. Dilute the SABC with SABC Dilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1μl of SABC into 99μl of SABC Dilution Buffer.)

Assay Procedure
Before adding reagents into wells, equilibrate TMB Substrate for 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!

2. Aliquot 0.1ml of 100ug/ml, 50ug/ml, 25ug/ml, 12.5ug/ml, 6.25ug/ml, 3.125ug/ml, 1.563ug/ml, standard solutions into the standard wells.

3. Add 0.1 ml of Sample/Standard Dilution Buffer into the control (zero) well.

1. Add 0.1 ml of properly diluted sample (Bovine serum, plasma, tissue homogenates and other biological fluids) into test sample wells.

2. Seal the plate with a cover and incubate at 37 °C for 90 minutes.

3. Remove the cover and discard the plate content, and wash plate 2 times with Wash Buffer. Do NOT let the wells dry completely at any time.

4. Add 0.1 ml Biotin-labeled antibody working solution into above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the sidewall.

5. Seal the plate with a cover and incubate at 37°C for 60 min.

6. Remove the cover, and wash plate 3 times with Wash Buffer, and let the wash buffer stay in the wells for 1 minute each time.

7. Add 0.1 ml of SABC Working Solution into each well, cover the plate and incubate at 37°C for 30 minutes.

8. Remove the cover and wash plate 5 times with Wash Buffer, and let the wash buffer stay in the wells for 1-2 minute each time.

9. Add 90μl TMB Substrate into each well, cover the plate and incubate at 37°C in dark within 15-30 minutes. (Note: This incubation time is for reference only, end user shall determine the optimal time.) It will turn blue in the first 3-4 wells (with most concentrated  Ig  standard solutions), the other wells may not display obvious color.

10. Add 50μl Stop Solution into each well and mix them thoroughly. The color changes to yellow immediately.

11. Read the O.D. absorbance at 450 nm in Microplate Reader immediately after adding the stop solution.

Regarding calculation, (the relative O.D.450) = (the O.D.450 of each well) – (the O.D.450 of Zero well). The standard curve can be plotted as the relative O.D.450 of each standard solution (Y) vs. the respective concentration of the standard solution (X). The  Ig concentration of the samples  can be interpolated from the standard curve. It is recommended to use professional software curve expert to 1.3, please  visit:  http://www.fn-test.com/services/software-download/  for more information.
Note: If the samples measured were diluted, multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution.
Summary

1. Wash plate 2 times before adding standard, sample and control (zero) wells!

2. Add 100μL standard or sample to each well and incubate for 90 minutes at 37°C

3. Aspirate and wash plates 2 times

1. Add 100μL Biotin-labeled antibody working solution to each well and incubate for 60 minutes at 37°C

2. Aspirate and wash plates 3 times

3. Add 100μL SABC Working Solution into each well and incubate for 30 minutes at 37°C

4. Aspirate and wash plates 5 times

5. Add 90μL TMB Substrate. Incubate 15 -30 minutes at 37°C

6. Add 50μL Stop Solution. Read at 450nm immediately

7. Calculation

Typical Data & Standard Curve
Results of a typical standard operation of a Ig ELISA Kit are listed below. This standard curve  was generated at our lab for demonstration purpose only. Users shall obtain standard curve as per experiment by themselves. (N/A=not applicable)
 

Specificity
This assay has high sensitivity and excellent specificity for detection of Ig . No significant cross- reactivity or interference between Ig and analogues was observed.
Note: Limited by current skills and knowledge, it is difficult for us to complete the cross- reactivity detection between Ig and all the analogues, therefore, cross reaction may still exist.
Recovery
Matrices listed below were spiked with certain level of Ig and the recovery rates were calculated by comparing the measured value to the expected amount of Ig in samples.

Linearity
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Ig and their serial dilutions. The results were demonstrated by percentage of calculated concentration to the expectation.
 

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

Stability
The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 10% within the expiration date under appropriate storage condition.
 

To minimize extra influence on  performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is strongly suggested that the same operator performs the whole assay from the beginning to the end.