1. Introduction to the Kit
This quantitative detection kit for bovine serum albumin (BSA) enzyme-linked immunosorbent assay (ELISA) uses monoclonal paired antibodies against BSA, which are respectively used as coating antibodies and detection antibodies to form a double antibody sandwich ELISA detection kit. It is used as a reference standard with the BSA standard of the American Bureau of Standards. This kit can detect a minimum of 1ng/mL of BSA.
2. Kit Components:
 

Name	Quantity (specification	Storage temperature
96-well slats coated with anti-BSA monoclonal antibody	1 bottle	4℃
BSA reference standards: 32ng/mL, 16ng/mL, 8ng/mL, 4ng/mL, 2ng/mL, 1ng/mL, 0ng/mL	各 1 bottle（0.4mL/bottle）	-20℃/-80℃
Internal reference of BSA (20ng/mL) :	1 bottle（0.4mL/瓶）
TMB substrate chromogenic solution	1 set（A 和 B 各 6.5mL）	4℃
Chromogenic substrate stop solution (2N, H2SO4) :	1 bottle（13mL）	4℃
Sample diluent	1 bottle（13mL）	4℃
Washing solution (10 times concentrated) :	1 bottle（50mL）	4℃
Instruction Manual	1 copy	4℃
Enzyme-labeled anti-BSA antibody	1 bottle（0.13mL）	-20℃/-80℃
Enzyme-labeled antibody diluent	1 bottle（13mL）	4℃

3. Required instruments:
Microplate reader (wavelength 450nm, 10μL, 100μL, 1mL pipettes, 300μl multi-channel pipettes, pipette tips, 37℃ constant temperature water bath.)
4. Operating Steps:
4.1 Dilute the concentrated washing solution 10 times with distilled water.
4.2 Take out the 96-well microplate coated with anti-BSA and place it at room temperature to warm up. Prepare two parallel Wells for each concentration of the standard substance and add 50μL per well to the microplate. Add the sample to be tested and the internal reference, two Wells each, 50μL per well. Then, dilute the enzyme-labeled antibody with the enzyme-labeled antibody dilution solution at a ratio of 1:100 and add it to each reaction well, with 100μL per well (dilute as much as needed, prepare as needed). The sample addition process must be completed within 15 minutes.
4.3 Place at room temperature of 20 to 24 degrees Celsius and react in the dark for 1 hour. Wash four times with the washing solution and pat dry.
4.4 Add 100μL of TMB substrate chromogenic solution per well with a pipette. Do not shake. React at room temperature of 20 to 24 ° C in the dark for 10 minutes.
4.5 Add 100μL of stop solution per well with a pipette and shake slightly for 5 seconds to mix well.
4.6 Read the value at a wavelength of 450nm on the microplate reader. 5. Precautions
5.1 If the BSA content in the sample is higher than 32ng/mL, the sample can be appropriately diluted.
5.2 If the sample is a freeze-dried preparation, it should be thoroughly dissolved and mixed with the sample diluent.
5.3 During the reaction, the entire plate should be placed in a clean and sealed space to prevent foreign objects from falling into the reaction holes.
After washing and drying, any residual air bubbles in each hole should be eliminated.
After opening, put the unused strips back into the sealed plastic bag with desiccant and store it in a sealed container at 4℃.

5.6 During the operation process, the pipette tips, washers (bottles), vessels, etc. used should all be strictly protected from contamination by environments and items with BSA.

6. Result Determination
6.1 Valid experimental parameters:
The experimental results are valid only if the following valid parameters are met:
The OD value of 0ng/ml is ≤0.1. The OD value of 32ng/ml is ≥0.80. Positive reference: 18-22ng/ml; R2≥0.98;
6.2 Calculation:

In EXCEL, a scatter plot is made with the standard concentration as the abscissa and the zeroing-out OD value as the ordinate. A trend line is added, and then the curve equation and R2 value are displayed. After zeroing the OD value of the test sample, it is substituted into the linear equation to calculate the corresponding concentration.

 

Standard concentration	OD1	OD2	OD3	Average value	Zero Elimination OD
0	0.069	0.062	0.064	0.065	0
1	0.111	0.109	0.113	0.111	0.046
2	0.13	0.127	0.131	0.129	0.064
4	0.191	0.192	0.193	0.192	0.127
8	0.332	0.319	0.327	0.326	0.261
16	0.583	0.588	0.596	0.589	0.524
32	1.039	1.025	1.054	1.039	0.974