BSA ELISA Kit

1、 Preparation

1. Kit preparation: put the components of the kit at room temperature for 30min and then start the operation
2. Consumables and equipment to be provided: microplate reader, thermostatic oscillator (or thermostatic incubator), plate washer (it is recommended not to cross use with other projects or directly use hand washing), vortex oscillator, timer, high-precision pipette and disposable suction head (0.5-10ul, 10-100ul, 30-300ul, 100-1000ul), sterile deionized water without heat source, absorbent paper, EP tubeDisposable gloves
3. Reagent preparation: (note: try not to weigh BSA powder or place an electronic balance that has weighed BSA in the laboratory, clean the experimental table before the experiment, and prepare reagents according to the requirements of the current experiment)

① buffer (1×)Preparation: take buffer (20×)Add deionized water to dilute 20 times for standby, for example: take buffer (20×)Add 10ml deionized water 190ml and mix well. (note: buffer (20×)If crystals are formed, they should be placed in a room temperature or 37 ℃ water bath and gently shaken, and then diluted after the crystals are completely dissolved)

② preparation of color developing solution: mix the color developing solution a, color developing solution B and other volumes, and place them away from light after mixing. (note: the time should not be too long, and it is usually prepared 10min before use. If the color developing solution has turned blue after mixing, do not use)

③ enzyme labeled antibody (1×)Preparation: take out the enzyme labeled antibody and put it on the ice box. According to the experimental dosage (100ul/ well), use buffer (1×)The enzyme labeled antibody (1×) is diluted 500 times. (note: enzyme labeled antibody should not be kept at room temperature for a long time. It is best to take it out and operate it on an ice box; enzyme labeled antibody (1×)It needs to be prepared and used immediately)

④ preparation of standard (note: a newly prepared standard solution needs to be used for each experiment): add 7ul of 5ug/ml standard to 63ul buffer (1×)Add 64ul of 500ng/ml standard to 936ul buffer (1×)Dilute it to 32ng/ml, and then dilute it according to the 2-fold ratio, as shown in the following figure:

1. Sample preparation: restore the sample to room temperature, and mix the sample before adding; if users need to dilute the sample or the matched high concentration standard in the box, they can use buffer (1×)Perform dilution; For cell samples, it is recommended to centrifuge at 3000 rpm/min for 5 min before testing, and take the supernatant for testing

II. Operation steps

all operations are carried out at room temperature. It is recommended that all sampling wells be re drilled for determination

1. Restore the components of the kit to room temperature for 30min, take out the strips required for the test from the aluminum foil bag that has been equilibrated to room temperature, mark the strip order with a marker, seal the remaining strips with a sealing film, and then put them back into the aluminum foil bag, seal them, and store them at 2-8 ℃. (note: the slats are easy to fall off during the plate washing process. Pay attention to make marks.)
2. Sample incubation: add the diluted standard and the sample to be tested into the enzyme plate (recommended adding order: standard well, blank well, sample well, standard is added according to the concentration gradient), 100ul/ well; After that, enzyme labeled antibody (1×) was added to each well, 100ul/ well, plate was sealed with plate sealing membrane, and then incubated at 37 ℃, 400-600rpm for 1H. (note: the sampling time should be controlled within 10min to avoid drift with time. If the plate is not sealed or incompletely sealed during the incubation process, the reaction liquid will evaporate, resulting in experimental errors.)
3. Plate washing: after incubation, carefully remove the plate sealing membrane, discard the liquid in the hole, and use buffer (1×)Wash the plate three times (250ul/ well), and pat the residual liquid in the sample well. (note: if the plate washing method is manual washing, add buffer (1×)After that, it can stand for 1min; Add buffer (1×) if the plate washer is used to wash the plateIt can vibrate slightly for 5S.)
4. Color development: add the pre configured color development solution to the enzyme labeled plate, 100ul/ well, seal the plate with sealing film, and stand at 37 ℃ in the dark for 20min
5. Termination: add termination solution, 100μ L/ well, read after the color is developed evenly (note: generally, the reading is completed within 20min after adding the stop solution)
6. Reading: put the microplate into the microplate reader, set the wavelength to dual wavelength 450/630nm, and read the absorbance value (note: it is recommended to set a 5-10S oscillation in the microplate reader reading program)

III. data processing

1. Calculation of absorbance correction value the absorbance correction value of each standard or sample is: od450nm-od630nm- absorbance value of blank control hole
2. Draw a standard curve with the concentration of standard as the abscissa (x) and the absorbance correction value of standard as the ordinate (y), It is recommended to use a four parameter logistic mathematical model to fit the equation:

Y= ((A-D) / (1+ (x/c) ^b)) +d

substitute the corrected value of sample absorbance into the formula to calculate the content of BSA in the sample. (note the dilution ratio)

1. If the OD value of the sample to be tested exceeds the OD value of the highest point of the standard curve, dilute the sample and re measure it

note: ① according to the Chinese Pharmacopoeia <9012>4、 According to the relevant requirements in ligand binding analysis, four parameter logistic mathematical model is recommended for the fitting equation. ② Try to use the recommended dual wavelength correction method, and use 630nm wavelength for correction. Od450-od630 is the corrected OD value, which can be directly used for calculation or blank correction according to the data quality. If there is no dual wavelength microplate reader, after reading OD450 data, the data quality should be judged first, and then blank correction should be carried out

example display:

(the following standard curve is for reference only, and should be subject to the standard curve drawn in each experiment)

product background:

bovine serum albumin (BSA) is a kind of globulin in bovine serum, which is widely used in biochemical experiments. For example, BSA is often used as a stabilizer in the storage solution and reaction solution of limiting or modifying enzymes, playing a “ Protect ” Or “ Carrier ” The activity of many enzymes can be greatly improved after adding BSA. Secondly, BSA can reduce nonspecific binding and is often used as a blocking agent in Western blot and ELISA

some biological products such as antibody drugs, vaccine drugs and CGT drugs will use fetal bovine serum in the production process, which contains a certain amount of BSA. BSA, as an exogenous protein, may cause immune reaction or affect the drug effect if it enters the human body with the drug product. Therefore, in the third part of the 2020 edition of the Chinese Pharmacopoeia, the detection requirements of BSA in some vaccines for human use are specified: for the verification of the stock solution, it should not be higher than 50ng/ml, and for the verification of the finished product, it should not be higher than 50ng/ dose

reaction principle:

this kit applies double antibody sandwich enzyme-linked immunosorbent assay technology to determine the trace residue of BSA in samples. A 96 well enzyme labeled plate was coated with capture antibody to make a solid-phase antibody, then the standard and detection sample were added, and then the horseradish peroxidase (HRP) - labeled detection antibody was added to form a sandwich conjugate of solid-phase antibody BSA detection antibody, which was washed after the reaction, and then the substrate was added for color reaction, and the substrate was converted from colorless to blue under the catalysis of HRP, And turn into the final yellow under the action of the stop solution. Its absorbance value (OD value) was measured at 450nm and 630nm wavelengths, of which 630nm was the correction wavelength. The OD value was positively correlated with the BSA content in the sample

guidance for the validation of analytical methods:

1. According to the guiding principles of the Chinese Pharmacopoeia <9101>≪ 9012>≪ 9401>And ichAccording to the relevant provisions on the validation of analytical methods in, it is recommended that all laboratories using the kit verify the following indicators of impurity determination: specificity, accuracy, precision, detection limit (optional), limit of quantification, linearity, range, and durability
2. When doing the linear verification of BSA raw material, it is recommended to dilute the BSA raw material to the linear range of the kit and then determine it. Dilute it to at least five concentrations according to a certain proportion. The detection concentration is linearly related to the dilution multiple
3. Specificity should be verified for other substances in the process. It is suggested to use high concentration and low concentration quality control samples and add increasing concentrations of relevant interfering substances for specificity investigation. The matrix without analytes should also be measured at the same time. The accuracy of the quality control sample should be within a certain range, and the measured value of the matrix without analytes should be lower than the lower limit of quantification
4. For the accuracy (spiked recovery) of samples, please refer to the guiding principles for technical review of in vitro diagnostic reagent analysis performance evaluation (accuracy recovery experiment)

product performance indicators:

1, detection limit: <0.5ng/ml

2, limit of quantification: 0.5ng/ml

3, linear range: 0.5-32ng/ml

4, accuracy (spiked recovery): 70%-130%

5, accuracy (measurement deviation): ≤ 15%

1. Repeatability (intra assay difference): ≤ 10%

specificity:

the type of sample verified has certain limitations. It is recommended that each user test the cross reactivity of known substances in their sample matrix in similar experiments

1. The container for measuring BSA content should be dedicated to prevent BSA contamination in the laboratory<2. The background value of this reagent kit (i.e. OD0ng/mL before calibration) is generally not greater than 0.2. If the background value is greater than 0.2, it is necessary to investigate the environment, utensils, consumables, etc., or use unopened bottled Wahaha water for water quality inspection
2. All components in the reagent kit must be restored to room temperature (20-25 ℃) before use<4. Each component needs to be thoroughly mixed before use to ensure the uniformity of the reagent. The standard sample needs to be briefly centrifuged for 5 seconds to concentrate all the liquid on the tube wall and lid at the bottom of the tube. After use, all reagents should be immediately returned to 2-8 ℃7. When the Flat noodles is dried after washing, pay attention to prevent the Flat noodles from falling off, and the sealing film should not be reused9. When reading, pay attention to checking whether the detection wavelength and fitting equation selection are correct
3. Only by strictly following the operating methods in the manual and using all the reagents matched with this kit can the best detection effect be guaranteed12. Our company is only responsible for the reagent kit itself and is not responsible for sample consumption caused by the use of the reagent kit. Users are advised to fully consider the possible usage of the sample and reserve sufficient samples before use
4. This kit is only for in vitro research use and is not intended for clinical diagnosis.