Antibodies are naturally produced immunoglobulins that are used to help the immune system fight off the inside and outside of cells. Immunofluorescence assay for antibody affinity at the cellular level is a very important indicator for evaluating antibody efficacy. The current major assay is to use flow cytometry to achieve relative quantification. Countstar ® FL can directly measure the antibody affinity by indirect immunofluorescence to detect the average fluorescence intensity of different antigen-antibody reactions, and also detect cell concentration and viability. In addition, cell images are available, and brightfield and fluorescent images are taken in real time during the experiment. Countstar ® FL provides customers with fast, accurate and reliable antibody affinity assays for antibody drug screening. Countstar ® FL (Figure 1) is an intelligent, intuitive cell analysis instrument for a variety of cell experiments, including cell transfection, apoptosis, cell surface markers, cell viability, antibody affinity, and cell cycle assessment. This system provides powerful fluorescence quantification results. An easy-to-use automated program guides you through the process of cell image acquisition to data analysis.
material:
Countstar products: Countstar® FL Cell Analyzer/Countstar® Sample Plate/0.2% Trypan Blue Staining Solution (CS0101001-50)
other materials
CHO cell line/DMEM cell culture medium (Hyclone-SH30243.01)/fetal calf serum (Hyclone-SH30084.03)/trypsin (Hyclone-SH30042.01), wherein the CHO cell line stably expresses X protein for evaluation Affinity of original and generic drugs
Primary antibody: 002-BMK1, 002-BMK2, AB1, AB2, AB3, AB4, AB5, AB6, AB7, AB8, AB9, PCSK-9 with different concentration gradients. Among them, 002-BMK1, 002-BMK2 can specifically bind to X protein (positive antibody), and PCSK-9 is a negative control antibody.
Secondary antibody: Alexa Fluor® 488 Goat anti-mouse IgG (Biolegend), 5 μg/mL
software
GraphPad Prism5®
method:
Cell culture conditions
1. CHO cells were cultured in complete medium with 500 m LDMEM medium, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate and 10% fetal bovine serum. Incubate for 48 hours at 37 ° C in a 5% CO 2 incubator;
2. Digest and collect cells.
Dyeing step
1. Determine the cell concentration using the trypan blue count;
2. Prepare 3*10^5 cells per reaction;
3. Centrifuge the cell sample at 400g for 3-5 minutes, remove the supernatant, and resuspend in 100 μL PBS;
4. Gently mix the cell sample, centrifuge at 400g for 3-5 minutes, remove the supernatant, and resuspend with 100 μL of Cellstaining buffer;
5. Add a different concentration of primary antibody to the cell sample, the highest concentration of each antibody is 10μg / mL, 4 times gradient dilution, the lowest concentration is 0.01μg / mL;
6. Incubate at 37 ° C for 60 minutes in the dark;
7. Centrifuge the cell sample at 400g for 3-5 minutes, remove the supernatant, and resuspend in 100 μL PBS;
8. Gently mix the cell sample, centrifuge at 400g for 3-5 minutes, remove the supernatant, and resuspend with 100 μL of Cellstaining buffer;
9. Add 5 μg/mL secondary antibody to each reaction group;
10. Incubate in the dark for 30 minutes at room temperature;
11. Centrifuge the cell sample at 400g for 3-5 minutes, remove the supernatant, and resuspend in 100 μL PBS;
12. Gently mix the cell sample, centrifuge at 400g for 3-5 minutes, remove the supernatant, and resuspend with 100 μL of Cellstaining buffer;
13. Mix the sample, add 20 μL to the cell counting plate, and check on the machine.
Note: The above operation can use EP tube or 96-well plate.
Image acquisition and data analysis
1. Select "Trypan Blue Count" for cell concentration and viability determination;
2. The single fluorescence module program is used to obtain an image of Alexa Fluor® 488 under the FITC channel;
3. Collect 1 or 3 fields of view per sample (the experiment detects 1 field of view);
4. The data result is directly presented during the detection process, and after the image acquisition and data analysis are completed, the data result is derived. The antibody affinity detection process is shown in Figure 2.
Figure 2. Antibody affinity detection process
Different antibody gradient dilution additive
A dose-effect experiment was performed using 96-well plates to determine the response of different antibodies to CHO cells, including the original drug, antibody drug, and negative control (Figure 3). The primary antibody concentration was indicated as indicated by the following table (10 μg/mL - 0.01 μg/mL).
Figure 3. Antibody concentration in 96-well plates. There are CHO cells (300,000), secondary antibody (5 μg/mL) and the indicated primary antibody (0.01, 0.039, 0.156, 0.625, 2.5, 10 μg/mL). The other holes are empty.
Countstar® FL system image acquisition
Countstar® FL's built-in antibody affinity detection program performs brightfield, acquisition of Alexa Fluor® 488 images under the FITC channel. The image of the bright field is used as a reference standard to segment the image of the fluorescence field to obtain the signal of the fluorescence field. The results of different concentration gradients AB4, highest concentration PCK9 and 002-BMK1 samples are shown in Fig. 3.
Figure 4. Cell images of 0.01, 0.039, 0.156, 0.625, 2.5, 10 μg/ml AB4 reacted with CHO cells.
The image shows that the higher the antibody concentration, the stronger the fluorescent signal. The 002-BMK1 antibody can specifically bind to the X protein, and the PCSK-9 is the negative control antibody. The image represents the strong fluorescence signal of the 002-BMK1 sample, PCSK-9 sample. There is basically no fluorescent signal.
Quantitative analysis of antibody affinity
The average fluorescence intensity exhibited by different antigen-antibody reactions directly quantifies the magnitude of antibody affinity. The CHO cell line stably expressing the X protein is used to analyze the antibody affinity, and the Countstar® FL can directly display the test results and export the data for analysis. The EC50 of the antibody was analyzed using GraphPad Prism5® (Fig. 5A). The test results show that the fluorescence intensity of the sample increases with the concentration of the primary antibody. The average fluorescence intensity curve represents the reactivity of each antibody at different concentrations. PCSK-9 is a negative control antibody, and the sample has almost no fluorescent signal. The results of LogEC50 of each antibody showed that the antibody affinity of the original drug was better than that of the generic drug, while the AB2, AB3, AB4, AB6 in the generic drug had better effect and could be further tested (Fig. 5B). The Countstar® FL system provides direct and reliable results for evaluating antibody affinity.
Figure 5. Quantitative analysis results of antibody affinity. A. Average fluorescence intensity in different antigen-antibody reactions under different concentration gradient antibodies. B. Comparison of analysis results between original and generic drugs.
to sum up:
The Countstar ® FL system provides a fast, direct, and easy way to evaluate antibody affinity. In addition, it is a device that combines electron microscopy, image processing, and automatic cell counting. In addition, Countstar ® FL offers an extended feature that users can customize to suit their needs. Countstar ® FL is a highly mobile device with a variety of preset programs that allow users to quickly access accurate, stable, and highly reproducible data. Each application module in the instrument is simple and easy to use, greatly simplifying the work of conventional laboratories and providing customers with high-quality scientific research data. Countstar ® FL provides a direct and reliable test result for customers conducting antibody affinity experiments.
China Daily chemical Suppliers
Here you can find the related products in Daily chemical, we are professional manufacturer of Daily chemical. We focused on international export product development, production and sales. We have improved quality control processes of Daily chemical to ensure each export qualified product.
If you want to know more about the products in Daily chemical, please click the Product details to view parameters, models, pictures, prices and Other information about Daily chemical.
Whatever you are a group or individual, we will do our best to provide you with accurate and comprehensive message about Daily chemical!
Hydrolyzed Keratin, Menthol, Daily Chemistry, Cosmetics Field
Xi'an Gawen Biotechnology Co., Ltd , https://www.ahualynbios.com