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Affinity of antibody detection on Countstar® FL

Affinity of antibody detection on Countstar® FL

 

Antibodies are nature’s molecular recognition machines designed to aid the immune system in destroying extracellular, as well as intracellular, threats. Detect affinity of antibody in cell level is an important indicator of monoclonal antibody detection using immunofluorescence method, and the current determination method is to use flow cytometry to achieve relatively quantitative. Here, the Countstar® FL can evaluate affinity of antibody using indirect immunofluorescence. The Countstar® FL present a direct quantitative value by detecting average fluorescence   intensity of different antigen-antibody reactions. It can monitor the concentration, viability of cell. The Countstar® FL has also the advantage in obtaining additional morphological information, provided by the permanent brightfield and fluorescence based image recordings during the whole process. The Countstar® FL offers a fast, sophisticated and reliable method for the affinity of antibody detection in antibody drug screening. The Countstar® FL system (Fig. 1) is a smart, intuitive cell analysis instrument that streamlines a wide variety of cellular assay including transfection, apoptosis, cell surface marker, cell viability, affinity of antibody and cell cycle assessments. The system provides robust fluorescence quantitative results. The easy-to-use, automated procedure guide you to complete a cellular assay form imaging and data acquisition.


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Figure 1. Countstar® FL system combines the functionalities of a digital microscope, an image cytometer and a cell counter in a single bench top instrument.



Materials:

Countstar products

Countstar® FL/Countstar® Chamber slides/0.2% Trypan blue solution (CS0101001-50)

 

Other materials

CHO cell line/DMEM (Hyclone-SH30243.01)/FBS (Hyclone-SH30084.03)/Trypsin (Hyclone-SH30042.01)

Using CHO cell line which stable express X protein to evaluate affinity of original drug and generic drug.

Preparing different concentrations of 002-BMK1, 002-BMK2, AB1, AB2, AB3, AB4, AB5, AB6, AB7, AB8, AB9, PCSK-9 as primary antibodies. Besides, 002-BMK1, 002-BMK2 antibodies can bind with X protein specifically, and PCSK-9 was a negative control antibody.

Using 5µg/mL Alexa Fluor® 488 Goat anti-mouse IgG (Biolegend) as secondary antibody.

 

Software used

GraphPad Prism5 

Methods:

Culture conditions

1.      CHO cells were plate in cell culture dish in medium containing DMEM, 2mML-Glutamine, 1.5g/L Sodium bicarbonate and 10% FBS. Incubate the plates for 48h at 37°C, 5% CO2 incubator.

2.      Harvest the cells.

 

Staining Procedure

1.      Determine the cell concentration by using trypan blue counting;

2.      Prepare 0.3million cells/action for next steps;

3.      Spin down cell sample at 400g for 3-5 minutes, aspirate medium, and then resuspend cells in 100µL PBS;

4.      Mix the samples gently and spin down cell sample at 400g for 3-5 minutes, aspirate medium, and then resuspend cells in 100µL staining buffer;

5.      Add different concentrations of primary antibodies to CHO cell line. The top concentration of primary antibodies was10µg/mL, 4  folds serial dilution, the bottom concentration was 0.01µg/mL;

6.      Incubate for 60min at 37℃ in the dark ;

7.      Spin down cell sample at 400g for 3-5 minutes, aspirate medium, and then resuspend cells in 100µL PBS;

8.      Mix the samples gently and spin down cell sample at 400g for 3-5 minutes, aspirate medium, and then resuspend cells in 100µLstaining buffer ;

9.      Add secondary antibody (5ug/mL) to CHO with each action;

10.  Incubate for 30min at RT (25℃) in the dark;

11.  Spin down cell sample at 400g for 3-5 minutes, aspirate medium, and then resuspend cells in 100µL PBS;

12.  Mix the samples gently and spin down cell sample at 400g for 3-5 minutes, aspirate medium, and then resuspend cells in 100µL staining buffer ;

13.  Mix the samples, load 20µL of samples into chamber and detect samples with Countstar® FL.

Note: Above operations can use EP tubes or 96-well plates.

 

Imaging and analysis with Countstar® FL

1.      The cell number and cell viability was detected by using “Trypan Blue Viability” directly.

2.      The signal-color application procedure was created by setting Green channel to image Alexa Fluor® 488 fluorescence.

3.      1or3 field(s) captured from each chamber.

4.      Data was displayed on the screen directly when testing. After imaging and initial analysis were completed, data were exported and analyzed. The process of affinity of antibody assay was described briefly in Figure 2. 


Figure 2. Process of affinity of antibody detection


Determining the does response curve for different antibodies

A dose-response experiment was set up in a 96-well plate to determine affinity of antibodies for CHO cells, including original drugs, generic drugs and negative antibody (Fig. 3). A range of reducing amounts of primary antibodies (10µg/mL-0.01µg/mL) were used across the plate as indicated.


Figure 3. 96-well plate map of antibodies conditions. Each well containing CHO cells (0.3 million), secondary antibody (5µg/mL) and primary antibodies (0.01, 0.039, 0.156, 0.625, 2.5, 10µg/mL) are indicated. Other wells untapped are unwritten.

 

Imaging and analysis with Countstar® FL imaging system

A signal-color application procedure was created by setting Green channel to image Alexa Fluor® 488 fluorescence, plus a bright field. Bright field picture reference segmentation was applied as a mask to sample the Alexa Fluor® 488 fluorescence signal. Example images of each concentration of AB4, images of highest concentration of PCSK9 and 002-BMK1 are shown in Figure 4. 



Figure 4. Images of 0.01, 0.039, 0.156, 0.625, 2.5, 10µg/ml of AB4 treated CHO cells. The images showed the higher antibody concentration, the stronger fluorescence signal. 002-BMK1 antibody can bind with Tim3 specifically, PCSK-9 was a negative control antibody. 002-BMK1 sample, all cells showed high fluorescence signal, representative image shown.PCSK-9 sample, showed almost no fluorescence signal, representative image shown.


Quantitative analysis for affinity of antibody with Countstar® FL imaging system

Different affinity of antibodies can be presented a direct  quantitative value by different average  fluorescence   intensity of antigen-antibody reactions. CHO cells which stable express X protein were applied to analyze affinity of antibodies arresting following treatment with different concentration of antibodies. The results were displayed in the database of Countstar® FL. Data was exported to analyse. Graphpad prism5 was implemented to evaluate the EC50 of antibodies (Fig. 5A). The fluorescence intensity increased as the primary antibodies concentrations increased. The average fluorescence intensity tendency showed that the reaction activity of each antibody at different concentrations, and PCSK-9 which was a negative control antibody showed almost no fluorescence intensity. LogEC50 of each antibody showed that the affinity of original drugs were better than generic drugs. AB2, AB3, AB4,AB6 are better among generic drugs, they can be used to do next assay (Fig. 5B). The Countstar® FL system can evaluate affinity of antibodies directly and reliable. 




Figure 5. Quantitative analysis results for affinity of antibody. A. Average fluorescence intensity in different antigen-antibody reactions varied with different concentrations of antibodies. B. Results about comparing affinity of original drugs and generic drugs.


Summary:

The Countstar® FL system provides a rapid, direct and easy means of evaluating affinity of antibodies. In addition, it combines multiple functionalities and is a compact, application-driven, automated cell imaging system providing robust quantitative results through the use of preconfigured biological applications. The Countstar® FL combines an extendable range of further applications in the same instrument. It is a compact and highly mobile equipment. The automated cell imaging and analysis system provides precise and reproducible quantitative results by using preconfigured biological application templates. Each pre-set assay is an easy-to-use, automated module that covers all steps of a specific biological assay to simplify routine cell laboratory tasks while providing high-quality scientific data. For quantifying affinity of antibodies, Countstar® FL supplies a direct and reliable method.