Now Bio-Rad's new array of SPR biosensor technology is expected to completely break the various bottlenecks of the existing methods, and achieve high-throughput data on concentration, affinity and reaction kinetics. This new array of SPR biosensors is called the ProteOn XPR36 system. As shown in the figure below, it has a 6-channel flow cell that can rotate 90°, and can produce 6 longitudinal or lateral channels on the chip. First, the flow cell is rotated into the longitudinal direction, and up to 6 different ligands can be marked on the chip in the longitudinal 6 channels. Then the flow cell is rotated 90° to become lateral, flowing through 6 analytes, when the analyte solution contacts the original mark. The interactions that occur at the time of ligand can be detected and recorded. High-throughput parallel analysis can be achieved by such an analysis. Using the ProteOn XPR36 system for antibody screening and optimization, Bio-Rad gave a new workflow that is completely different from the traditional method, as shown below. According to this procedure, the expression levels and kinetic data of 6 clones can be determined for each cycle. When all clones have been tested, we can draw a kinetic map containing all the clones, as shown below.    The advantage of this process is that the initial screening of a large amount of antibody information, basically no need to pick the clone for the second step of screening can directly pick out the clones we need to purify or further analysis to determine its subtype, antigen recognition epitope Wait. Some people may ask some questions, and now answer these questions as follows: 1 . How many clones can I filter in one day? A: About 200 . This number may sound frustrating, but don't forget that this is a screening that involves a lot of information. It's a step-by-step process that takes more steps than traditional methods. 2 . Do you need a lot of capture reagents and antigens? A: Not much at all, the capture reagent consumes a few micrograms, and the antigen consumption depends on the number of clones screened. If 1000 clones are screened, the antigen consumption usually does not exceed 1 mg. 3 . Is the cost of the entire process high? A: Not at all high, only one chip can complete the screening of thousands of clones, and the total cost of adding other consumption is only a little higher than ELISA . If you consider the kinetic data after the traditional method is selected, the cost will be lower.
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In the first step, the capture reagents, ie, molecules that can bind to the monoclonal antibody, such as Anti-mouse IgG , Protein A/G, etc., are labeled on six channels in the lateral direction. If the Fab fragment of the antibody is screened, Anti-(Fab') 2 can be labeled. After the marking is completed, the channel is rotated 90 ° into the longitudinal direction and the second step is entered.
The second step is to capture the monoclonal antibody from the cell culture medium and flow up to 6 clones at a time. If a mAb is expressed, the instrument will show an upward curve and the higher the concentration, the faster the curve will rise. If a standard curve is defined, the concentration value can be calculated. After this step is completed, the antibody or antibody fragment is bound to the capture reagent.
In the third step, the channel was turned back to the lateral direction, and five different concentrations of antigen molecules were added plus one buffer as a negative control. This step can draw six kinetic response curve shown in the figure bottom right, representing the results of antibody and antigen-reactive clones 6, we could then calculate the 6 antibodies and antigen affinity and kinetics data constant.
In the fourth step, regeneration is carried out with phosphoric acid or a glycine solution of pH 2.0 , and the antigen is washed together with the antibody from the capture reagent to prepare for the detection of the next batch of monoclonal antibodies.
The ordinate on the upper graph represents the dissociation rate constant ( k off ), the abscissa represents the binding rate constant ( k on ), and the oblique line is the iso-affinity line, that is, the clonal affinity distributed on the same affinity line is the same. We can select clones in specific regions as needed, and then perform antibody characterization.
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Antibody development and research using the array SPR sensor ProteOn XPR36 system
For antibody development and research, whether it is a complete monoclonal antibody molecule or Fab, scFv, we need to understand the expression level, affinity, reaction kinetics (especially the off-rate k off ) of the antibody molecule and other properties of the antibody such as subtype , specificity, etc. Screening for antibodies using traditional ELISA methods usually only shows which clones are expressed first, and then selects clones with higher expression levels for further study. This method of screening antibodies, although fast, but at best can only know the approximate expression level, all the rest of the information must be further studied, put a lot of pressure on the subsequent work, and often lost although the expression level is not high, but Very good clones with good antibody affinity, specificity, etc.
Later, people tried to use some new methods to screen antibodies or antibody fragments, the most common of which is SPR technology. Since the advent of SPR biosensor technology, its flux has been a problem. Measuring antigen-antibody reaction kinetic data usually takes several hours, far from meeting the requirements of antibody screening, and the price of products with higher flux is expensive. It can almost deter the world's most extravagant laboratories, so most laboratories with SPR sensors just use it for late antibody characterization. The ELISA method was also used in the early screening stage.