Using antigen arrays, researchers at the European Molecular Biology Laboratory have developed a high-throughput method for producing monoclonal antibodies.
In a study published in the Oct. 27 issue of Proteomics, researchers led by Federico De Masi and Alan Sawyer showed that the method was able to generate 68 monoclonal antibodies within six weeks after immunizing eight mice with 10 antigens each.
"Normal antibody production takes five to nine months for a single antibody, depending on how immunogenic the antigen is," said De Masi, who is now a postdoc in Martha Bulyk's laboratory at Brigham & Women's Hospital. "By using antigen microarrays, we can basically detect the antibody of the animal at a much earlier stage, and the screening is faster than with an ELISA or Western blot."
The EMBL's patented antibody production method has been commercialized by Taiwan-based Abnova, which signed a collaboration agreement with EMBL in December 2004.
As with many other antibody production methods, the EMBL method begins with immunizing a mouse with an antigen of interest. Researchers then wait four weeks for the mouse to generate an immune response before sacrificing it.
After B-cells have been harvested from the spleen of the mouse, researchers generate a library of thousands of hybridoma cell lines — immortal cell lines that produce one type of monoclonal antibody each — by fusing the B cells with cells from a mouse cancer cell line.
"The greatest challenge in creating the array is that we had to find the right concentration of the antigens, and the right chemical modification of the slide to get the microarray functional. Then each sample that we spot has a different density and viscosity, so there was a lot of tweaking that had to be done. It took some time to find the right conditions that were highly reproducible for the whole spectrum of antibodies."
The key to the new antibody production method is that screening of hybridoma cell lines is done using a chemically modified glass slide coated with the antigen of interest. Because this array method is more sensitive for detecting antibodies than ELISA or Western blot methods, researchers do not have to wait as long for mice to generate an antibody response before sacrificing the animals.
While traditional monoclonal antibody production methods require waiting six weeks to three months before sacrificing the immunized mouse, the EMBL method requires only four weeks of waiting.
After hybridomas have been spotted onto the antigen-coated slide, the slide is washed, incubated with a fluorescent secondary antibody that recognizes mouse antibodies, then washed again. If an antibody has stuck to the antigen-coated slide, then it can be visualized through the fluorescent secondary antibody.
"The greatest challenge in creating the array is that we had to find the right concentration of the antigens, and the right chemical modification of the slide to get the microarray functional," said De Masi. "Then each sample that we spot has a different density and viscosity, so there was a lot of tweaking that had to be done. It took some time to find the right conditions that were highly reproducible for the whole spectrum of antibodies."
To make the monoclonal antibody production process even more high throughput, EMBL researchers decided to immunize mice with more than one antigen at a time. They first tried two antigens per mouse, then five, then 10.
In a large-scale experiment, De Masi and his colleagues immunized eight mice with 10 antigens each. After the mice were sacrificed and hybridomas were created, the researchers screened the hybridomas on 10 slides that were coated with one antigen each. In the end, the researchers were able to generate antibodies against 68, or 85 percent, of the antigen targets, within six weeks of primary immunization.
Aside from using the arrays to cut down on antibody production time, EMBL researchers also saved on time by using custom-built robots to automate the hybridoma production process.
"The robots take care of changing cell medium, fusion of B-cells, plating onto plates, [and] incubation," said De Masi. "Otherwise, we'd have to have somebody 24 hours per day taking care of these cells."
Currently, the EMBL's monoclonal antibody core facility will produce monoclonal antibodies to an antigen for about 2,500 euros ($3,017) per antigen, De Masi said. Monoclonal antibodies against peptides cost a little more — 3,000 euros ($3,591) per peptide — because it is harder to raise a good immune response against a peptide, which is generally shorter than an antigen.
The EMBL generally leaves quality testing of antibodies up to the researchers who have requested them, De Masi said.
"Generally what's done is we obtain five to 10 strong affinity hybridomas and we send it to the investigator so that he or she can choose the one that he or she likes," said De Masi. "Depending on what the investigator needs the antibody for, the antibody might be fantastic for one application, but useless for another. For example, with some of our antibodies that we did test, they gave some signal on the microarray, a beautiful Western blot band, but no signal on an ELISA."
Abnova president and CEO Wilber Huang said that his company's collaboration with EMBL furthers its effort to become a leader in high-throughput antibody production.
"Custom service and partnership now form the core of our business. We are concurrently developing a comprehensive antibody bio-tool catalog for the drug discovery and diagnostic industry," said Huang, following the formation of the collaboration agreement. "As such, we are focusing on several key areas of human proteins: kinase, apoptosis, receptors, stem cell and plasma proteome. The collaboration with EMBL is the latest step in our effort to realize these goals."
— Tien-Shun Lee ([email protected])