One of the quality control procedures performed when developing monoclonal antibodies is to perform an assay to determine the isotype of the hybridoma supernatant after clonal isolation. Several methods can be used such as Ochterlony and ELISA. One reason for knowing the isotype is to help determine whether to perform antibody purification using cell culture supernatant or ascites. Isotype can also be used to reject unwanted IgMs or the sometimes unstable IgG3s. Two separate tests to determine isotype were compared in our lab: Mouse Immunoglobulin Isotyping ELISA Kit from BD Biosciences, and IsoGold Rapid Mouse-Monoclonal Isotyping Kit™ from BioAssay Works®.
The assay currently used by our lab is with the BD kit; although the test is very reliable, it is also cumbersome and time-consuming. There is a coating step in which we choose to incubate overnight (alternatively, it can be performed in 1 hour at 37°C), and several steps following the coating which require incubations. A complete test requires approximately 4 hours including incubations and lab preparation. Also, testing just one cell supernatant from a hybridoma is costly since at least an entire ELISA plate is used for the testing. Although not required, a plate washer makes the assay much more efficient to perform, and a plate reader is necessary for producing quantitative results. The test results can be read visually because of the color change produced by the enzyme-substrate reaction; in this case a plate reader is not required. If we were producing a large quantity of clones, this test would be most useful. One kit contains enough reagents for 80 samples.
Recently, our lab was introduced to the IsoGold™ Rapid Mouse-Monoclonal Isotyping Kit from BioAssay Works®. This colloidal gold-based immunoassay can screen hybridoma cell supernatants or monoclonal antibody ascites fluid for its isotype. This test can be performed in 5 minutes, saving a lot of precious laboratory time. Also, neither a plate washer nor reader is required. The results are read by eye and can be removed from the kit cassette and taped in to the notebook. Therefore, if quantitative results are required, this test could not be used. But after performing a dozen of these tests, it was very easy to determine the isotype. The colloidal gold lines are very distinct. In most cases, a qualitative assay is all that is needed.
Figure I shows the result from the IsoGold test with cell supernatant of isotype IgG1. This produced an optical density (OD) reading of 1.182 in the BD ELISA kit.
Figure II shows the result from the IsoGold test with cell supernatant of isotype IgG2a. This produced an OD of 1.178 in the BD ELISA kit.
We performed an analysis in which 5 different hybridoma cell supernatants were compared in the IsoGold™ and the BD kits. Myeloma cell supernatant was also tested as a negative control. The results are listed in Table I: Side-by-Side Comparison of BioAssay’s Mouse IsoGold Kit versus BD Pharmingen™ Isotype ELISA Kit. As shown, the same results were obtained with both kits. One of the advantages of using the IsoGold test is that as soon as a hybridoma is stabilized, the supernatant can be quickly tested to determine its isotype with confidence. With the ELISA test, we usually wait until there are at least 8 hybridomas finalized before performing the experiment.
Each kit described above has advantages and caveats. For large-scale analysis, the BD isotyping kit is probably more affordable. When testing fewer samples, the time savings in the lab using the IsoGold Kit is also a savings in labor. The IsoGold test requires no equipment or materials, but the BD kit requires ELISA plates and performs optimally with an ELISA plate washer and reader. The plates can be washed manually, although this requires more time. Both tests can be visually read, but the BD kit can also be read using a plate reader. The IsoGold kit is a rapid test and requires minimal preparation and only 5 minutes incubation once sample is added to the strip. It is also very easy to use. The BD kit requires at least 4 hours for completing the assay.
It is best to choose an isotyping kit based on your needs, and the summary above highlights two choices that are being used in our laboratory.
Scientist
Immunology
Tetracore, Inc.