Tion of antigen solution can be applied onto nitrocellulose paper at the corresponding position of the 96-well plate by a micropipet (approximately 0.5 #l per spot) or a fine brush. Alternatively, instead of using the mini-manifold block, higher concentra-Ġ022-1759/90/$03.50 © 1990 Elsevier SciencePublishers B.V. After passing the solution through the nitrocellulose paper, it was air-dried and then incubated with 1% gelatin (Sigma, MO) in PBS overnight at room temperature (Fig.lA). A nitrocellulose paper (11.5 x 7.5 cm, BioTrace NT, Gelman, MI) was placed in a minimanifold block (Atto, Tokyo) and 0.1 #g of CSPG in 100/~1 of PBS was added into each well. Cells were grown in RPMI 1640 (Gibco, New York) containing 10% fetal calf serum (Hyclone, UT) and H A T (10 -5 hypoxanthine, 4 x 10 -8 M aminopterin and 1.6 x 10 -6 M thymidine, Sigma, MO) according to Galfr6 and Milstein (1981). After five injections with 2 weeks intervals, the immunized mice were killed and spleen cells were isolated and fused with myeloma cells, P3 x 63 Ag 6. Female B A L B / c mice were immunized with 50 # g / m o u s e / i n j e c t i o n of chondroitin sulfate proteoglycan (CSPG) isolated from human aorta. It can be used as a standard method for hybridoma screening during monoclonal antibody production in many laboratories. The procedure is simple and can be employed without any special equipment. This method provides high sensitivity of the nitrocellulose strip method with background information and simplicity in handling as the mini-manifold method. We describe here a large scale dot blot ELISA using the 96-well culture plate as an incubation chamber.
The second disadvantage is that the method requires as many expensive minimanifold blocks as the number of 96-well plates to be screened at the same time. The disadvantage of this method is again the lack of information about the background. Abbreviations: ELISA, enzyme-linkedimmunoadsorbent assay CSPG, chondroitin sulfate proteoglycan PBS, phosphate-bufferedsaline SDS, sodium dodecyl sulfate. Alton Jones Cell Science Center, 10 Old Barn Road, Lake Placid, NY 12946, U.S.A. This method allows to use one sheet of nitrocellulose paper for as many as 96 hybridoma supernatants withoutĬorrespondence to: R. (1983) developed a method using a mini-manifold block as an incubation chamber. To make the handling of the nitrocellulose paper easier, Pardue et al. (1982) claimed that the dot blot ELISA provides higher sensitivity than the plastic plate ELISA. The main advantage of this method is the possibility of distinguishing between the background and the signal distinctively. This method uses nitrocellulose paper strips (3 × 3 mm) with spots of antigen immersed separately into each well of a 96-well plate filled with culture supernatant of the growing hybridomas. Alternatively, a dot blot ELISA using the nitrocellulose paper was developed by Hawkes et al. One disadvantage of this plastic plate ELISA is, however, the lack of information about the background which often varies significantly between hybridomas. The ELISA based on the binding of protein and plastic plate is widely used for this purpose. Screening of positive hybridoma is one of the most critical steps during monoclonal antibody production. (Received28 March 1990, revised received, accepted )ĭear Editors, Monoclonal antibody is one of the most important tools in modern biology and medicine (Harlow and Lane, 1988). Alton Jones Cell Science Center, 10 Old Barn Road, Lake Placid, N Y 12946, U.S.A. Journal of Immunological Methods, 132 (1990) 147-149Įlsevier JIM 05689 L e t t e r to the editorsĪ large scale dot blot ELISA using the 96-well culture plate Witold Piwowarczyk and Ryoichi Matsuda Iv.
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