A modified hybridoma technique for production of monoclonal antibodies having desired isotypes

Abstract

In the present study, we describe a modified hybridoma technique for production of monoclonal antibodies (mAbs) having a desired isotype. Mice were immunized with the antigen of interest. After having reached a high antibody titer, cells expressing IgM or IgG molecules were isolated from spleen cells of the immunized mice using a Magnetic Cell Sorting System. The isolated cells were fused with myeloma cells using the conventional fusion protocol. With the isolated IgM(+) spleen cells, more than 75% (85 ± 7%; means ± SD) were IgM producing cells and a large number of IgM mAbs specific to the protein of interest were obtained. With the isolated IgG(+) spleen cells, 41 ± 40% of the generated hybridomas produced IgG antibody and no IgM producing hybridoma was generated. A large number of IgG mAbs specific to the protein of interest could be produced. The results indicate that the generated hybridomas produce corresponding antibody isotypes as expressed on the surface of their starting cells. The technique that we have developed will be very useful for production of desired mAbs having a specific isotype.

Production of Monoclonal Antibodies against Human Trefoil Factor 3 and Development of a Modified-Sandwich ELISA for Detection of Trefoil Factor 3 Homodimer in Saliva

“…The IgG mAbs usually have higher affinity compared to the IgM isotype and are more advantageous for applications in immunological methods [ 43 ]. In order to produce mAbs with the desired isotypes, we modified the standard hybridoma technique for the production of mAbs having a desired isotype [ 39 ]. In the present study, we aimed to generate IgG mAbs specific for the TFF3 peptide.…”

“…After a high level of antibody titer was detected, the mouse was IP boosted with 50 μg dimeric TFF3 in the absence of Freund’s adjuvant. Five days after the last boosting, the mouse was sacrificed and its spleen cells were collected and fused with myeloma cells by modified hybridoma technique [ 39 ]. In brief, the spleen cells were subjected to isolate cells expressing surface IgG using a Magnetic Cell Sorting System (MACS) (Miltenyi Biotec, Bergish Gladbach, Germany).…”

“…In this study, we sought to develop an immunoassay for the determination of TFF3 homodimer in saliva. We have produced mAbs specific for human TFF3 peptides using our modified hybridoma technique [ 39 ]. Using the generated mAb, a modified-sandwich ELISA was developed which could be employed for the detection of TFF3 homodimer in human saliva.…”

Functionalized Parylene Films for Enhancement of Antibody Production by Hybridoma Cells

“…The diluted purified antibodies or culture supernatants were added to each well, and the mixture was incubated at RT for 1 h. Then, 100 μL of HRP-conjugated antimouse polyclonal antibody (from goat) (100 ng/mL) was added to each well and incubated at RT for 1 h. To quantify the antibodies against ochratoxin A, the microplate was treated with a TMB solution for 3 min. The enzyme reaction between HRP and TMB was quenched by treatment with 100 μL of 2 M sulfuric acid. − The amount of antibodies was reported by measurement of optical density at a wavelength of 450 nm using a microplate reader from molecular devices (CA, USA) (Figure S1). For the analysis of sensitivity and specificity of anti-OTA monoclonal antibody, the antibody after purification at different time was performed.…”