Optimal conditions for the generation of monoclonal antibodies using primary immunisation of mouse splenocytes in vitro under serum-free conditions

Boer, Mark de; Ossendorp, Ferry; Duijn, G. Van; Voorde, G.H.J. ten; Tager, J. M.

doi:10.1016/0022-1759(89)90168-3

Cited by 14 publications

(8 citation statements)

References 23 publications

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“…Briefly, mice were immunized s.c. at 3-week intervals with recombinant chicken Prl (50 lg per animal per injection, total of four injections), and activated splenocytes were fused with myeloma cells by electrofusion (De Boer et al 1989). Hybridomas were screened for antibody production by an immunocytochemical protocol on sagittal paraffin sections of Bouin-Hollande sublimate-fixed chicken pituitary glands (Berghman et al 1989).…”

Section: Production Of Monoclonal Antiserum For Chicken Prlmentioning

confidence: 99%

Independent differentiation of mammotropes and somatotropes in the chicken embryonic pituitary gland

Zheng

Nakamura

Maseki

et al. 2005

Histochem Cell Biol

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It has been reported that mammotropes in a rodent pituitary gland are derived from somatotropes via somatomammotropes (SMTs), cells that produce both growth hormone (GH) and prolactin (Prl). However, no studies have been done on the transdifferentiation of somatotropes in the chicken pituitary gland. In this study, in order to determine the origin of mammotropes, we studied detail property of appearance of chicken somatotropes, mammotropes and pit-1 cells and then evaluated the existence of SMTs in the chicken embryonic pituitary gland. Immunohistochemical analysis revealed that GH-immunopositive (GH-ip) cells appeared on embryonic day (E) 14 and were mainly distributed in the caudal lobe, while Prl-immunopositive (Prl-ip) cells appeared in the cephalic lobe of the pituitary gland on E16. In situ hybridization (ISH) and RT-PCR analysis showed that expression of GH and Prl mRNA starts at E12 in the caudal lobe and at E14 in the cephalic lobe respectively. Pit-1 mRNA was first detected on E5 by RT-PCR, and pit-1 mRNA-expressing cells were found in the cephalic lobe on E8. Then with the ontogeny of the chicken, these cells spread into both lobes. Using a double staining method with ISH and immunohistochemistry, we could not detect the existence of SMTs in the chicken embryonic pituitary gland even in the marginal region of either lobe. These results suggest that chicken somatotropes and mammotropes independently appear in different lobes of pituitary gland and that transdifferentiation from somatotropes to mammotropes is not the central route for differentiation of mammotropes in the embryonic chicken pituitary gland.

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Section: Production Of Monoclonal Antiserum For Chicken Prlmentioning

confidence: 99%

Independent differentiation of mammotropes and somatotropes in the chicken embryonic pituitary gland

Zheng

Nakamura

Maseki

et al. 2005

Histochem Cell Biol

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“…injection of 50 g of chicken FSH in PBS 4 and 2 days prior to fusion. A suspension of activated splenocytes was prepared, and a portion was fused with SP2/0 myeloma cells by electrofusion [22]. The fused cells were plated into a total of thirty 96-well plates at a density of 20 000 cells per well in the presence of 100 U recombinant human interleukin-6 (Boehringer-Mannheim, Indianapolis, IN) to support single-cell growth.…”

Section: Mab Productionmentioning

confidence: 99%

Immunohistochemical Evidence That Follicle-Stimulating Hormone and Luteinizing Hormone Reside in Separate Cells in the Chicken Pituitary1

Proudman

Vandesande

Berghman

1999

Biology of Reproduction

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As is the case in other tetrapod species, the chicken gonadotropins LH and FSH consist of a common alpha subunit and a hormone-specific beta subunit. Gonadotrophs containing LH were shown earlier to be distributed throughout both the caudal and cephalic lobes of the chicken anterior pituitary, but the cellular distribution of FSH in avian species is still uncertain. The purpose of this study was to determine the cellular distribution of FSH-containing chicken gonadotrophs by use of FSH-specific monoclonal antibodies (mAbs). Three new mAbs toward chicken FSH were proven hormone specific by immunodetection of purified hormones on dot blots and by dual-label immunohistochemistry (IHC) on sagittal sections of chicken pituitaries. A rabbit antibody was used to detect chicken LH. Results showed that LH-containing gonadotrophs were densely distributed throughout the anterior pituitary, whereas gonadotrophs containing FSH were much less numerous; in addition, while also present in both lobes, FSH-positive cells were largely absent from the outer margin of the gland. Dual-label IHC revealed that LH and FSH reside almost exclusively in separate gonadotrophs. The identity of FSH-containing cells was further confirmed through use of an antibody to the chicken alpha subunit, which showed that FSH immunoreactivity was always colocalized with the alpha subunit. Our results suggest the possibility that production and secretion of LH and FSH may be regulated differently in chickens than in most other species studied to date.

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“…Many workers favour serum-free medium for in vitro immunization (Reading, 1982;Van Ness et al, 1984;Darfler, 1987;De Boer et al, 1989), but when spleen cells from mouse 3473 were in u/fro-boosted in serum-free medium only 16% of the cells survived the 4 day culture period (Table 4.10). In contrast, there was a 46% survival rate when spleen cells from mouse 3365 were in vitro-boosted in 15% FCS medium.…”

Section: Fusions With In V7tro-boosted Spleen Cellsmentioning

confidence: 99%

Monoclonal Antibodies

1994

Hybridoma

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Optimal conditions for the generation of monoclonal antibodies using primary immunisation of mouse splenocytes in vitro under serum-free conditions

Cited by 14 publications

References 23 publications

Independent differentiation of mammotropes and somatotropes in the chicken embryonic pituitary gland

Independent differentiation of mammotropes and somatotropes in the chicken embryonic pituitary gland

Immunohistochemical Evidence That Follicle-Stimulating Hormone and Luteinizing Hormone Reside in Separate Cells in the Chicken Pituitary1

Monoclonal Antibodies

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