| || |
Special Internet Prices.
Fast & Guaranteed Worldwide Delivery!
Secure & FAST Online Ordering.
Our Drugstore Is The Most Trusted Online Drug Supplier.
Related post: Several characteristics of the cells secreting these molecules have also
been examined, particularly with a view to obtaining appropriate mutants for
further fusion studies between various hybridomas.
ZOl AI 00171-03 LIG
Genetic Studies on Rabbit Immunoglobulins and Other Serum Proteins
Initial steps in the purification of the rabbit immunoglobulin a3 heavy
chain produced by 702 has shown that the majority of ELISA-positive material
for rabbit immunoglobulin, and RIA-positive material for aS allotype, can be
obtained by passing either ascites fluid or cell culture supernatant through
DEAE-cel lulose at low ionic strength and constant pH. These findings were
confirmed and quantitative data concerning the levels of rabbit
Immunoglobulin were obtained using direct binding of radiolabeled
DEAE-cel lulose fractions to immunoadsorbents. Ascites fluid produced by
injection 10 7D2 cells into nude mice was found to be 100 fold more
concentrated with respect to rabbit immunoglobulin chain than that present
in 5X concentrated tissue culture supernatant obtained at a maximum cell
density of 2 X 10 cells/ml.
Rabbit immunoglobulin chain isolated from 702 ascites fluid by DEAE-
cellulose ion exchange chromatography was radiolabeled Desogen Price and adsorbed onto
ant1-a3 Sepharose. Adsorbed material was eluted and analyzed by SOS-PAGE.
Material isolated from 7D2 ascites fluid when analyzed under non-reducing
conditions on 5% SDS gel gave a single peak with a molecular weight of
130,000. The same sample, when reduced gave two peaks on 10% SDS gels with
molecular weights of 53,000 and 22,000. The 7D2 a3 positive product
quantitatively binds to goat anti-mouse L chain antibodies but not to any
anti-rabbit L chain antisera. The chain composition of the 130,000 mol . wt.
material is still unknown. It may be possible that 2 rabbit H chains are
joined in some fashion to a single mouse L chain.
The b4 light chain produced by cell line 12F2 has been isolated by
procedures similar to those used for the 7D2 heavy chain. DEAE-cel lulose
chromatography of ascites fluid, followed by immunoadsorbent and/or gel
filtration chromatography yields a fairly pure product with a normal L chain
molecular weight of 22,000, A mouse L chain is secreted by 12F2 along with
the rabbit L chain but the two chains are not covalently associated and do
not appear to be noncovalently associated either. Studies on the Isolation
of the al lotype-negative, ELISA-positive L chain from cell line 2C4 have
just been initiated.
Propagation of an L chain producing hybridoma cell line (1D4P5) in
ascites fluid produced sufficient material for preliminary structural
analyses. Affinity purification on an anti-b5 Sepharose column yield
approximately 1 mg of L chain which was used for sequence analysis. Twenty
N-terminal residues were identified and these confirmed that this was a
rabbit L chain. A minor sequence presumably from an Internal cleavage
(position 138) was characteristic of the b5 allotype.
Characterization and modification of rabbit-mouse hybrldomas . Wh i 1 e
the fusion of mouse myeloma cells to rabbit splenocytes has proven a useable
method to obtain at least a limited class of stable cell lines secreting
rabbit products, the range of products available is limited by the chromosomal
instability in that only one Ig chain has been stabilized per cell line and
ZOl AI U0171-03 LIG
a great deal of work is required to obtain each line. These problems would
be solved directly if a suitable rabbit myeloma cell were available.
Unfortunately all attempts to obtain rabbit B cell lines have been
unsuccessful in our hands. Viral transformation with a wide range of
lymphotropic transforming viruses has not produced growth and the only known
rabbit B cell tumor, the hereditary lymphosarcoma described by Fox at the
Jackson Laboratory, could not be established in culture in experiments
carried out in collaboration with Dr. George Moore at the Denver General
The most promising approach to altering the available repertoire of
rabbit-mouse hybrids is to use stabilized rabbit-mouse hybridomas as parental
lines for further fusions. Fusions may be done between hybridomas and
rabbit splenocytes or between two hybridomas. Such fusions would yield
cells producing complete IgG molecules and they might yield cells secreting
the desired products at much higher rates. All that is required is the
introduction of appropriate drug on metabolic sensitivities into the stable
hybridomas. Sensitivity to HAT medium, which is the only thing required for
fusion to splenocytes, is immediately available because, when Buy Desogen HAT medium is
removed after hybridoma formation, the instability of rabbit chromosomal
material assures a rapid and spontaneous loss of the rabbit HPRTase, with
reversion to HAT sensitivity. Rabbit-mouse hybrids of H-chain-secreting and
L-chain-secreting types have been cloned in 8-azaguanine to ensure HAT-
sensitivity. Fusions using these clones will be done as soon as reagents
are ready for a chain-specific ELISA screening assay. Fusions between two
hybridomas require separate, complementary drug sensitivities in the fusion
partners. In addition to HPRTase deficiency, APRTase and TKase deficiencies
are easily introduced into these cells.
Another type of experiment which will be done is to fuse a hybridoma to
cytoplasts obtained from a hybridoma secreting the same type of
immunoglobulin chain (H or L) but of a different allotypic specificity. In
some schemes Desogen Cost for the regulation of allotype expression, cytoplasmic factors
could be present which would induce synthesis of a new allotypic specificity
upon cybrid formation. This will be tested using appropriate chloramphenicol-
resistant mutants which have been cloned.
MITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)
U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
Related links: buy prilosec cheap, nexium 40 mg buy online, cost atarax 25 mg, how much does propecia cost, actos 15 mg tablet #30, Ondansetron Online, buy lisinopril no prescription, buy generic priligy uk, tricor order forms, buy albuterol online cheap
| || |