Rat Hybridoma Research Articles

Minor modifications to the structure of tunicamycin lead to loss of the biological activity of the antibiotic

Three analogues of tunicamycin, each with minor alterations in structure in different regions of the molecule, have been employed to study the effects of such modifications upon the biological activity of the antibiotic. The data indicate that any modification of structure results inloss of the ability of the antibiotic to inhibit N-glycosylation of proteins. In contrast to tunicamycin itself, none of the analogues had any deleterious effects upon cellular macromolecule synthesis, nor upon the kinetics of export of de novo synthesized IgM or IgG molecules from treated rat hybridoma cells. In addition, the incorporation of tritiated sugars into acid-precipitable macromolecules was not inhibited. Endoglycosidase H digestion of isolated IgG molecules further suggested that the analogues employed did not interfere with qualitative glycosylation at the level of N-acetylglucosamine transferases I and II in the golgi apparatus. The data are consistent with the interpretation that tunicamycin has very precise structural requirements for expression of inhibitory effects upon protein glycosylation, and that small variations of structure can lead to loss of its inhibitory effects.

5] Somatic cell genetic analysis of myelomas and hybridomas

The study and genetic manipulation of hybridoma cells growing in culture have acquired great practical significance. As with parental myeloma cells, mouse, rat, and human hybridomas grow as suspended cells. The best hybridomas produce about 50 μg of antibody/106 cells/24 hr and usually have doubling times of 17–19 hr. They are commonly grown in petri or tissue culture dishes in CO2 incubators but may also be propagated in large amounts in any of a variety of culture vessels. A few mouse myelomas were originally made with derivatives of MPC 11 but these have similar characteristics. There are still considerable problems with making human monoclonal antibodies and an ideal fusion parent has not yet been found. The chapter discusses mouse and rat myelomas having similar characteristics.

An immunodominant domain in adenovirus type 2 early region 1A proteins

Six independent rat hybridoma cell lines producing monoclonal antibodies to human subgroup C adenovirus early region 1A (E1A) proteins were isolated. Competition binding experiments revealed that each of the monoclonal antibodies was directed against the same epitope or overlapping cluster of epitopes on the E1A proteins. Viral E1A deletion mutants and deleted forms of E1A proteins expressed in Escherichia coli were used to localize the antibody recognition sites to sequences between amino acids 23 and 120, encoded within the first exon of the E1A gene. Similarly, polyclonal antisera raised against the trpE-E1A fusion protein, as well as against the native, biologically active E1A protein, were also directed primarily against this immunodominant region.

EXPRESSION OF IMMUNOGLOBULIN LAMBDA CHAINS IN THE LABORATORY RAT

We immunized a BALB/c mouse with the lambda-bearing rat IgG1 myeloma IR31, fused its spleen cells with the hybridoma parent line P3.X63.Ag8.653, and isolated a monoclonal antibody (G33/11) directed against rat immunoglobulin lambda chains. We used this antibody to classify two existing rat hybridomas as lambda-bearing proteins (D4.37HL.252 and PC61.5), and isolated one new lambda-bearing rat IgM hybridoma, G36/1. All the normal inbred rat sera that were tested contained lambda-bearing Ig as detected by G33/11, at levels ranging from 1.5% to 13% of the total serum Ig, the mean value being 7.9%. This antibody will be valuable for broadening our understanding of the immunogenetics of the rat, and for the characterization of monoclonal antibodies made in this species.

Rat monoclonal antibodies VI. Production of IgA secreting hybridomas with specificity for the 2,4-dinitrophenyl (DNP) hapten

A simple method to obtain rat hybridomas producing specific IgA antibodies is reported. By fusing the IR983F rat myeloma cell line with mesenteric lymph node cells from LOU/C rats immunized via the Peyer's patches with DNP- Salmonella typhimurium, twenty hybrids secreting monoclonal IgA antibodies specific for DNP were produced and maintained as highly secreting transplantable ascitic tumors. The monoclonal IgA antibodies were easily purified by affinity chromatography on a DNP-immunosorbent and were found to comprise both monomers and polymers.

Monoclonal antibody ARC MAC 50.1 binds to a site on the phytochrome molecule which undergoes a photoreversible conformational change

Monoclonal antibody from rat hybridoma cell line ARC MAC 50.1 binds more strongly to Pfr than to Pr in a sandwich ELISA of phytochrome in crude Avena sativa L. extracts or highly purified Avena phytochrome. Discrimination is not a consequence of differential modification of Pr and Pfr during the assay. Loss of a 6 kDa peptide from the N-terminal end of the phytochrome apoprotein during purification does not prevent discrimination. Neither is preferential binding to Pfr a consequence of a steric interaction between ARC MAC 50.1 and the antibody used with it in the sandwich ELISA. It is concluded that the binding site for ARC MAC 50.1 undergoes a reversible conformational change upon photoconversion and may thus represent a functional region of the phytochrome molecule.

6] Production of IgA-secreting rat × rat hybridomas

6] Production of IgA-secreting rat × rat hybridomas

Human, rat or mouse hybridomas secrete high levels of monoclonal antibodies following transplantation into mice with severe combined immunodificiency disease (SCID)

Mice with severe combined immunodeficiency disease (SCID) have been investigated for their ability to grow xenogenic hybridomas of mouse, rat and human origin. Two rat×mouse hybridoma lines (187.1.10 and 3B9) and 1 mouse×mouse hybridoma (2D9) grown in pristane-treated SCID mice as ascites tumors showed a 100–200-fold increase in monoclonal antibody levels over the amount produced in vitro with a total yield up to 0.5 g of antibody per animal. A human×human hybridoma, CLL-11-D1, exhibited a 1000-fold increase in human immunoglobulin levels in ascites (1.3 mg/ml) as compared to that obtained in tissue culture. Analyses of the antibody protein in the SCID ascites produced by these hybridomas using protein electrophoresis, SDS polyacrylamide gel electrophoresis and high resolution isoelectric focusing indicated the antibodies were monoclonal and free from any contaminating immunoglobulins. Yields of monoclonal antibodies of over 90% purity could be obtained from the ascites by a single ammonium sulfate precipitation step. This study indicates that SCID mice provide several significant advantages over other in vivo methods for the production of pure monoclonal antibodies of human, rat, or mouse origin.

Rat monoclonal antibodies. IV. Easy method for in vitro production

A simple technique for in vitro production of monoclonal antibodies is described. Two to 5 × 10 6 hybridoma cells are diluted in 300 ml of medium in a 2 litre ‘roller bottle’ and incubated for 2 weeks without subsequent adjustment of the cell concentration or renewal of the medium. The method is based on the observation that when hybridoma cell cultures are maintained in the same medium, although cell growth stops after a few days and the percentage of dead cells increases rapidly, the amount of monoclonal antibody present in the culture medium increases up to the 15th day, the yield reaching more than 10 mg per flask. The method can be used both for rat and mouse hybridoma cell culture.

Monoclonal antibody production to a B16 melanoma associated antigen

Eight different rat hybridoma cell lines have been produced which secrete MAbs identifying the melanoma-specific B700 antigen. This antigen has been previously shown to have significant sequence homology to the mammalian serum albumins, but antibodies specifically raised against B700 cross-react only with murine albumin. Each of these MAbs has been shown by Western immunoblotting to recognize an independent epitope on the B700 protein; it is concluded not only that the B700 protein is a highly immunogenic antigen, but also that the sequence homologies between the B700 antigen and murine albumin must occur extensively throughout the molecule.

Monoclonal antibodies to antigens in the peribacteroid membrane from Rhizobium-induced root nodules of pea cross-react with plasma membranes and Golgi bodies.

Three rat hybridoma lines that produced monoclonal antibodies reacting with the peribacteroid membrane from Pisum sativum were isolated, and these all appeared to recognize the same antigenic structure. Using one of these monoclonal antibodies, AFRC MAC 64, electron microscopy of immunogold-stained thin sections of nodule tissue revealed that the antigen, present in the peribacteroid membrane, was also found in the plant plasma membranes and in the Golgi bodies, but not in the endoplasmic reticulum. When peribacteroid membrane proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose by electro-blotting, it was found that MAC 64 bound to a series of protease-sensitive bands that migrated in the mol. wt. range 50-85 K. The epitope was sensitive to periodate oxidation and its structure may therefore involve the carbohydrate component of a membrane glycoprotein. We suggest that this structure originates in the Golgi apparatus and is subsequently transferred to the peribacteroid membranes and plasma membranes. The monoclonal antibody also reacted with peribacteroid membranes from nodules of Vicia and lupin, and with plasma membranes and Golgi membranes from uninfected plant cells, including root tip cells from onion (Allium cepa), indicating that the antigen is highly conserved in the plasma membranes of plant cells.

Immunohistological screening in the selection of monoclonal antibodies: the use of isotype-specific antiglobulins

This paper describes the use of the immunoperoxidase technique for the screening of rat hybridoma culture supernatants on tissue sections. By combining the avidin-biotin system with mouse monoclonal antibodies specific to different rat immunoglobulin isotypes, it is possible to resolve the specificity patterns of complex mixtures of monoclonal antibodies from uncloned culture wells. This strategy is particularly useful in the derivation of monoclonal antibodies to cell surface antigens.

Rat hybridoma antibodies against canine parvovirus.

Hybrid cells have been produced by fusion between rat myeloma cells IR983F and spleen cells from a LOU rat immunized with canine enteritis parvovirus. Four stable cell lines were selected each secreting a monoclonal antibody directed against this virus. By indirect immunofluorescence, the antigenic determinants recognized by these antibodies were found to be present not only in canine parvovirus infected cells but also in cells infected by the related parvoviruses of mink enteritis and feline panleukopenia. Two of the monoclonal antibodies inhibit hemagglutination by the canine virus and neutralize its infectivity. Hybrid cells were transplanted into histocompatible animals, and large amounts of the antibodies were obtained.

Production of IgA secreting hybridomas: a monoclonal rat antibody of the IgA class with specificity for RT1 c

We report a method for the production of rat hybridomas secreting IgA antibodies with biological activity. By fusing the rat myeloma Y3.Ag.1.2.3 with mesenteric lymph node cells taken from animals that had been immunised via the Peyer's patches with allogeneic cells we have obtained a hybridoma secreting monoclonal antibodies of the IgA class with specificity for RT1 c.