A Rapid Method for Antigen-Specific Hybridoma Clone Isolation.

Using an enzyme-linked immunosorbent assay (ELISA) and limited dilution methods to screen and clone antigen-specific hybridoma cells is extremely time-consuming and labor-intensive. This work features a simple and rapid cell surface fluorescence immunosorbent assay (CSFIA), designed for the detection and isolation of antigen-specific hybridoma clones. In this assay, antigens are first anchored to the hybridoma cell surface through a dual-functioning molecular Oleyl-PEG4000-NHS. Specific antibodies secreted from hybridoma cells are then captured by the antigens on the cell surface. Positive hybridoma cells are stained using a fluorescently labeled anti-mouse IgG-Fc antibody. After the addition of a methylcellulose semisolid medium, positive clones are easily picked using a pipet. These positive cell clones can be used to produce monoclonal antibodies after direct expansion. Using this method, positive hybridoma clones against both malachite green and porcine epidemic diarrhea virus are selected with high efficiency. Compared to the ELISA-based method, the CSFIA-based method achieved the capability of isolating >2-fold more hybridoma clones in <25% of the corresponding processing time. In brief, the CSFIA-based method is highly efficient and inexpensive with a simple and direct operation, which is an excellent candidate method for antigen-specific positive clone isolation in a monoclonal antibody preparation.

T cell proliferation induced by anti-self-I-A-specific T cell hybridomas. Evidence of a T cell network.

Allo-I-A-reactive T cell hybridomas were generated from MLR-activated lymphoblasts. Cloned hybridomas T1.203, T1.321, and T1.426 were stimulated by I-Ab determinants, as shown by their ability to secrete IL-2 in response to a panel of MHC-recombinant mice. T2.146, T2.205, and T3.116 were found to be specific for I-Ak determinants using a similar panel of MHC-recombinant mice. Inhibition of IL-2 secretion by anti-I-A mAb confirmed these data. Some I-Ab-specific hybrids stimulated the proliferation of T cells from C57BL/6 (H-2b) mice. Similarly, some I-Ak-specific hybrids stimulated the proliferation of T cells from C3H/HeJ (H-2k) mice. These hybrids expressed no detectable surface I-A, and stimulation of T cells was not inhibited by anti-I-A mAb. These results are consistent with the hypothesis that normal mice possess a population of T cells responsive to idiotypic determinants on anti-MHC class II T cell receptors.

Cellular basis for neonatally induced T-suppressor activity. Primary B cell maturation is blocked by suppressor-helper interactions restricted by loci on chromosome 12.

The cellular mechanism and genetic restriction of neonatally induced HA-specific suppressor T (Ts) cells have been examined. The in vivo effect of these Ts cells on antibody production, primary B cell proliferation, B cell surface marker changes, and helper T (Th) cell priming during primary responses to HA have been determined. The results indicate that, although antigen-induced B cell proliferative responses and surface marker changes occur in the presence of Ts cells, differentiation to Ig secretion, and long-lived memory B cell production are prevented. Further, antigen-specific Th cell priming is completely ablated by Ts cells, suggesting that Ts act by preventing the delivery of Th signals required for both the later stages of primary B cell maturation, and the formation of memory B cell populations. Finally, in vivo cell mixing experiments using congenic mice indicate that this Ts-Th interaction is restricted by loci on mouse chromosome 12.

Multiple autoantigen binding capabilities of mouse monoclonal antibodies selected for rheumatoid factor activity.

We report that approximately 1/4 of monoclonal rheumatoid factors produced by hybridomas derived from fusions of spleen cells from MRL/lpr/lpr mice with systemic lupus erythematosus (SLE) and arthritis exhibited multiple reactivities with other autoantigens, including dDNA , histones, and/or cytoskeletal-cytoplasmic elements. The patterns of reactivities of most of these clones differed, indicating that each had a separate B cell ancestor. Studies with eluted antibodies demonstrated that a single species of antibody molecules was responsible for the observed multiple reactivities. Inhibition experiments suggested that an antibody combining site may be large enough to accommodate dissimilar epitopes. These findings may provide further insights into the generation and extent of antibody diversity as well as the etiopathogenesis of systemic autoimmune diseases.

Restricted immunoglobulin variable region gene usage by normal Ly-1 (CD5+) B cells that recognize phosphatidyl choline.

5-15% of lymphocytes in the peritoneums of normal adult B10.H-2aH-4bp/Wts (2a4b) mice are CD5+ (Ly-1) B cells that recognize phosphatidyl choline (PtC), a phospholipid component of all mammalian cells. We produced a set of IgM-secreting hybridomas from the peritoneal cells of normal, adult 2a4b mice. We found that this set of hybridomas shows a similarly high frequency of antibodies specific for PtC (21 of 86) that also react with bromelain-treated mouse erythrocytes. Restriction fragment analysis of Ig gene rearrangements and analysis of expressed Ig idiotypes reveal that these cells use a restricted set of variable region genes to generate the PtC-specific antibodies. The Ig genes used by the PtC-specific hybridomas appear to be the same as those found in the PtC-specific Ly-1 B cell lymphomas, CH27 and CH34.

Comparison of different ELISAs for the detection of monoclonal antibodies to human interferon-alpha. Implications for antibody screening.

Three enzyme-linked immunosorbent assays (ELISA) were compared in the initial screening of some 400 hybridoma supernatants for antibodies to a recombinant human interferon-alpha subtype, 4a (IFN-alpha 4a). In these assays, (i) the antigen was coated directly to polystyrene microtitre plates (ELISA-PS), (ii) the antigen was coated directly to nitrocellulose (ELISA-NC), or (iii) the antigen and mouse antibody were reacted in solution and the resulting complex immobilized to a solid support precoated with polyclonal rabbit anti-IFN-alpha antibody (ELISA-SW). The ELISA-PS detected eight antibodies, the ELISA-NC 15 and the ELISA-SW 18. The interferon specificity of the MAbs detected by each of the ELISAs was confirmed by neutralization of IFN-alpha antiviral activity and Western immunoblotting analysis. The results suggest that in ELISAs, the presentation of an antigen and its recognition by antibodies is substantially influenced by the method used in the immobilization of antigen and the type of solid support used. The ELISA-SW proved optimal for screening hybridoma supernatants for antibodies to IFN-alpha 4a, and is recommended for screening for antibodies to other soluble antigens.

Comparison of ascites production for monoclonal antibodies in BALB/c and BALB/c-derived cross-bred mice.

BALB/c male mice were mated with either Swiss-Webster or MF1 females to produce first generation cross-bred offspring. Hybridoma cell lines, from the fusion of P3-NS1-Ag4/1 myeloma cells with spleen cells sensitised to the porcine coronavirus causing transmissible gastroenteritis, were injected intraperitoneally into these mice to produce ascitic fluid containing monoclonal antibodies. Mice of 11 weeks of age weighing between 26 and 34 g were used. The volume of ascites produced by mice injected with four of the five hybrid cell lines tested was greater in the cross-bred offspring than in the BALB/c parent. The fifth cell line gave comparable volumes in the MF1 cross-breed and BALB/c parent but a lesser volume in the Swiss-Webster cross-breed. The antibody titres of the ascites as determined by virus neutralisation, radioimmune and indirect immune fluorescence assays, did not differ significantly between mouse types. The ability to use all offspring from a litter of cross-bred mice, irrespective of sex, and the increased volume of ascitic fluid formed in each mouse, permits fewer animals to be used for the production of ascites in these strains, thereby offering considerable economic and ethical advantages over the use of BALB/c mice.

Requirements for the generation of memory B cells in vivo and their subsequent activation in vitro for the production of antigen-specific hybridomas.

We have studied the conditions required for the activation in vitro of memory B cells generated in vivo. BALB/c mice were immunised by a single injection of antigen emulsified in Freund's complete adjuvant. Splenocytes were isolated after different time intervals and cultured in a serum-free medium in the presence of antigen and thymocyte-conditioned medium. After 3 days the splenocytes were fused with myeloma cells. A minimum time interval of more than 2 weeks between priming in vivo and stimulation in vitro was required in order to obtain antigen-specific IgG-secreting hybridomas. After a time interval of 4 weeks or longer most of the antigen-specific hybridomas secreted IgGs. During stimulation in vitro the presence of antigen and of T cells was found to be essential for obtaining an antigen-specific IgG response. The addition of thymocyte-conditioned medium enhanced the IgG response.

Isolation of rare isotype switch variants in hybridoma cell lines using an agarose gel microdrop-based protein secretion assay.

Using gel microdrop (GMD) encapsulation technology and fluorescence-activated cell storing (FACS), we have developed a rapid, sensitive, and reliable method for discriminating and recovering rare isotypic switch variants in hybridoma cell lines. Using the GMD-based IgSwitch assay, a novel approach for isolating subpopulations of IgG-secreting hybridoma cells present at a frequency of approximately 1-10 in 10(6), we successfully isolated spontaneous and in vitro-induced isotypic switch variants in less than half the time required for conventional sublining. The effectiveness and specificity of the assay are demonstrated.

Monoclonal antibodies to human amnion.

Mouse hybridoma cell lines secreting monoclonal antibodies to human amnion were established. The reactivities of eight of these monoclonal antibodies (GB1, GB3, GB4, GB5, GB6, GB9, GB10 and GB11) on human skin and term extra-embryonic tissues, which included reflected amniochorions, basal plates, placentae, chorionic plates and umbilical cords, are reported. GB1, GB4, GB5, GB6, GB9 and GB11 showed various reactivity patterns on the epithelial cells of amnion, chorion and skin at different stages of differentiation. In addition, GB9 and GB11 showed extracellular reactivities; GB9 detected chorionic villi which were usually surrounded by fibrinoid and GB11 reacted with fibrinoid structures in the placentae and chorion laeve. GB10 recognized connective tissues in fetal mesenchyme and adult dermis. This study demonstrates the expression of many shared antigens between tissues derived from the extra-embryonic ectoderm and adult skin. These monoclonal antibodies will provide useful tools for further investigations of epithelial differentiation and transformation.

The CODATA/IUIS Hybridoma Data Bank: development of a hybrid system to handle complex data relationships.

System design for the Hybridoma Data Bank, a database of comprehensive information on immunoreagents for use by scientists in diverse disciplines, is described. Unique problems include: use of nomenclature from diverse fields that is neither static nor standard; the need for two representations of the database--textual for readability and numeric for complex search capabilities, analysis and data compression; and a method of translating between the two representations of the database.

A human-hybridoma system based on a fast-growing mutant of the ARH-77 plasma cell leukemia-derived line.

A rapidly-growing, HAT-sensitive cell line LICR-LON-HMy2 has been derived from the ARH-77 human plasma cell leukemia-derived line. It lacks the enzyme hypoxanthine phosphoribosyltransferase (EC 2.4.2.8). Hybrids have been reproducibly made for more than a year and in independent laboratories with lymphocytes from tonsils, lymph nodes and peripheral blood and tonsil lymphocytes cultured with antigens. The parent line and hybrids are very robust in culture and double in 20-30 h. Hybrids clone easily and have stable karyotypes, most with modal numbers in the sixties. Stable Ig secretion patterns have been observed over 20-30 passages, and after cloning. It was estimated that about half the hybrids produce new immunoglobulin chains in addition to the parent cell line's IgG1 (kappa light chain). High, but not limiting, density hybridoma cultures (approximately 5 x 10(5) cells/ml) typically produce 0.25-2 micrograms/ml immunoglobulin per day, but some hybrids produce more. A high-secreting variant of LICR-LON-HMy2 has been derived. The LICR-LON-HMy2 line is human and is distinct from the HAT-sensitive human B cell-lines SKO-007 and GM 1500-6TG Al 2. It is available for distribution.

Alloantigen presentation by B cells: two types of alloreactive T cell hybridomas, B cell-reactive and B cell-nonreactive.

T cell-depleted, Sephadex G-10-passed unstimulated splenic B cells from C57BL/6 mice stimulated splenic T cells from CKB mice to produce IL 2 and to proliferate. The stimulatory ability of the unstimulated B cells was eliminated by 4000 rad irradiation of the unstimulated stimulator B cells. LPS-activated B cells could stimulate responder T cells more efficiently than unstimulated B cells. For further analysis of allostimulation by B cells, we established a series of alloreactive T cell hybridomas. Forty-five percent of these alloreactive T cell hybridomas could be stimulated to produce IL 2 by either macrophage-dendritic cells or unstimulated B cells. Fifty-five percent of these alloreactive T cell hybridomas could be stimulated by macrophage-dendritic cells but not by unstimulated B cells. T cell hybridomas that were not reactive with unstimulated B cells were also nonreactive to LPS-activated B cells. Analysis of two representative I-Ab-reactive T cell hybridoma clones, B cell-reactive clone CB-11.4 and B cell-nonreactive clone HTB-9.3, revealed again that the stimulatory ability of unstimulated B cells was sensitive to 4000 rad irradiation in the activation of CB-11.4 clone and that CB-11.4 could be stimulated more efficiently by LPS-activated B cells than by unstimulated B cells, but HTB-9.3 could not be stimulated by LPS-activated B cells. Thus, there may be two distinct types of T cells in the alloreaction: B-cell-reactive and B cell-nonreactive.

Receptors for immunoglobulin isotypes (FcR) on murine T cells. I. Multiple FcR expression on T lymphocytes and hybridoma T cell clones.

T cell receptors for the Fc portion of the various isotypes of mouse immunoglobulins (FcR) were examined by rosette formation, using as indicator cells erythrocytes coated with monoclonal antibodies of all known isotypes of serum immunoglobulins. Three populations of mouse T cells were studied: normal thymocytes, activated T cells (ATC), generated by educating thymocytes in lethally irradiated allogeneic hosts, and hybridoma T cells, derived from somatic hybridization of ATC with the FcR-negative thymoma BW.5147. We found that many different FcR could be distinguished by their specificity for a single isotype or for a combination of several isotypes; ATC and hybridoma T cells expressed several such receptors that, at least in cloned cells, could be demonstrated to be borne by individual cells; hybridoma T cells of independent origin bore indistinguishable receptors whereas ATC expressed markedly different FcR and upon overnight incubation at 37 degrees C, immunoglobulins were found to bind onto the cell surface, even though no corresponding constitutive FcR was detected. The same was observed with hybridoma T cells and with thymocytes. It follows that a single T cell can express several FcR. Altogether, these FcR are capable of binding all known isotypes of serum immunoglobulins. They differ from one T cell to another.

Cytolytic T cell hybridomas. III. The antigen specificity and the restriction specificity of cytolytic T cells do not phenotypically mix.

We have obtained cytolytic T-T hybrids by fusing an H-2Kk restricted clone specific for the hapten 3-(p-sulfophenyldiazo)-4-hydroxyphenyl acetic acid (SP) with an H-2Dd-restricted clone specific for the hapten fluorescein (FL). Several hybrid clones express both parental specificities but fail to lyse SP-coupled H-2Dd and FL-coupled H-2Kk target cells. We also fused the H-2Kk-restricted, SP-specific clone with a clone which recognizes FL in the context of any class I major histocompatibility complex antigen. Again several hybrids show both parental specificities but fail to recognize SP coupled to target cells which are not recognized by the parental SP-specific clone. These findings indicate that the observed cytotoxic T lymphocyte specificities for haptens on the one hand and polymorphic as well as nonpolymorphic class I major histocompatibility complex antigenic determinants on the other hand are not carried by independent proteins.

Fine specificity of antigen recognition by T cell hybridoma clones specific for poly-18: a synthetic polypeptide antigen of defined sequence and conformation.

Antigen-specific T cell blasts to poly-18, a polypeptide antigen of defined sequence and conformation, were generated from lymph nodes of antigen-primed BALB/cCr mice. These blasts were fused with the BW5147 thymoma to obtain anti-poly-18-reactive T cell hybridomas. All of the hybridomas were IAd-restricted and secreted IL2 in the presence of IAd/poly-18. On the basis of fine specificity analysis, these hybridomas were classified into two groups. Group A hybridomas recognized a minimal peptide sequence of Glu-Tyr-Lys-(Glu-Tyr-Ala)3-Glu-Tyr-Lys, whereas Group B needed the sequence Glu-Tyr-Ala-(Glu-Tyr-Ala)3-Glu-Tyr-Lys/Ala for activation. Three critical residues were identified in Group A hybridomas: the alanine residue at position 9, the carboxy terminal lysine, and the lysine at position 3. In Group B hybridomas, the alanine at position 3 was found to be the critical residue. We suggest that the amino acid residue at position 3 (lysine/alanine) is the T cell receptor contact residue on the poly-18 antigen in BALB/cCr mice.

Studies on immunity in hybridoma-bearing mice. B. Immunity against the hybridoma. II. The phenotype and specificity of the immune cell.

When appropriate numbers of anti-dinitrophenyl (DNP) immunoglobulin (Ig) E-secreting hybridoma (B 53) cells were injected s.c. into normal BALB/c mice, some of the recipients rejected the tumors. These mice were shown to be immune to B 53 as they withstood, without any ill effect, the i.p. injection of lethal doses of B 53 cells. In previous studies, it was shown that the spleen cells of these mice protected against the growth of B 53 cells. In this study, the characteristics and specificity of the immune spleen cells were examined. The cells responsible for this immunity were shown to be T cells that express Ly-2 on their surface. These cells were shown in in vivo and in vitro assays to limit the growth of the immunizing hybridoma, as well as some but not all BALB/c plasmacytomas and hybridomas.

Elicited antibody nature of human monoclonal protein with anti-streptolysin O activity--analysis with monoclonal anti-idiotype antibody.

Sera from 7 patients with multiple myeloma having antistreptolysin O (ASO) activity in high titers were detected by a streptolysin O (SLO) inhibition assay. However, activity was in low titer when assayed by a passive agglutination assay. The discrepancy between these 2 assays raised some doubts as to whether these monoclonal proteins (M.protein) bond to SLO in the same manner as elicited antibodies. Immunochemical analysis and idiotope analysis using monoclonal antibody to one of these M.proteins strongly suggest that M.protein with ASO activity bind to SLO in a manner similar to elicited antibody. The discrepancy between the 2 assays might be due to differences in the antigenic structure of different forms of the SLO molecule.

Ek beta mutant antigen-presenting cell lines expressing altered Ak alpha molecules.

Antigen-presenting cells (APC) expressing mutant Ek beta and Ak alpha proteins were isolated after chemical mutagenesis of TA3 cells and negative immunoselection for altered Ek beta molecules. Mutant clones were analyzed for biosynthesis, assembly, and cell surface expression of altered Ia molecules, and were assayed for antigen-presenting function by using a variety of T cell clones. Three types of mutants were detected: type 1, which had lost expression of the Ek beta chain and produced altered Ak alpha chains; type 2, which also expressed altered Ak alpha chains, and which expressed Ek beta proteins that had lost reactivity to the 17.3.3 and 74D monoclonal antibodies (mAb), but retained reactivity to other anti-Ek beta mAb; and type 3, which had lost expression of both Ek beta and Ak beta: Ak alpha surface molecules. Thus, all of the mutant clones that produced modified Ak alpha proteins also displayed either total loss or serologic modification of the Ek beta molecule. Ek beta:E alpha-reactive T cell clones were not stimulated when type 1 or type 3 cells were used as APC, but all such T cells were fully reactive with type 2 mutant APC. Most Ak beta:Ak alpha-reactive T cell clones could respond to type 1 and 2 APC, and none were responsive to type 3 APC. However, two autoreactive Ak beta:Ak alpha-specific T cell hybridomas were stimulated only very weakly by type 1 and type 2 cells expressing modified Ak alpha proteins. These results demonstrate that Ia mutations can have highly selective effects on antigen presentation to T cells as well as on mAb binding, and thus suggest that individual Ia molecules may be composed of many different functional subsites.