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At least 19 recordsLinked to original sources
PubMed1976

Modified hemolytic plaque technique for the detection of bluetongue virus antibody-forming cells.

A hemolytic plaque assay was developed for the detection of antibody-forming cells to bluetongue virus (BTV). Sheep erythrocytes (SRBC), onto which BTV had been absorbed, served as the indicator of lysis due to the presence of BTV antibody-forming cells. The ratio of BTV to SRBC was found to be critical for optimum hemolytic plaque formation. For routine use, 50 mul of 12% BTV SRBC, 0.1 ml of a spleen cell suspension, and 0.5 ml of 0.5% agarose in a balanced salt solution were mixed and plated on a microscope slide precoated with 0.1% aqueous agarose. Slides were incubated for 1 h at 37 C in a humidified incubator and subsequently flooded with 0.4 ml of a 1:15 dilution of complement. Incubation was continued for a further 2 h before the hemolytic plaques were scored. It was not possible to establish BTV serotype specificity by this technique.

Animals↗
PubMed1978

A singular perturbation approach to diffusion reaction equations containing a point source, with application to the hemolytic plaque assay.

Many cells secrete factors which diffuse and bind to receptors on neighboring cells. These processes can be described by a nonlinear diffusion equation with a point source and spatially distributed binding reaction. We show via perturbation analysis how approximate solutions can be obtained for such equations when the binding reaction is fast compared to diffusive transport. We base our analysis on an example which is of great practical importance in immunology, the hemolytic plaque technique.

Binding Sites↗
PubMed1979

B cell activity in rat bone marrow and spleen during primary and secondary response: dependence on strength of antigen.

B cell activity was studied in the spleen and the bone marrow of inbred rats after applying xenogeneic erythrocytes of different antigenic strength (mouse or sheep). The hemolytic plaque technique of Cunningham was used. In contrast to the studies of other authors with other species, these experiments showed that in the rat the kinetics of IgM and IgG responses in bone marrow and spleen are principally similar during a primary as well as during a secondary reaction, varying only with the strength of the antigen.

Animals↗
PubMed1979

Enhancement and suppression in production of IgM-antibody in mice treated with purified saponins.

Effects of purified saponins on antibody production against SRBC antigen were investigated in adult mice by using the direct hemolytic plaque technique of Cunningham and Szenberg. Intravenous injection of ginsenoside-Rb1, -Rc, senegin-III, -IV or platycodin-D caused increase in antibody-producing cells. However, the same treatment with ophiopogonin-D, ginsenoside-Rd or glycoside-H2 suppressed antibody production. Immuno-suppression due to the latter three substances seemed not to be associated with cytocidal effect. Ophiopogonin-B and platycodin-C had no effects.

Animals↗
PubMed1975

A hemolytic plaque assay for the detection of direct and indirect antibody-forming cells to keyhole limpet hemocyanin.

A procedure is described for the in vitro enumeration of individual lymphoid cells producing antibody against Keyhole Limpet Hemocyanin (KLH) by a modification of the hemolytic plaque technique. Optimal conditions for the coupling of KLH to sheep erythrocytes by chromic chloride are described. Plaque-forming cells are detected in a liquid monolayer slide chamber assay system. The kinetics of the in vivo primary and secondary PFC responses of both rabbit popliteal lymph node cells and mouse spleen cells are described. Both direct (IgM) and indirect (IgG) plaque-forming cells can be enumerated. The method can also be used to detect an in vitro anamnestic response in dispersed rabbit lymph node cell suspensions. The method is simple, extremely sensitive and the results correlate with those previously obtained in which newly synthesized antibody was detected in similar systems using the coprecipitation assay.

Animals↗
PubMed1979

A hemolytic plaque assay for activated murine T cells.

In an earlier report, it was shown that murine spleen cells cultured with concanavalin A (Con A) released into the culture supernatants helper and suppressor substances for antibody production. The present communication describes the production of rabbit antisera against culture supernates from Con A-activated spleen cells and their use in a plaque assay for mitogen-activated T cells. The plaque assay, utilizing SRBC to which Staphylococcal protein A had been coupled, the developing anti-supernatant antiserum and guinea pig complement, readily detected secreting T cells. The T-cell nature of the plaque-forming cells (PFC) was established principally by the following: (a) the majority of lymphocytes in the centers of plaques were Thy-1-positive by fluroescence; (b) spleen cells depleted of B cells by incubation in plastic dishes coated with rabbit anti-mouse Ig antibody gave greatly enriched PFC responses; (c) anti-Thy-1 and anti-Lyt-2.2 treatment of spleen cells almost completely depleted PFC; (d) T-cell mitogens (Con A and phytohemagglutinin) but not B-cell mitogens (lipopolysaccharides) induced PFC responses; (e) T cells maintained in culture for 10 d with Con A and T-cell growth factor yielded PFC. Kinetic and dose response studies showed that high doses of mitogen induced rapidly appearing T-PFC and the responses peaked at day 1--2 of culture. Lower doses of mitogen-induced PFC required longer periods of incubation for detection, indicating that cell activation and secretion may be different dose-dependent activities of mitogens. Another noteworthy finding was that the antiserum reacted with surface antigens of T-PFC, indicating that secreted products are expressed on the membranes of T cells, offering the possibility of isolating populations of cells with specific secretory potential. Although the precise nature of the T-cell products detected by the antiserum used in this assay are unresolved, 10% of the target-cell-adherent population from spleen cells of BALB/c mice sensitized to L929 cells formed plaques. This suggests that the antiserum has significant activity against the products of cytotoxic T cells, a finding which accords with the activity of anti-Lyt-2.2 serum against mitogen-induced T-PFC. The method clearly offers new possibilities for the analysis of T cells and their products and should provide an important approach to the clonal analysis of lymphokine production.

Animals↗
PubMed1979

Stimulation of humoral immunity by peptidoglycan monomer from Brevibacterium divaricatum.

Peptidoglycan monomer (PGM), a water soluble and nontoxic disaccharide pentapeptide unit obtained from Brevibacterium divaricatum, was administered intravenously into mice, and the humoral immune response to sheep erythrocytes was assayed by means of Jerne's technique for plaque-forming cells (PFC) in the spleen. The PFC response was evidently stimulated. The counts were increased to practically the same extent over a great range of doses of PGM (from 25 to 1600 microgram per animal), and the effect was present in the mice immunised with optimal, as well as in those immunised with suboptimal, doses of antigen. The magnitude of the immunostimulation depend only on the timing of PGM administration: it was maximal if PGM was injected 1 or 2 days after the antigen. In vitro, in a 4-day culture of spleen cells, PGM did not stimulate PFC formation. We conclude that stimulation of the humoral immune response to sheep red blood cell antigens by PGM probably occurs without cell multiplication and probably involves more than simply a contact of immunocompetent cells with PGM.

Adjuvants, Immunologic↗
PubMed1977

Polyclonal antibody secretion in human lymphocytes induced by killed staphylococcal bacteria and by lipopolysaccharide.

Preparations of Staphylococcus aureus strains Cowan 1 and Wood 46 and of lipopolysaccharide (LPS) were found to act as polyclonal B-cell-activating substances for human splenic and blood lymphocytes. All three substances induced polyclonal antibody secretion in blood and spleen cell cultures, as tested against fluorescein isothiocyanate-coupled sheep erythrocytes by a modification of the local hemolysis-in-gel assay. Antibodies were of IgM class, as shown by inhibition of plaque formation by anti-IgM but not by anti-IgG or anti-IgA antisera. All these substances also consistently induced the formation of intracellular immunoglobulin and increased DNA synthesis in stimulated spleen cells. In blood lymphocytes Staph. aureus Cowan 1 induced a consistent increase in DNA synthesis, whereas Staph, aureus Wood and LPS often gave low or no increase in DNA synthesis. Peak antibody formation was observed on day 3 in spleen cells and on day 6 in blood lymphocyte cultures. Stimulation into high-rate immunoglobulin secretion occurred with all PBAs also in B-cell-enriched cell suspensions but not in T-cell-enriched cells. Optimal responses were, however, always noted in unseparated cell suspensions. It is concluded that preparations of killed bacteria can be useful tools for the clinical evaluation of both specific and nonspecific antibody-forming ability in cells from different groups of patients.

Antibody Formation↗
PubMed1979

The plasma cell differentiation antigen PC.1 is absent in CH3/Tif and present in C3H/HeJ.

(C3H/TifxDBA/2)F1 mice, immunized with viable BALB/c plasmacytoma MOPC315 cells, produce antibodies directed against a cell-surface antigen. The strain and tissue distribution of this antigen was identical to that of the plasma cell differentiation alloantigen PC.1. The antigen is absent in the mouse strain C3H/Tif but is present in the closely related substrain C3H/HeJ. This is the third difference between surface structures of the B-cell lineage of C3H/Tif and C3H/HeJ mice.

Animals↗
PubMed1979

Aging increases expression of LPS-induced autoantibody-secreting B cells.

Escherichia coli lipopolysaccharide (LPS), a polyclonal B cell activator, has been employed to achieve in vitro stimulation of autoantibody-secreting B cells in young adult and aged mice of long-lived strains as assayed in a hemolytic plaque technique to syngeneic mouse erythrocytes. Aged 21- to 24-month-old C57BL/6J and (C57BL/10Sn x C3H/HeDiSn)F1 mice were found to express 3 to 4 times as many LPS-induced plaque-forming cells (PFC) to autologous erythrocytes than did younger 6-month-old animals. With the use of cyclophosphamide (CY), a significant enhancement of auto-PFC production in young mice occurred, approaching levels found in non-CY-treated old mice. Thus, autoreactive clones of lymphocytes exist in the spleens of young adult mice, but under normal circumstances produce little autoantibody. The situation in aged members of these strains, therefore, does not seem to involve an actual increase in numbers of autoreactive B cells, but may possibly involve some form of deregulation, permitting increased age-related expression of autoreactive lymphocyte clones.

Aging↗
PubMed1977

B-memory cells can be stimulated by antigen in vitro to become IgG antibody-secreting cells.

Whereas DNP-KLH primed mouse spleen cells fail to show the high IgG anti DNP levels characteristic of an anamnestic immune response when cultured for 5 days under Mishell-Dutton conditions, we show here that such a response can be observed after 8--10 days in vitro using modified Marbrook culture vessels. The kinetics of this secondary response in vitro resemble that described elsewhere for the adotpive secondary immune response in irradiated recipients, and show that insignificant numbers of IgG PFC can be expected before 6 days of culture. This timing is governed by the maturation state of B cells at the start of culture, and was not accelerated by the addition of recently boosted carrier primed spleen cells. We conclude that the deficit in IgG PFC numbers that characterizes Mishell-Dutton cultures of cells primed 2--4 months previously is due to the shortage of time during which such cultures can maintain lymphocyte division and maturation.

Animals↗
PubMed1979

Separation of mouse T cell subsets by a fluorescent activated cell sorter using fluorescence-labeled peanut agglutinin.

Nylon non-adherent spleen T cells obtained from concanavalin A-injected mice were labeled with fluorescein-labeled peanut agglutinin and separated into peanut agglutinin-positive (PNA+) cells and peanut agglutinin-negative (PNA-) cells by a fluorescent activated cell sorter. PNA+ cells were found to exert marked suppressive effect on primary anti-sheep red blood cells antibody response, but PNA- cells did not affect the antibody response. From these results and from the sugar-binding specificity of PNA, suppressor T cells are supposed to possess abundant galactosyl residues exposed on the cell surface, which are not masked by sialyl residues.

Animals↗
PubMed1979

A review of laboratory methods for identification of group B streptococci (Streptococcus agalactiae).

A selected review is presented of the history of Group B streptococcal infections and the laboratory tests used to identify Group B streptococcus (Streptococcus agalactiae), an opportunistic pathogen which causes serious infections in newborn infants and compromised patients in other age groups. Its isolation may be increased by growth in a selective medium. The methods for Group B streptococci identification include the hydrolysis of sodium hippurate, the CAMP reaction, pigment production, and antibiotic disk susceptibility. Also, immunological tests, such as Lancefield's classical precipitin test, immunofluorescence staining, counterimmunoelectrophoresis, and coagglutination are available.

Bacteriological Techniques↗
PubMed1979

Xenogeneic bone marrow stimulating effect in vitro on antibody-producing cells.

It was described earlier that in the tissue cultures of immune mouse LN cells the number of antibody producing cells was increased 2-3-fold when syngeneic of allogeneic nonimmune BM cells were included in the cultures. In these experiments, parallel mixed cultures were set up of mouse immune LN cells with non-immune BM cells of either syngeneic or xenogeneic origin (rat, pig and chicken). Xenogeneic BM also increased the number of PFC in the mixed cultures. The intensity of the stimulation effected by xenogeneic BM of all three species tested was comparable to that induced by syngeneic BM. The number of PFC in all types of the mixed cultures was significantly higher (P less than 0.005) than in the LN cell cultures alone. The differences between the effect of syngeneic and xenogeneic BM and between the xenogeneic BM cells of different origin were not statistically significant (P less than 0.05).

Animals↗
PubMed1978

Viral antibodies in cerebrospinal fluid of multiple sclerosis and control patients: comparison between radioimmunoassay and conventional techniques.

Cerebrospinal fluid antibodies to measles, rubella, vaccinia, herpes simplex, and varicella-zoster viruses in four patient study groups (clinically definite multiple sclerosis [MS], early probable MS, optic neuritis, and control patients with other neurological diseases) were assayed by radioimmunoassay, complement fixation, hemagglutination-inhibition, or complement-enhanced plaque reduction methods. Antibodies were more frequently found and at higher dilutions by radioimmunoassay than by other techniques. Measles virus antibody, the most frequently found antibody, was present in the cerebrospinal fluid of 72% of MS patients and 5% of control patients. The differences between the numbers of MS patients and control patients with antibodies to other viruses were not as marked. Thus, 58% of MS patients versus 21% of control patients had antibody to rubella virus, 20 versus 3% had antibody to vaccinia virus, 50 versus 33% had antibody to herpes simplex virus, and 25 versus 8% had antibody to varicella virus. Sixty-seven percent of MS patients and 26% of control patients had antibodies to two or more viruses in their cerebrospinal fluid.

Adolescent↗