Antibody-producing Cells In Spleen Research Articles

Preparation of Antispermidine/Spermine-N1-Acetyltransferase Monoclonal Antibodies.

Spermidine/spermine N1-acetyltransferase (SSAT) is a catabolic regulator of polyamines, ubiquitous molecules essential for cell proliferation and differentiation. Anti-SSAT antibodies (monoclonal antibodies [mAbs]) of high titer were prepared by immunizing BALB/c mice with multifocal intradermal injections and by fusing high-titer antibody-producing spleen cells with myeloma cells of SP2/0 origin. Four mAbs were selected for further characterization as classes and subclasses. Antibodies were produced by these three clones with high affinities ranging from 10(9) to 10(11) M(-1). These clones were found to be of the immunoglobulin IgG1 subclass with kappa light chain. They could recognize SSAT as determined by Western blot and immunohistochemistry. The specificity of one clone, 4H6, was studied by using the small interfering RNA (siRNA) on SSAT. 4H6 was also compared with the commercial antibody. The produced mAbs will be a useful tool for further investigation of SSAT functions in organisms.

Preparation of Novel Anti-Ski Monoclonal Antibodies

Ski is an avian sarcoma virus oncogene homolog best known for inhibiting TGF beta signaling through its association with the SMAD proteins. Anti-Ski antibodies (MAbs) of high titer were prepared by immunizing BALB/c mice with multifocal intradermal injections and fusing high titer antibody producing spleen cells with myeloma cells of SP2/0 origin. Three MAbs were selected for further characterization as classes and subclasses. Antibodies were produced by these three clones with high affinities ranging from 10(9) to 10(11)/m. These clones were found to be of the immunoglobulin IgG1 and IgG2b subclass with kappa light chain. They could recognize Ski as determined by Western blot analysis. The produced MAbs will be a useful tool for further investigation of Ski functions in organisms.

α1- and β-Adrenoblockers Effects on Immunogenesis in Rats under Thermoneutral Conditions and after Cooling of Various Extent

Experiments with rats showed that ionophoretic delivery of alpha1-adrenoblocker into the skin does not change effects of superficial and deep cooling and did not affect the amount of the antibody producing cells in spleen and a blood level of circulating antibodies under thermoneutral conditions. Meanwhile alpha1-adrenoblocker abolished inhibitory effect of fast deep cooling on antigen-binding properties of spleen cells and peritoneal extraction cells, and markedly stimulated it. beta-Adrenoblocker had no effect on immunogenesis under thermoneutral conditions and changes modulating effect of cooling on immune response. It abolished immunosuppressive effect of deep cooling and enhanced the stimulating effect of superficial cooling. It was true both for antigen-binding processes in spleen and peritoneal cavity and for antibody production in spleen. The results obtained indicate an involvement of alpha1--and beta-adrenoreceptors in immune response inhibition after deep cooling. Adrenoblockers effects on various immune competent cells are ambiguous and depend on temperature.

Post-stress chemosignals affect cells from immunocompetent organs in laboratory mice of three inbred strains

Quantity of antibody producing cells and changes in bone marrow dividing cells of mouse males were studied after the exposure with chemosignals from intact or stressed donor mouse males. Inbred CBA, BALB/c and C57BL/6 strains were used. It is shown that excreted volatiles decrease quantity of antibody producing cells in spleen and at the same time raise the level of mitotic disturbances in bone marrow cells of recipient mice. Pheromone effect depends on genotype and physiological state of the recipients. For the first time we describe here the influence of mouse female pheromone 2,5-dimethylpyrazine on analyzed features. Biological meaning of the discovered effects is discussed.

Preparation and identification of anti‐melatonin monoclonal antibodies

Anti-melatonin monoclonal antibodies (MAbs) of high titer were prepared by coupling melatonin to bovine serum albumin with formaldehyde and by immunizing BALB/c mice with multifocal intradermal injections and by fusing high titer antibody producing spleen cells with myeloma cells of SP2/0 origin. Five MAbs were selected for further characterization as classes and subclasses. After four successive limiting dilutions, antibodies were produced by these five clones with high affinities ranging from 10(9) to 10(11)/m. These clones were found to be of the immunoglobulin Ig G1 and IgG(2b) subclass with kappa light chain. A systematic study of cross-reactions with seven compounds (indole, aromatic and imidazole derivatives) showed that the antibody had a high specificity for melatonin, low reactivity with 6-hydroxymelatonin and N-acetyl-5-hydroxytryptamine, and no detectable reactivity with tryptamine, l-tryptophan, 5-methoxytryptamine and N-acetyl-L-tryptophan. The roles of the indole nucleus and the side chain in the determination of the antigenic properties of the molecule are discussed. One of the MAbs, 4C9D7, was used to establish a competitive enzyme-linked immunosorbent assay for the detection of melatonin in supernatant.

Preparation and identification of anti-2, 4-dinitrophenyl monoclonal antibodies

2, 4-Dinitrophenyl (DNP) is a widely used hapten in molecular biology and immunoassay fields. Considering that 2, 4-dintrophenylhydrazine (DNPH) could be used as DNA probe and bind with protein carbonyl to form a stable 2 4-dinitrophenyl (DNP) hydrazone product, on which the level of oxidative stress could be validated with a sensitive noncompetitive ELISA, we prepared DNP-aminocaproic acid and NHS-aminocaproic acid-dinitrobenzene and the conjugates between DNP and carrier proteins such as bovine thyroglobulin (BTG) and bovine serum albumin (BSA). High titer antibody producing spleen cells were removed and fused with myeloma cells of SP2/0 origin. Using a conventional immunization protocol, twenty stable murine monoclonal antibodies (MAbs) producing cell lines to DNP were generated. The donor mouse produced antiserum with a high titer of 1/1,280,000. Five MAbs were selected for further characterization as class and subclass. After four successive limiting dilutions, antibodies were produced by five clones with high affinities ranging from 10 10 to 10 11 M − 1 . These clones were found to be of IgG 1 subclass with κ and λ light chain. Competitive ELISA and SPR-based sensing system for the detection of DNPH are both used to confirm the specificity of MAb (4D 9A 9C 2C 2).

Effect of Lipopolysaccharide (LPS) Injection on the Immune Responses of LPS-Sensitive Mice

The effect of lipopolysaccharide (LPS) on humoral and cell-mediated immunity was assessed using LPS-sensitive C3H/HeN mice. A single injection of LPS significantly decreased the anti-sheep red blood cells (SRBC) antibody titers, but not the number of anti-SRBC antibody producing spleen cells. In contrast, double LPS injection did not significantly decrease the anti-SRBC titers and even increased the number of anti-SRBC antibody producing spleen cells. Similarly, single LPS injection significantly suppressed the swelling of the footpad, but double LPS injection caused milder suppression. These results suggest that a difference in the level and timing of exposure to LPS may influence the immune response to infection or vaccination.

Immunomodulatory Activity of Naturally Occurring Monoterpenes Carvone, Limonene, and Perillic Acid

The immunomodulatory activity of some naturally occurring monoterpenes were studied in Balb/c mice. Administration of various monoterpenes such as carvone (100 μmoles/Kg body wt/dose/animal), limonene (100 μmoles/Kg body wt/dose/animal) and perillic acid (50 μmoles/Kg body wt/dose/animal) were found to increase the total white blood cells (WBC) count in Balb/c mice. The maximum total WBC count in carvone treated animals was observed on the 12th day (16560 cells/cmm) while in limonene (13783 cells/cmm) and perillic acid (14437 cells/cmm) treated animals the maximum count was observed on the 9th day after the drug treatment. Administration of terpenoids increased the total antibody production, antibody producing cells in spleen, bone marrow cellularity and α-esterase positive cells significantly compared to the normal animals indicating its potentiating effect on the immune system.

Preparation and identification of specific and high-affinity monoclonal antibodies against morphine.

A C6-hemisuccinate derivative of morphine was prepared and conjugated to bovine serum albumin and ovalbumin. High titer antibody producing spleen cells were removed and fused with myeloma cells of SP2/0 origin. Using a conventional immunization protocol generated twenty-six stable murine monoclonal antibodies (MAbs) producing cell lines to morphine. The donor mouse produced antiserum with a high titer of 1/640,000. Twelve MAbs were selected for further characterization since they showed high sensitivities (53 pg/well to inhibit 50% of the tracer) in improved group-selective immunoassay (IGSI). The assay, which maintains high sensitivity, high precision, and a wide range of optical density (OD) values, was developed using the conjugate M-6-S-OVA to screen and characterize the anti-morphine MAbs. After four successive limiting dilutions, antibodies produced by 12 clones had high affinities ranging from 10(9) to 10(10) M(-1). These clones were found to be of Ig(G) class and IgM class with kappa and lambda light chain. Subclass determination showed that the clones produced IgG1, IgG2a, IgG3, and IgM types of antibody. One clone (2F8B11F2A12) was used to establish the calibration curve with a sensitivity of 400 pg/mL covering up to 25.6 ng/mL in urine.

Characterization of a monoclonal antibody against neopterin using an enzyme-linked immunosorbent assay with penicillinase as label.

An active ester derivative of neopterin was prepared using 4-(N-maleimidomethyl) cyclohexan 4-carboxilic acid N-hydroxy succinimide ester (MCH-NHS), conjugated to bovine serum albumin (BSA) and injected for antibody production (for both monoclonal and polyclonal antibodies). High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. Neopterin was conjugated to the enzyme penicillinase by a one-step glutaraldehyde method, which was used as tracer. A novel enzyme immunoassay was developed using this conjugate to screen and characterize the monoclonal antibody (MAb) produced in these experiments. After limiting dilutions, it was found that antibody produced by one clone with a Ka value of 7.6 x 10-7 mol/L was specific for a number of structurally related molecules. This clone was found to be of IgG class and IgG2a subclass. The standard curvewas constructed with a sensitivity of 10 pg/well (100 pg/mL) covering up to 1 ng/mL.

A single strain of Mycobacterium bovis bacillus Calmette-Guérin (BCG) grown in two different media evokes distinct humoral immune responses in mice.

Two attenuated bacillus Calmette-Guérin (BCG) preparations derived from the same Moreau strain, Copenhagen but grown in Sauton medium containing starch and bacto-peptone (onco BCG, O-BCG), or asparagine (intradermal BCG, ID-BCG), exhibited indistinguishable DNA sequences and bacterial morphology. The number of viable bacilli recovered from spleen, liver and lungs was approximately the same in mice inoculated with the vaccines and was similarly reduced (over 90%) in mice previously immunized with either BCG vaccine. The humoral immune response evoked by the vaccines was, however, distinct. Spleen cell proliferation accompanying the growth of bacilli in tissue was significantly higher in mice inoculated with O-BCG. These cells proliferated in vitro upon challenge with the corresponding BCG extract. Previous cell treatment with mAb anti-CD4 T cells abolished this effect. Anti-BCG antibodies, as assayed either in serum by ELISA or by determining the number of antibody-producing spleen cells by the spot-ELISA method, were significantly higher in mice inoculated with ID-BCG. Anti-BCG antibodies were detected in all immunoglobulin classes, but they were more prevalent in IgG with the following distribution among its isotypes: IgG1>(IgG2a = IgG2b)>IgG3. When some well-characterized Mycobacterium tuberculosis antigens were used as substitutes for BCG extracts in ELISA, although antibodies against the 65-kDa and 96-kDa proteins were detected significantly, antibodies against the 71-kDa, 38-kDa proteins and lipoarabinomannan were only barely detected or even absent. These results indicate that BCG bacilli cultured in Sauton-asparagine medium permitted the multiplication of bacilli, tending to induce a stronger humoral immune response as compared with bacilli grown in Sauton-starch/bacto-peptone-enriched medium.

Preparation and characterization of specific and high-affinity monoclonal antibodies against morphine.

A C6-hemisuccinate derivative of morphine was prepared and conjugated to bovine serum albumin. High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. A C3-hemisuccinate derivative of morphine was prepared and conjugated to enzyme penicillinase used as a tracer molecule. A novel enzyme-linked immunoadsorbent assay was developed using this conjugate to screen and characterize the monoclonal antibody produced in these experiments. After two successive limiting dilutions, antibodies produced by 5 clones with good affinities ranging from 10(8) to 10(12) M(-1) and less cross-reaction (least for codeine and other structurally related molecules) were selected. These clones were found to be of IgG class with kappa light chain. Subclass determination showed that two of the clones produced IgG2b and three of them produced IgG1 type of antibody. Affinity purifications were performed for the selected clone (MOR-I). Purified antibody was coated onto the wells of microtiter plate. The standard curve was constructed with a sensitivity of 100 pg/mL covering up to 10 ng/mL in buffer and urine. The slope of the standard curve for selected clone in buffer and urine was calculated to be -0.7 and -0.64, respectively.

The effect of various incorporated doses of131I on immunological reactions of the organism

Administration of radioisotope131I at 148 kBq/g body weight results in an inhibition of the primary immune response and in a decrease of proliferating activity of mouse lymphocytes in response to alloantigen stimulation. The number of antibody-producing spleen cells for immunization of mice with sheep erythrocytes diminished after administration of131I at 74 kBq/g animal weight.

Suppression of immune response to Lol p I by administration of idiotype

Background: Allergic diseases are characterized by an increased production of specific IgE antibodies. Suppression of IgE antibody production may be accomplished through idiotypic manipulation. Objective: Using an animal model, we explored the effects of anti- Lol p I monoclonal antibody administration on the subsequent IgE and IgG antibody response against Lol p I. Methods: Mice were treated with an anti- Lol p I monoclonal antibody (290A-167), which resulted in the production of anti-idiotypic antibodies as evidenced by their ability to bind to the Fab fraction of 290A-167 and to inhibit the binding of rabbit polyclonal anti-idiotypic antibodies to 290A-167. The animals were then immunized with Lol p I adsorbed onto alum, and the immune response to the protein was analyzed. Results: Antigen-specific IgG 1 and IgE responses were strongly suppressed as determined by immunoassay. Suppression of anti- Lol p I IgE antibodies was confirmed by a reduction of end-point titers measured by passive cutaneous anaphylaxis. The suppression of antigen-specific antibody was accompanied by a reduction of anti- Lol p I antibody-producing spleen cells. Conclusion: These data indicate that pretreatment with 290A-167 can strongly downregulate the IgE response to the main allergen of ryegrass pollen, which is associated with an increase in anti-idiotypic antibodies. This approach could provide rapid, long-term hyposensitization in patients with grass pollen allergy. (J A LLERGY C LIN I MMUNOL 1995;95:751-8.)

A群連鎖球菌4型菌を凝集する単クローン抗体について

BALB/C mice were immunized with a partially purified M-protein fraction prepared from hot acid-extracts of a type 4, group A streptococcus, strain SS91. Two samples of monoclonal antibodies (MAb) were obtained from hybridoma cells of antibody producing spleen cells fused with NS-1 myeloma cells. Both MAb were of the subclass IgG1 having kappa-type light chains. The MAb agglutinated trypsin-digested cells of type 4 strains, but not of types 1, 2, 18, 28 and 41. This type 4-cell agglutination was inhibited by extracts of type 4 cells; strongly by hot acid-extract and partially by trypsin-extract. Hot acid-extract of type 41 cells had no inhibitory effect. Sandwich-enzyme-linked immunosorbent assay of the MAb and partially purified M-protein preparation combination against commercial T-typing sera showed that only T-type 4 antiserum reacted with the combination system. From these data, we thought that the MAb preparations were not directed to M-protein but to T-protein of type 4, group A streptococci.

Monoclonal antibody technology

The development, production, limitations, and uses of monoclonal antibody (MoAb) technology are presented. The first MoAbs were developed in 1975 using a process whereby the antibody-producing spleen cells of mice that had been immunized against sheep red blood cells were fused with the cells of a mouse myeloma cell line, producing hybridomas. These hybridoma cells are used to produce MoAbs, which are antibodies that will bind to only one specific target site on an antigen. Large quantities of MoAbs are grown, either in cell cultures or in the peritoneum of mice, and harvested. Although large quantities of MoAbs can be produced, these techniques are limited because of the potential for contamination by mouse viruses and the inability of the hybridomas to yield sufficient quantities of MoAbs. MoAbs are currently used in diagnostic techniques, including pregnancy tests and drug assays, as well as in tests for detecting viral and bacterial infections and cancer. MoAbs, coupled with dyes or radioactive isotopes, can be used in imaging techniques. Other possible applications of MoAbs include tissue typing, purification, therapy of cancer and autoimmune diseases, and treatment of drug toxicities. As the use of MoAbs in health care increases, pharmacists will need to have a good understanding of the functions and applications of these agents.

The immune response of the mouse to lymphocytic choriomeningitis virus. IV. Enumeration of antibody-producing cells in spleens during acute and persistent infection.

The solid-phase immunoenzymatic technique for the enumeration of single rat cells producing antibodies against ovalbumin has been adapted to mouse cells producing antibodies against lymphocytic choriomeningitis (LCM) virus. After intravenous (i.v.) infection with 10(3) infectious units, IgM- and IgG-producing cells appeared on days 4 and 5, respectively. They rose to high numbers on days 7, 8, and 9 and were still detectable on day 38. After a second i.v. inoculation of 10(7) infectious units, antibody-producing cells (APC), most of which made IgG, appeared faster and reached much higher numbers. Mutatis mutandis, very similar results were obtained with mice inoculated with vesicular stomatitis virus. Antibodies were specific as to virus, but probably corresponded to more than one viral antigen. Relatively low numbers of APC were also detected in the spleens of NMRI strain carrier mice, which develop severe immune complex disease in later life, but not in spleens of persistently infected young mice of strains that remain free of late immune complex disease, namely CBA/J, C3H/HeJ, and gray house mice; APC were detected in spleens of aging CBA/J but not gray house mice.

Monoclonal antibodies binding renal renin.

Somatic-cell fusion of normal antibody-producing spleen cells with cells from a plasmacytoma culture results in a culture of hybrid cells from which a monoclonal line may be selected. These lines are immortal and may be amplified as tumors in syngeneic animals to produce large quantities of antibodies characterized by molecular homogeneity. We report the application of this technique to the production of antibodies binding canine renin. Balb/c mice were immunized with pure canine renal renin and their spleen cells fused with the NS-1 myeloma line. In two separate fusions, nine clones of cells were isolated that bound canine renal renin but did not cross-react with a number of protein antigens tested. One of these antibodies cross-reacted with renins of several different species, including human renin. Binding inhibition studies carried out with one of these monoclonal antibodies demonstrated a dissociation constant for renin of 10(-7) M. These monoclonal antibodies have great potential in answering significant questions concerning the structure, biosynthesis, tissue localization, and physiologic actions of renin.

Alterations of immune response of DBA/2 mice by procarbazine treatment.

Bacterial lipopolysaccharide (LPS) and sheep red blood cells (SRBC) were used as antigens to investigate the in vivo dose- and time-dependent effects of procarbazine (PCZ) treatment on antibody responses of adult male DBA/2 mice. The mice were exposed to different doses of PCZ (100, 300, and 600 mg/kg) and then immunized with LPS or SRBC at days 0, 7, 14, and 21. Procarbazine treatment had essentially no effect on the number of LPS antibody-producing cells in spleens of mice in the entire range of PCZ dose and time, while the circulating antibody to LPS in serum diminished in mice treated with 600 mg/kg dose. In contrast, PCZ treatment altered the plaque-forming cell (PFC) response of the mice to T-cell dependent antigen SRBC. At 100 mg/kg dose, a slight stimulation of immune response was noticed. However, at higher doses (300 and 600 mg/kg), the number of antibody-producing cells to SRBC was decreased. This decrease was 87% at the highest dose compared to the untreated mice. Besides the antibody-producing cells in spleen, the antibody titer to SRBC in serum was also significantly diminished. This markedly reduced immune response, however, was released with the passage of time and was almost completely recovered by day 21 after the PCZ treatment of the mice. In addition, a very significant decrease in the number of cells per spleen was observed at 600 mg dose. It is suggested that PCZ exerts its immunosuppressive effects by essentially affecting T cells, both by decreasing the antibody synthesis as well as the number of cells.

In vitro studies of the natural humoral antitumor reactivity of BALB/c and C57BL/6J mice

Antitumor antibodies were produced in vitro by spleen cells harvested from 12- to 18-month-old C57BL/6J and BALB/c mice with a naturally occurring complement-dependent serum cytotoxicity for tumor cells. The antibodies were of the IgM class and had a complement-dependent cytotoxic reactivity on EL4 cells, which was not absorbed by normal thymus cells. No natural antibodies were produced by untreated spleen cells from 1- or 3-month-old mice of the same two strains, which had no natural serum reactivity. However, after a treatment with an anti-Thy-1 serum and complement before culturing, spleen cells from 3-month-old mice, but not 1-month-old mice, produced the natural antitumor cytotoxic antibodies in vitro. When antibody-producing spleen cells from 12-month-old mice were cultured in vitro together with spleen cells from the unreactive 3-month-old syngeneic mice, inhibition of the antibody production occurred. This inhibiting capacity increased between 1 and 6 months of age and was abrogated by a treatment with an anti-Thy-1 serum and complement or with an anti-Ly-2 serum, whereas the passage of the inhibiting spleen cells on an anti-IgG column did not modify the inhibiting capacity. The in vitro data confirm previous in vivo findings on the nature, specificity and systems of regulation of the natural antitumor cytotoxic antibodies.