ovalbumin and Hemolysis

It is urgently needed to develop novel adjuvants for improving the safety and efficacy of vaccines. Metal-organic frameworks (MOFs), with high surface area, play an important role in drug delivery. With perfect biocompatibility and green preparation process, the γ-cyclodextrin metal-organic framework (γ-CD-MOF) fabricated with cyclodextrin and potassium suitable for antigen delivery. In this study, we modified γ-CD-MOF with span-85 to fabricate the SP-γ-CD-MOF as animal vaccine adjuvants. The ovalbumin (OVA) as the model antigen was encapsulated into particles to investigate the immune response. SP-γ-CD-MOF displayed excellent biocompatibility

Topics: Adjuvants, Vaccine; Animals; Animals, Outbred Strains; Bone Marrow Cells; Cell Survival; Chemistry, Pharmaceutical; Cytokines; Female; gamma-Cyclodextrins; Hemolysis; Immunoglobulin G; Metal-Organic Frameworks; Mice; Ovalbumin; Random Allocation; RAW 264.7 Cells; Spleen

Protein-based vaccines offer a number of important advantages over organism-based vaccines but generally elicit poor CD8(+) T cell responses. We have previously demonstrated that pH-responsive, endosomolytic polymers can enhance protein antigen delivery to major histocompatibility complex class I (MHC-I) antigen presentation pathways thereby augmenting CD8(+) T cell responses following immunization. Here, we describe a new family of nanocarriers for protein antigen delivery assembled using architecturally distinct pH-responsive polymers. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize linear, hyperbranched, and core-crosslinked copolymers of 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) that were subsequently chain extended with a hydrophilic N,N-dimethylacrylamide (DMA) segment copolymerized with thiol-reactive pyridyl disulfide (PDS) groups. In aqueous solution, polymer chains assembled into 25 nm micellar nanoparticles and enabled efficient and reducible conjugation of a thiolated protein antigen, ovalbumin. Polymers demonstrated pH-dependent membrane-destabilizing activity in an erythrocyte lysis assay, with the hyperbranched and cross-linked polymer architectures exhibiting significantly higher hemolysis at pH ≤ 7.0 than the linear diblock. Antigen delivery with the hyperbranched and cross-linked polymer architecture enhanced in vitro MHC-I antigen presentation relative to free antigen, whereas the linear construct did not have a discernible effect. The hyperbranched system elicited a four- to fivefold increase in MHC-I presentation relative to the cross-linked architecture, demonstrating the superior capacity of the hyperbranched architecture in enhancing MHC-I presentation. This work demonstrates that the architecture of pH-responsive, endosomolytic polymers can have dramatic effects on intracellular antigen delivery, and offers a promising strategy for enhancing CD8(+) T cell responses to protein-based vaccines.

Topics: Acrylamides; Animals; Antigen Presentation; CD8-Positive T-Lymphocytes; Cross-Linking Reagents; Endosomes; Hemolysis; Histocompatibility Antigens Class I; Humans; Hydrogen-Ion Concentration; Mice; Micelles; Nanoparticles; Ovalbumin; Polymers; Vaccines

A Cu-mediated azide-alkyne click chemistry protocol was employed for the synthesis of a focused library of novel 1,2,3-triazolyl conjugates bearing various carbohydrate-steroid/triterpenoid entities. The immunogenicity of these compounds was examined initially by ex vivo assays. The lead compound 15g was further subjected to in vivo evaluation in BALB/c mice immunized with ovalbumin. These in vivo biological studies revealed an increase in B cell-mediated proliferation, higher expression levels of IL-2, TNF-α, IL-12, and IFN-γ indicating Th1 activation, together with an enhanced OVA-specific antibody (IgG) response compared to alum, affirming adjuvanticity of these glycolipids. The primary indications of response skewed toward Th1 immunity induced by the new triazoyl analogs indicate the potential of these molecules for possible application as adjuvants.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; B-Lymphocytes; Cell Proliferation; Cells, Cultured; Drug Design; Glycolipids; Hemolysis; Immunization; Immunoglobulin G; Interferon-gamma; Interleukin-12; Lymphocyte Activation; Male; Mice, Inbred BALB C; Molecular Structure; Ovalbumin; Rats, Wistar; Structure-Activity Relationship; Th1 Cells; Triazoles; Tumor Necrosis Factor-alpha

Immunological adjuvants are agents that enhance specific immune responses to vaccines. At present more studies are needed to identify a suitable adjuvant for a particular vaccine with maximum safety and efficacy. In this study, six soyasaponins (Aa, Ab, Af, Ba, Bb, and Bb') and three soyasaponin Ab-derivatives (AbDs) were selected to evaluate their toxicities and adjuvant activities. The haemolytic activity assay was performed to evaluate the toxicity of the tested soyasaponins and AbDs. Immunoadjuvant activity was investigated in vivo and in vitro using a splenocyte proliferation assay and sera indirect ELISA. Our results demonstrated that soyasaponins and AbDs showed a slight haemolytic effect to 0.5% red blood cell. Except for the Af and Ba groups, other soyasaponins and AbDs groups stimulated by concanavalin A (ConA) and lipopolysaccharide (LPS) showed a greater proliferative response at appropriate doses (0.01-10 μg/ml) compared with the control and Alum groups. Anti-OVA IgG, IgG1, IgG2a, and IgG2b were significantly enhanced by the soyasaponins (Ab, Ba, Bb, and Bb'), QS and AbDs groups (p < 0.05 or p < 0.01). In addition, three AbDs indicated a tendency of immunoadjuvant potential improvement after structural modification. Moreover, Ab-D2 showed adjuvant activity at the lowest injection dose among the three AbDs. In conclusion, these results suggested that soyasaponins together with their derivatives may represent viable candidates for effective vaccine adjuvants due to their higher immune response and lower or non-haemolytic effects.

Topics: Adjuvants, Immunologic; Allergens; Animals; Cell Proliferation; Erythrocytes; Female; Hemolysis; Immunoglobulin G; Mice; Mice, Inbred ICR; Ovalbumin; Saponins; Spleen

The lipid core peptide (LCP) system has successfully been used in development of peptide-based vaccines against cancer and infectious diseases (such as group A streptococcal infection). CD8(+) T cells are important targets for vaccines, however developing a vaccine that activates long-lasting immunity has proven challenging. The ability of LCP vaccines to activate antigen-specific CD8(+) and/or CD4(+) T cell responses was tested using compounds that contained two or four copies of OVA257-264 and/or OVA323-339 peptides conjugated to LCP, which are recognised by OTI (CD8(+) specific) and OTII (CD4(+) specific) T cells, respectively. The LCP-ovalbumin vaccines developed in this study were synthesised in 30% yields and showed no significant haemolytic effect on red blood cells (below 4% haemolysis when tested with compounds at up to 100μM concentrations). Promising in vivo data in mice suggested that this LCP-ovalbumin vaccine system could act as a novel and potent vehicle for the stimulation of robust antigen-specific CD8(+) T cell responses.

Topics: Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Erythrocytes; Female; Hemolysis; Humans; Mice; Mice, Inbred C57BL; Ovalbumin; Vaccines, Subunit

A new steroidal saponin was isolated from the bulbs of Allium ampeloprasum L. var. porrum. On the basis of chemical evidence, comprehensive spectroscopic analyses, and comparison with known compounds, its structure was established as (3β,5α,6β,25R)-3-{(O-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→2)-O-[O-β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranosyl)oxy}-6-hydroxyspirostan-2-one (1). Results of the present study indicated that 1 exhibited haemolytic activity in the in vitro assays, and immunological adjuvant activity on the cellular immune response against ovalbumin antigen.

Topics: Adjuvants, Immunologic; Animals; Erythrocytes; Hemolysis; Humans; Magnetic Resonance Spectroscopy; Male; Mice; Molecular Conformation; Onions; Ovalbumin; Saponins; Spirostans; Steroids

In this study, the saponins (PCS) from the roots of Pulsatilla chinensis were evaluated for its haemolytic activity, acute toxicity and tested for potential adjuvant activity in mice immunized with ovalbumin (OVA) compared with that of Quil A saponin. The haemolytic activity of PCS was determined using 0.5% rabbit red blood cell with values of 15.41 and 7.42% at concentrations of 500 and 250microg/mL, respectively. The saponins were tested for their toxicity by lethality in mice and were found to be less toxic at the same dose than their counterpart Quil A. ICR mice were immunized subcutaneously with OVA 100microg in phosphate-buffered saline (PBS) alone or with OVA 100microg in the presence of Quil A (10microg) or PCS (50, 100 or 200microg) twice at a 2-week interval. Four weeks later, the ConA-, LPS-, and OVA-induced splenocyte proliferation, OVA-specific antibodies levels (IgG, IgG1, and IgG2a) in serum, IL-2, and TNF-alpha were significantly enhanced by PCS at a high dose compared to that induced by Quil A. The P values of various testing activities in saponin-treated groups were obviously differential to that in the OVA-immunized mice (p<0.05 or p<0.01). This finding suggested that PCS might have an effect on Th1 and Th2 helper T cells. In conclusion, the results indicated that PCS showed slight side effects and at an appropriate dose could be used as a vaccine adjuvant to increase immune responses.

Topics: Adjuvants, Immunologic; Animals; Antibodies; Cell Proliferation; Cells, Cultured; Hemolysis; Immunity, Humoral; Interleukin-2; Male; Mice; Mice, Inbred ICR; Ovalbumin; Plant Roots; Pulsatilla; Saponins; Spleen; Tumor Necrosis Factor-alpha; Vaccines

While many infectious diseases are controlled by vaccine strategies, important limitations continue to motivate the development of better antigen delivery systems. This study focuses on the use of a pH-sensitive polymeric carrier based on poly(propylacrylic acid) (PPAA) to address the need for more potent CD8 cytotoxic T-cell (CTL) responses. An MHC-1/CD8 CTL cell model system with ovalbumin as the protein antigen was used to test whether PPAA could enhance the delivery of ovalbumin into the MHC-1 display pathway. Ovalbumin was conjugated to poly(propylacrylic acid-co-pyridyldisulfide acrylate) (PPAA-PDSA) by disulfide exchange to make reversible conjugates that could be reduced by the glutathione redox system in the cytosol of antigen presenting cells. The PPAA-PDSA ovalbumin conjugates displayed the pH-sensitive membrane disruptive properties of the parent polymer as determined by their hemolysis activities (sharply active at the endosomal pH values of 6-6.5). The polymer-ovalbumin conjugates exhibited strong 22-fold increases in the MHC-1 presentation and ovalbumin-specific CTL activation compared to free ovalbumin. No CTL activation was observed with control conjugates of ovalbumin and poly(methylacrylic acid) (PMAA) that do not display membrane disruptive activies, suggesting that it is the membrane destabilizing properties of the polymer that result in increased MHC-1 display and CTL activation. Further mechanistic studies quantitated the time course of stable intracellular localization of radiolabeled conjugates. 52% of initially internalized PPAA-conjugated ovalbumin remained in the cells after 4 h, compared to less than 10% of ovalbumin or PMAA-ovalbumin. These results showing enhanced cytosolic delivery and MHC-1 presentation for the PPAA-antigen conjugates suggest that they warrant future characterization as a CD8-enhancing vaccine delivery system.

Topics: Acrylates; Animals; Antigen Presentation; Carbon Radioisotopes; CD8-Positive T-Lymphocytes; Cell Line; Cell Survival; Cytosol; Disulfides; Drug Carriers; Endocytosis; Erythrocytes; Exocytosis; Glutathione; Hemolysis; Histocompatibility Antigens Class I; Hydrogen-Ion Concentration; Lymphocyte Activation; Macrophages; Ovalbumin; Polymers; Receptors, Antigen, T-Cell; Vaccines

As a primary drug for the treatment of acute lymphoblastic leukemia (ALL), encapsulation of L-asparaginase (ASNase) into red blood cells (RBC) has been popular to circumvent immunogenicity from the exogenous protein. Unlike existing methods that perturbs RBC membranes, we introduce a novel method of RBC-incorporation of proteins using the membrane-translocating low molecular weight protamine (LMWP). Confocal study of fluorescence-labeled LMWP-ovalbumin, as a model protein conjugate, has shown significant fluorescence inside RBCs. Surface morphology by scanning electron microscopy of the RBCs loaded with LMWP-ASNase was indistinguishable with normal RBCs. These drug loaded RBCs also closely resembled the profile of the native erythrocytes in terms of osmotic fragility, oxygen dissociation and hematological parameters. The in vivo half-life of enzyme activity after administering 8 units of RBC/LMWP-ASNase in DBA/2 mice was prolonged to 4.5+/-0.5 days whereas that of RBCs loaded with ASNase via a hypotonic method was 2.4+/-0.7 days. Furthermore, the mean survival time of DBA/2 mice bearing mouse lymphoma cell L5178Y was improved by approximately 44% compared to the saline control group after treatment with the RBC loaded enzymes. From these data, an innovative, novel method for encapsulating proteins into intact and fully functional erythrocytes was established for potential treatment of ALL.

Topics: Animals; Antineoplastic Agents; Asparaginase; Biological Transport; Cell Line, Tumor; Chemistry, Pharmaceutical; Drug Carriers; Drug Compounding; Enzyme Stability; Erythrocyte Transfusion; Erythrocytes; Feasibility Studies; Hemolysis; Mice; Mice, Inbred DBA; Microscopy, Confocal; Microscopy, Electron, Scanning; Molecular Weight; Osmotic Fragility; Ovalbumin; Pilot Projects; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protamines; Sheep

The haemolytic activities and adjuvant potentials of Platycodon grandiflorum saponin (PGS) and its fractions on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were evaluated. PGS was subjected to silica gel column chromatography to afford four fractions, and two fractions PGSC and PGSD selected for testing for activities because of containing dominant saponin peaks. PGS, PGSC, and PGSD showed a slight haemolytic effect, with their HD50 value being 37.91+/-2.24, 21.30+/-1.22, 37.58+/-1.86 microg/ml against 0.5% rabbit red blood cell, respectively. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing Alum (200 microg), Quil A (10 microg), PGS (50, 100 or 200 microg), PGSC, or PGSD (25, 50 or 100 microg) on days 1 and 15. Two weeks later (day 28), concanavalin A (Con A)-, pokeweed (PWM)-, and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. PGS and PGSC significantly enhanced the Con A-, PWM-, and OVA-induced splenocyte proliferation in OVA-immunized mice at three doses (P<0.01 or P<0.001). However, no significant differences (P>0.05) were observed among the OVA group, OVA/Alum group and OVA/PGSD group. OVA-specific IgG, IgG1, and IgG2b antibody levels in serum were significantly enhanced by PGS, PGSC, and PGSD compared with OVA control group (P<0.05, P<0.01, or P<0.001). Moreover, the adjuvant effects of PGSC (50 or 100 microg) on the OVA-specific IgG, IgG1, and IgG2b antibody responses to OVA in mice were more significant than those of Alum. In conclusion, PGS seem to be a promising balanced Th1 and Th2 directing immunological adjuvants which can enhance the immunogenicity of vaccine.

Topics: Adjuvants, Immunologic; Animals; Antibodies; Cell Proliferation; Chromatography, Gel; Erythrocytes; Female; Hemolysis; Immunization, Secondary; Immunoglobulin G; Inhibitory Concentration 50; Lymphocytes; Mice; Mice, Inbred ICR; Molecular Structure; Ovalbumin; Plant Roots; Platycodon; Rabbits; Saponins; Spleen; Th1 Cells; Th2 Cells

In this study, saponins (ARS) extracted from the rhizoma of Anemone raddeana were evaluated for their haemolytic activities and its potential ability as adjuvant on the cellular and humoral immune responses of ICR mice against ovalbumin. The haemolytic activity of ARS was determined using 0.5% rabbit red blood cell. ARS showed a slight haemolytic effect, with its haemolytic percents being 16.50 and 3.56% at the concentrations of 500 and 250 microg/ml, respectively. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 mug dissolved in saline containing Alum (200 microg), QuilA (10 and 20 microg) or ARS (50, 100 or 200 microg) on Days 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)- and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. ARS significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice especially at a dose of 100 microg (P<0.01 or P<0.05). The OVA-specific IgG, IgG1 and IgG2b antibody levels in serum were also significantly enhanced by ARS compared with OVA control group (P<0.01 or P<0.05). Moreover, no significant differences (P>0.05) were observed between enhancing effect of ARS and QuilA on the OVA-specific IgG2b antibody responses to OVA in mice. The results suggest that ARS showed a slight haemolytic effect and enhanced significantly a specific antibody and cellular response against OVA in mice.

Topics: Adjuvants, Immunologic; Anemone; Animals; Antibody Formation; Cell Proliferation; Drugs, Chinese Herbal; Hemolysis; Immunity, Cellular; Lymphocyte Activation; Male; Medicine, Chinese Traditional; Mice; Ovalbumin; Saponins

Platycodin D2 (PD2), a saponin isolated from the root of Platycodon grandiflorum, was evaluated for its haemolytic activity and adjuvant potential on the cellular and humoral immune responses to ovalbumin (OVA) in ICR mice. The HD(50) values of PD2 and Quil A were 18.57+/-1.37 and 5.76+/-0.23 microg/ml against 0.5% rabbit red blood cell, respectively, implicating the less haemolytic activity for PD2 than Quil A. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing Alum (200 microg), or PD2 (25, 50, 75 or 100 microg) on Day 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS-) and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. PD2 significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice (P<0.05, P<0.01 or P<0.001). OVA-specific IgG, IgG1, IgG2a, and IgG2b antibody titers in serum were also significantly enhanced by PD2 compared with OVA control group (P<0.05, P<0.01 or P<0.001). Further, the effects of PD2 on mRNA expression of cytokines and transcription factors in Con A-stimulated mice splenocytes were evaluated by RT-PCR analysis. PD2 significantly promoted the mRNA expression of cytokines IL-2, IFN-gamma, IL-4, and IL-10 and transcription factors T-bet and GATA-3 (P<0.05, P<0.01 or P<0.001). In conclusion, the results suggest that PD2 could be safely used as adjuvant eliciting Th1 and Th2 immune responses.

Topics: Adjuvants, Immunologic; Animals; Antibodies; Antibody Formation; Cell Proliferation; Cytokines; Female; Hemolysis; Immunity, Cellular; Immunization; Lymphocyte Activation; Mice; Ovalbumin; Saponins; Th1 Cells; Th2 Cells; Transcription Factors; Triterpenes

In this study, the saponins (JGS) extracted from the rhizoma of Japanese ginseng were evaluated for their haemolytic activities and their potential ability as adjuvants on the immune responses to ovalbumin (OVA) in mice. The haemolytic activity of JGS was determined using 0.5% rabbit red blood cell, with its HD(50) value being 177.78+/-6.77microg/mL. ICR mice were immunized subcutaneously with OVA 100microg alone or with OVA 100microg dissolved in saline containing Alum (200microg), QuilA (10 and 20microg) or JGS (50, 100 or 200microg) on Days 1 and 15. Two weeks later (Day 28), concanavalin A (ConA)-, lipopolysaccharide (LPS)-, OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. JGS significantly enhanced the ConA-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice especially at a dose of 100microg (P<0.05 or P<0.01). The OVA-specific IgG, IgG1 and IgG2b antibody levels in serum were also significantly enhanced by JGS compared with OVA control group (P<0.01). The results suggest that JGS showed a slight haemolytic effect and enhanced significantly a specific antibody and cellular response against OVA in mice.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Antibody Specificity; Cell Proliferation; Hemolysis; Immunity, Cellular; Japan; Male; Mice; Mice, Inbred ICR; Ovalbumin; Panax; Saponins

The combined adjuvant effect of ginsenoside Rg1 and aluminum hydroxide (alum) on immune responses to ovalbumin (OVA) in mice was investigated. BALB/c mice were subcutaneously (s.c.) inoculated twice with OVA alone or in combination with Rg1, alum, or Rg1 plus alum. Samples were collected 2 weeks after the boosting for the measurement of anti-OVA immunoglobulin G (IgG) isotypes in sera and gamma interferon (IFN-gamma) and interleukin-5 (IL-5) produced in singular splenocyte cultures. Delayed-type hypersensitivity (DTH) responses were measured in mice immunized as described above. After 10 days, the mice were injected s.c. with OVA at the footpads. Thereafter, the thickness of the footpads was measured once daily for 5 days. The results indicated that alum enhanced mainly Th2 (IgG1 and IL-5) responses (P < 0.05), while Rg1 enhanced both Th1 (IgG1 and IL-5) and Th2 (IgG2a, IFN-gamma, and DTH) responses (P < 0.05). The highest immune responses were found in the mice injected with OVA solution containing both alum and Rg1. In addition, the hemolytic activity of Rg1 was much lower than that of Quil A. Therefore, Rg1 deserves further studies in order to tailor desired immune responses when a mixed Th1/Th2 immune response is needed.

Topics: Adjuvants, Immunologic; Aluminum Hydroxide; Animals; Female; Ginsenosides; Hemolysis; Hypersensitivity, Delayed; Immunoglobulin G; Injections, Subcutaneous; Interferon-gamma; Interleukin-5; Lymphocytes; Mice; Mice, Inbred BALB C; Ovalbumin; Spleen; Th1 Cells; Th2 Cells

Platycodin D (PD), platycodin D3 (PD3), and platycoside E (PE) were the platycodigenin-type saponins isolated from the root of Platycodon grandiflorum. All shared a platycodigenin skeleton and the same sugar side chains attached to C-28 of the aglycone, only differ from one another by the number of glycosyl units in sugar moieties attached to C-3. To assess the potential contribution of the glycidic chains to the biological activities and elucidate the structure-activity relationships of the platycodigenin-type saponins, these three saponins were evaluated for their haemolytic activities and adjuvant potentials on the cellular and humoral immune responses of mice against ovalbumin (OVA). Among three saponins, the ranking of the haemolytic activity was PD>PD3>PE (P<0.001). PD and PD3 could significantly enhance mitogen- and OVA-induced splenocyte proliferation in the OVA-immunized mice (P<0.001). The order of increasing OVA-stimulated splenocyte proliferation was PD>PD3>PE (P<0.05, P<0.01, or P<0.001). The sera OVA-specific IgG, IgG1, IgG2a and IgG2b antibody levels in the OVA-immunized mice were significantly enhanced by PD and PD3. However, PE only significantly promoted the production of the sera OVA-specific IgG2a and IgG2b antibody in the OVA-immunized mice. Adjuvant potentials of PD on antibody responses were higher than those of PD3 and PE (P<0.05, P<0.01, or P<0.001). Meanwhile, PD also significantly enhanced the mRNA expression of cytokines IL-2, IFN-gamma, IL-4, and IL-10 and transcription factors T-bet and GATA-3 in mice splenocyte induced by Con A (P<0.05, P<0.01, or P<0.001). These results suggested that the number of sugar residues in the glycidic chains attached to C-3 of aglycone could affect the haemolytic and adjuvant activities of platycodigenin-type saponins, and that PD had immunological adjuvant activity, and simultaneously elicited a Th1 and Th2 immune response by regulating gene expression of Th1/Th2 cytokines and transcription factors.

Topics: Adjuvants, Immunologic; Animals; Antibodies; Cell Proliferation; Cells, Cultured; Cytokines; Epoxy Compounds; Female; Gene Expression Profiling; Hemolysis; Immunoglobulin G; Lymphocytes; Mice; Mice, Inbred ICR; Molecular Structure; Ovalbumin; Plant Roots; Platycodon; Propionates; Saponins; Spleen

Ginsenoside Rh(4) (1), a saponin isolated from the roots of Panax notoginseng (Burk.) F. H. Chen, was evaluated for its haemolytic activity and adjuvant potential on specific antibody and cellular response to ovalbumin (OVA) in mice. Compound 1 showed a slight haemolytic effect, its concentration inducing 50% of the maximum haemolysis (HD(50) value) being 407+/-12 microg/ml using a 0.5% suspension of red blood cells. Compound 1 significantly increased the concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-induced splenocyte proliferation in OVA-immunized mice especially at a dose of 25 microg (P<0.05, P<0.01, or P<0.001). The OVA-specific serum IgG, IgG1, and IgG2b antibody levels were also significantly enhanced by 1 at a dose of 25 microg compared to the OVA control group (P<0.05 or P<0.01). Moreover, the enhancing effect of 1 on the OVA-specific IgG2b antibody responses to OVA in mice was more significant than that of Alum (Al(OH)(3) gel; P<0.01). These results suggest that 1 could be safely used as adjuvant with low or non-haemolytic effect.

Topics: Adjuvants, Immunologic; Animals; Dose-Response Relationship, Drug; Female; Ginsenosides; Hemolysis; Immunoglobulin G; Lymphocyte Activation; Mice; Mice, Inbred ICR; Ovalbumin; Panax notoginseng; Quillaja Saponins; Saponins

In this study, the haemolytic activities of Bupleurum chinense saponins (BCS) and its adjuvant potentials on the immune responses of ICR mice against ovalbumin (OVA) were evaluated. BCS showed a slight haemolytic effect, with its haemolytic percents being 3.32% and 1.19% at the concentrations of 500 and 250 microg/ml, respectively. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing aluminium hydroxide gel (Alum, 200 microg), QuilA (10 and 20 microg) or BCS (50, 100 or 200 microg) on Days 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)- and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. BCS significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice especially at a dose of 100 microg (P<0.05 or P<0.001). OVA-specific IgG, IgG1 and IgG2b antibody levels in serum were also significantly enhanced by BCS compared with OVA control group (P<0.01 or P<0.001). Moreover, no significant differences (P>0.05) were observed between enhancing effect of BCS and QuilA on the OVA-specific IgG2b antibody responses to OVA in mice. In conclusion, the results suggest that BCS could be safely used as adjuvant with low or non-haemolytic effect.

Topics: Adjuvants, Immunologic; Aluminum Hydroxide; Animals; Antibodies; Bupleurum; Cells, Cultured; Erythrocytes; Hemolysis; Immunoglobulin G; Injections, Subcutaneous; Lymphocytes; Male; Mice; Mice, Inbred ICR; Ovalbumin; Quillaja Saponins; Rabbits; Saponins

In this study, the haemolytic activities of Glycyrrhiza uralensis saponins (GLS) and its adjuvant potentials on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were evaluated. We determined the haemolytic activity of GLS using 0.5% rabbit red blood cell. Haemolytic percents of GLS-treated red blood cell were 11.20 and 5.54% at the concentrations of 500 and 250 microg/ml, respectively. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing Alum (200 microg), QuilA (10 and 20 microg), or GLS (50, 100, or 200 microg) on Days 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-stimulated splenocyte proliferation and OVA-specific serum antibodies were measured. GLS significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice at a dose of 100 microg (P<0.025). OVA-specific IgG, IgG1, and IgG2b antibody titers in serum were also significantly enhanced by GLS compared with OVA control group (P<0.025). Moreover, no significant differences (P>0.05) were observed between enhancing effect of GLS and QuilA on the OVA-specific IgG, IgG1, and IgG2b antibody responses to OVA in mice. The results suggest that GLS showed a slight haemolytic effect and enhanced significantly a specific antibody and cellular response against OVA in mice, and deserved further researches to be developed as immunological adjuvant.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies; Antibody Formation; Erythrocytes; Glycyrrhiza uralensis; Hemolysis; Immunoglobulin G; Injections, Subcutaneous; Lymphocyte Activation; Lymphocytes; Male; Mice; Mice, Inbred ICR; Ovalbumin; Quillaja Saponins; Rabbits; Saponins

The further purification of the total saponins from the roots of Panax notoginseng by using ordinary and reversed-phase silica-gel, as well as Sephadex LH-20 chromatography afford seven adjuvant active protopanaxatriol-type saponins (PTS), ginsenosides-Rh1 (Rh1),-Rh4 (Rh4),-Rg1 (Rg1),-Re (Re), notoginsenosides-R1 (R1),-R2 (R2),-U (U). These saponins were evaluated for their haemolytic activities and adjuvant potentials on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA). The effect of the substitution pattern of these PTS on their biological activities was investigated and structure-activity relationships were established. Among seven PTS, the haemolytic activity of Rh1 was higher than that of other six compounds (p<0.001) The HD50 values of Rh4 and U were significantly bigger than those of R2, Rg1 and Re (p<0.05 or p<0.01). Seven PTS could significantly increase the concanavalin A (Con A)-, lipopolysaccharide (LPS)- and OVA-induced splenocyte proliferation in the OVA-immunized mice (p<0.01 or p<0.001). The OVA-specific IgG, IgG1, IgG2a and IgG2b antibody levels in serum were also significantly enhanced by seven PTS compared with OVA control group (p<0.01 or p<0.001). The structure-activity relationship studies suggested that the number, the length and the position of sugar side chains, and the type of glucosyl group in the structure of PTS could not only affect their haemolytic activities and adjuvant potentials, but have significant effects on the nature of the immune responses. The information about this structure/function relationship might be useful for developing semisynthetic tetracyclic triterpenoid saponin derivatives with immunological adjuvant activity, as well as a reference to the distribution of the functional groups composing the saponin molecule.

Topics: Animals; Antibody Formation; Dose-Response Relationship, Drug; Hemolysis; Immunity, Cellular; Immunologic Factors; In Vitro Techniques; Lymphocyte Activation; Male; Mice; Mice, Inbred ICR; Ovalbumin; Panax; Plant Roots; Sapogenins; Saponins; Structure-Activity Relationship; Triterpenes

In this study, the haemolytic activities of Achyranthes bidentata saponins (ABS) and its adjuvant potentials on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were evaluated. We determined the haemolytic activity of ABS using 0.5% rabbit red blood cell. The concentration inducing 50% of the maximum haemolysis (HD50) for ABS was 164.59+/-13.41 microg/ml. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing Alum (200 microg), QuilA (10 and 20 microg) or ABS (50, 100 or 200 microg) on Days 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)- and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. ABS significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice especially at a dose of 100 microg (P<0.05 or P<0.025). OVA-specific serum IgG, IgG1 and IgG2b antibody titers were also significantly enhanced by ABS compared with OVA control group (P<0.05 or P<0.025). The results suggest that ABS showed a slight haemolytic effect and enhanced significantly a specific antibody and cellular response against OVA in mice.

Topics: Achyranthes; Adjuvants, Immunologic; Animals; Antibody Formation; Hemolysis; Immunoglobulin G; Lymphocyte Activation; Male; Mice; Mice, Inbred ICR; Ovalbumin; Saponins

Pods of Acacia concinna (Leguminosae) contain several saponins. In this study, four saponin fractions which were acetone fraction (AAC), aqueous fraction (WAC), hydromethanolic fraction (HAC) and methanolic fraction (MAC) were generated and their haemolytic activities and surface activities were determined in comparison with quillaja saponin (QS). There were no significant differences between the haemolytic activities of MAC and QS. However, the surface tensions of MAC was significantly lower than QS (p < 0.001). Furthermore, the immunomodulatory effect and the adjuvant potential of MAC on the cellular and humoral immune response of BALB/c mice against ovalbumin were investigated. The splenocyte proliferations induced by MAC were significantly higher than QS at the concentrations of 200, 400, 800 and 1000 microg/ml (p < 0.05). BALB/c mice were immunized subcutaneously either with OVA 20 microg alone or with OVA 20 microg combining with QS (10 microg) or MAC (10 and 40 microg). Ten days after the second immunization, concanavalin A (Con A)-, pokeweed mitogen (PWM)-, and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. The results suggested that MAC (40 microg) could activate T and B cells. In addition, OVA-specific IgG, IgG1 IgG2a and IgG2b antibody levels in serum were significantly enhanced by MAC (40 microg) as compared with OVA control group (p < 0.001). This finding suggested that MAC might be effect on Th1 and Th2 helper T cells. In conclusion, the results indicated that MAC at a dose of 40 microg could be used as vaccine adjuvant to increase immune responses.

Topics: Acacia; Adjuvants, Immunologic; Animals; Cell Proliferation; Cells, Cultured; Erythrocytes; Hemolysis; Immunoglobulin G; Methanol; Mice; Mice, Inbred BALB C; Ovalbumin; Plant Extracts; Rabbits; Saponins; Spleen

Notoginsenoside K (1), a saponin isolated from the roots of Panax notoginseng (Burk.) F. H. Chen, was evaluated for its haemolytic activity and adjuvant potential on specific antibody and cellular response to ovalbumin (OVA) in mice. Compound 1 showed a slight haemolytic effect, its concentration inducing 50% of the maximum haemolysis (HD50 value) being 318+/-13 microg/ml, on a 0.5% suspension of red blood cells. Compound 1 significantly increased the concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-induced splenocyte proliferation in OVA-immunized mice (P<0.05, P<0.01, or P<0.001). The OVA-specific serum IgG, IgG1, and IgG2b antibody levels were also significantly enhanced by 1, especially at a dose of 25 mug compared to an OVA control group (P<0.001). Moreover, the enhancing effect of 1 on the OVA-specific IgG2b antibody responses to OVA in mice was more significant than that of Alum (AlOH gel; P<0.01). These results suggest that 1 exhibits a slight haemolytic activity and a significant adjuvant effect on specific antibody and cellular response against OVA in mice.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Cell Proliferation; Erythrocytes; Female; Hemolysis; Immunity, Cellular; Immunization; Mice; Mice, Inbred ICR; Ovalbumin; Panax notoginseng; Plant Roots; Rabbits; Saponins; Spleen

The further purification of the total saponins from the roots of Panax notoginseng (Burk.) F. H. Chen by ordinary and reversed-phase silica-gel, as well as Sephadex LH-20 chromatography afforded two adjuvant active dammarane-type saponins, ginsenoside Re (1) and notoginsenoside R1 (2). These two saponins were evaluated for haemolytic activities and adjuvant potentials on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA). The concentrations inducing 50% of the maximum haemolysis (HD50), using 0.5% red blood cell suspensions, were 469.6+/-16.9 and 420.4+/-22.9 microg/ml for 1 and 2, respectively. Compounds 1 and 2 significantly increased the concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-induced splenocyte proliferation in the OVA-immunized mice (P<0.05, P<0.01, or P<0.001). The OVA-specific IgG, IgG1, and IgG2b antibody titres in serum were also significantly enhanced by 1 and 2 compared with OVA control group (P<0.05, P<0.01, or P<0.001). The results indicate that 1 and 2 showed a slight haemolytic activity and significant adjuvant effect on specific antibody and cellular immune response against OVA in mice, and that the type of the terminal sugar of the sugar chain at C(6) of protopanaxatriol could not only affect their haemolytic activities and adjuvant potentials, but have significant effects on the nature of the immune responses. The information about this structure-function relationship might be useful for developing semisynthetic dammarane-type saponin derivatives with immunological adjuvant activity.

Topics: Adjuvants, Immunologic; Animals; Antibodies; Cell Proliferation; Cells, Cultured; Erythrocytes; Ginsenosides; Hemolysis; Magnetic Resonance Spectroscopy; Male; Mice; Molecular Structure; Ovalbumin; Rabbits; Spleen

In this study, the haemolytic activities of Astragalus membranaceus saponins (AMS) and its adjuvant potentials on the cellular and humoral immune responses of ICR mice against OVA were evaluated. We determined the haemolytic activity of AMS using 0.5% rabbit red blood cell. AMS showed a slight haemolytic effect, with its haemolytic percent being 0.66% at the concentration of 500 microg/ml. Furthermore, the adjuvant potentials of AMS at three dose levels on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were investigated. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing Alum (200 microg), QuilA (10 and 20 microg) or AMS (50, 100 or 200 microg) on Day 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)- and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. AMS significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice especially at a dose of 100 microg (P<0.05 or P<0.001). OVA-specific IgG, IgG1 and IgG2b antibody titers in serum were also significantly enhanced by AMS compared with OVA control group (P<0.01 or P<0.001). Moreover, no significant differences (P>0.05) were observed between enhancing effect of AMS and QuilA on the OVA-specific IgG, IgG1 and IgG2b antibody responses to OVA in mice. In conclusion, the results suggest that AMS could be safely used as adjuvant with low or non-haemolytic effect.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Astragalus propinquus; Cell Proliferation; Hemolysis; Immunity, Cellular; Mice; Ovalbumin; Saponins

Four protopanaxadiol-type saponins (PDS), ginsenosides-Rb(1), -Rd, notoginsenosides-K, -R(4) isolated from the roots of Panax notoginseng were evaluated for their haemolytic activities and adjuvant potentials on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA). The effect of the substitution pattern of these PDS on their biological activities was investigated and structure-activity relationships were established. Among four PDS, the ranking of the haemolytic activity was K>R(4)>Rb(1)>Rd (P<0.01 or <0.001). Rd, Rb(1), and K could significantly enhance mitogen- and OVA-induced splenocyte proliferation in the OVA-immunized mice (P<0.001), with the order in terms of stimulation index being Rd>Rb(1)>K>R(4). OVA-specific IgG, IgG1, IgG2a and IgG2b antibody levels in the OVA-immunized mice were significantly enhanced by four PDS. Adjuvant potentials of Rd on antibody responses were higher than those of other three PDS. Meanwhile, Rd also significantly enhanced the production of the Th1 and Th2 cytokines in OVA-immunized mice (P<0.05 or <0.01). The structure-activity relationship studies suggested that the length of sugar side chains at position C-20 and the linkage of glucose moiety at position C-3 of protopanaxadiol could affect the haemolytic and adjuvant activities of PDS. The information about this structure/function relationship might be useful for developing semisynthetic tetracyclic triterpenoid saponin derivatives with immunological adjuvant activity, as well as a reference to the distribution of the functional groups composing the saponin molecule.

Topics: Adjuvants, Immunologic; Animals; Ginsenosides; Hemolysis; Mice; Mice, Inbred ICR; Ovalbumin; Panax; Plant Roots; Plants, Medicinal; Saponins; Spleen; Structure-Activity Relationship

In this study, the haemolytic activities of Panax notoginseng saponins (PNS) and its adjuvant potentials on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were evaluated. We determined the haemolytic activity of PNS using 0.5% rabbit red blood cell. PNS showed a slight haemolytic effect, with its haemolytic percents being 11.59 and 3.60% at the concentrations of 500 and 250 microg/ml, respectively. Furthermore, the adjuvant potential of PNS at three dose levels on the cellular and humoral immune responses of ICR mice against ovalbumin were investigated. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing aluminum hydroxide gel (Alum) (200 microg), Quil A (10 and 50 microg) or PNS (50, 100 or 200 microg) on days 1 and 15. Two weeks later (day 28), concanavalin A (Con A)-, pokeweed (PWM)- and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. PNS significantly enhanced the Con A-, PWM-, and OVA-induced splenocyte proliferation in the OVA-immunized mice at a dose of 100 microg (P < 0.05 or P < 0.025). OVA-specific IgG, IgG1 and IgG2b antibody levels in serum were significantly enhanced by PNS compared with OVA control group (P < 0.025). Moreover, enhancing effect of PNS on the OVA-specific IgG2b antibody responses to OVA in mice were more significant than that of Quil A (P < 0.025). In conclusion, the results suggest that PNS could be safely used as adjuvant with low or non-haemolytic effect.

Topics: Adjuvants, Immunologic; Aluminum Hydroxide; Animals; Drug Evaluation, Preclinical; Hemolysis; Immunization; Immunoglobulin G; Lymphocyte Activation; Male; Mice; Mice, Inbred ICR; Ovalbumin; Panax; Quillaja Saponins; Rabbits; Saponins

We have identified saponins in the root of Polygala senega L., a plant indigenous to the Canadian prairies, which display immunopotentiation activity to protein and viral antigens. By two-step extraction and hemolytic activity-guided fractionation by silica flush chromatography six saponin fractions were generated and their HPLC profiles determined. Two dominant fractions, designated as PS-1 and PS-2, were tested for adjuvant activity in mice immunized with ovalbumin, and hens immunized with rotavirus. The resulting adjuvant activity was compared with that of Quil A saponin. The P. senega saponins increased specific antibody levels to the antigens, in both mice and hens. In mice, there was a preferential increase of the IgG2a subclass, and upon in vitro secondary antigen stimulation, high IL-2 and IFN-gamma levels were observed in spleen cell cultures from P. senega saponins-immunized animals. The saponins were tested for their toxicity by lethality in mice and were found to be less toxic at the same dose than their counterpart Quil A. The results of this study indicated the potential of P. senega saponins as vaccine adjuvants to increase specific immune responses.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Viral; Canada; Chickens; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Enzyme-Linked Immunosorbent Assay; Female; Hemolysis; Mice; Ovalbumin; Plant Roots; Plants, Medicinal; Quillaja Saponins; Rotavirus; Saponins

Recombinant (r) Mycobacterium bovis strains were constructed that secrete biologically active listeriolysin (Hly) fusion protein of Listeria monocytogenes. The r-BCG strains pAT261:Hly or pMV306:Hly expressed plasmid multicopies or chromosomal single copies of the hly gene, respectively. Human and murine macrophage-like cell lines were infected with r-BCG pAT261:Hly and pMV306:Hly strains. Interestingly, intracellular persistence of both r-BCG strains was reduced in macrophages as compared with the parental BCG strain. By immunogold labeling Hly was detected in membrane structures and within the phagosomal space of macrophages. In addition, Hly was localized within cytoplasmic vacuoles outside the mycobacteria-containing phagosome of host cells infected with r-BCG pAT261:Hly or r-BCG pMV306:Hly. Hly fusions consistently colocalized with a lysosome-associated membrane glycoprotein, suggesting that membrane-attack conformation of Hly was not altered. Although r-BCG pAT261:Hly and r-BCG pMV306:Hly microorganims apparently did not egress into the cytoplasmic compartment of host cells, they both improved major histocompatibility complex class I presentation of cophagocytosed soluble protein as compared with wild-type BCG microbes. These data suggest that Hly secretion endows BCG with an improved capacity to stimulate CD8 T cells. Because CD8 T cells play a major role in protection against tuberculosis such Hly secreting r-BCG constructs are antituberculosis vaccine candidates.

Topics: Amino Acid Sequence; Animals; Antigen-Presenting Cells; Bacterial Toxins; BCG Vaccine; Cell Membrane Permeability; Genetic Vectors; Heat-Shock Proteins; Hemolysin Proteins; Hemolysis; Histocompatibility Antigens Class I; Listeria monocytogenes; Macrophages; Mice; Molecular Sequence Data; Mycobacterium bovis; Ovalbumin; Recombinant Fusion Proteins; Recombinant Proteins

Saponins extracted from the seed of Chenopodium quinoa (quinoa) were studied for their ability to act as mucosal adjuvants upon their intragastric or intranasal administration together with model antigens in mice. Quinoa saponins, co-administered intragastrically or intranasally with cholera toxin or ovalbumin, potentiated specific IgG and IgA antibody responses to the antigens in serum, intestinal and lung secretions. The potentiating effect of the saponins appeared, to some extent, mediated by increased permeability of the mucosa, allowing increased uptake of the antigen. The intragastric administration of 99mTc-radio-labeled human serum albumin together with quinoa saponins revealed an increased presence of the radiolabeled protein in blood, liver, spleen and lungs of mice. This study indicates the potential of quinoa saponins as adjuvants for mucosally administered vaccines.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Administration, Oral; Animals; Antibody Formation; Chenopodiaceae; Cholera Toxin; Edible Grain; Enzyme-Linked Immunosorbent Assay; Female; Hemolysis; Immunity, Mucosal; Immunoglobulin A, Secretory; Immunoglobulin G; Intestinal Mucosa; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; Permeability; Saponins; Seeds; Sheep

Aluminum hydroxide (Al) and calcium phosphate (Ca) gels have been used as vaccine adjuvants for many years. We investigated mechanism of the hemolytic activities of both adjuvant materials. The hemolytic activity of each gel depended on the gel dose. The adsorption capacities and the hemolytic activities of both adjuvants decreased as the concentration of phosphate increased in a gel-washing solution. A positive correlation between the hemolytic activity and the adsorption capacity was found in Al-gel. A disruptive effect of Ca-gel on membrane of erythrocytes was shown by electron microscopy. Ca-gel required more than 10 times as much pre-adsorbed ovalbumin as did Al-gel to inhibit the hemolysis. These results suggest that the hemolytic activity of both adjuvant materials depended mainly on the adsorption ability, and it may be useful to control the adsorption ability of adjuvants to reduce their hemolytic activity.

Topics: Adjuvants, Immunologic; Adsorption; Aluminum Hydroxide; Animals; Calcium Phosphates; Erythrocyte Membrane; Female; Gels; Guinea Pigs; Hemolysis; Microscopy, Electron, Scanning; Ovalbumin; Phosphates

A Modified Microcomplement Fixation Test (MMFT), useful for detecting and quantifying soluble immune complexes (ICs) and their components in chromatographic separations, is described. This method is based on the addition of excess specific antibody to the ICs against any of their components in order to increase the ICs' anticomplementarity. The concentration of ICs is expressed as the sample dilution which fixes 50% of the added Complement. The MMFT was applied to profiles of ICs obtained in vivo and in vitro. MMFT allows great sensitivity, good reproductibility and the detection of noncomplement fixing ICs without any interference of free antigen (Ag) or free antibody (Ab).

Topics: Animals; Antibodies; Antigen-Antibody Complex; Antigens; Complement Fixation Tests; Hemolysis; Ovalbumin; Rabbits; Rats

The results of this study demonstrate that inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC) of human peripheral blood mononuclear cells (PBMC) by ovalbumin (OA)-IgG anti-OA immune complexes (IC) can be reversed by normal human serum (NHS) or serum from a patient with congenital deficiency of the second component of complement (C2 def-HS) lacking activity of the classical complement (C) pathway. On the other hand, NHS that had been inactivated by heating at 56 degrees C for 30 min (HI-NHS) or NHS depleted of the alternative C pathway activity by absorption with zymosan (Zy-NHS) did not restore the ADCC of IC-blocked PBMC. These results suggest that the alternative pathway of C plays a very important role in the re-establishment of ADCC of PBMC blocked with IC. The recovered activity was susceptible to a new inhibition when re-exposed to IC, demonstrating that the NHS effect depends on the recovery of the functional activity of the receptor for the FC fragment of IgG on PBMC and not on the induction of non-specific cytotoxicity. The ability of NHS to restore the cytolytic potential of IC-inhibited PBMC was dependent on the time of exposure of PBMC to IC before the addition of the unblocking agent (NHS). After prolonged reaction of PBMC with IC, the blocked cells were unable to recover their ADCC activity by incubation with NHS. Unblocking of the Fc receptor of PBMC by C may be a physiological way to prevent permanent impairment of the immune mechanisms that depend on its function in the free state.

Topics: Antibody-Dependent Cell Cytotoxicity; Antigen-Antibody Complex; Blood; Cells, Cultured; Complement C2; Complement Pathway, Alternative; Hemolysis; Humans; Immunoglobulin G; Leukocytes; Ovalbumin; Zymosan

Topics: Animals; Antibodies; Complement C8; Complement C9; Complement System Proteins; Erythrocyte Membrane; Erythrocytes; Guinea Pigs; Hemoglobins; Hemolysis; Humans; Inulin; Ovalbumin; Rabbits; Ribonucleases; Serum Albumin, Bovine; Sheep; Sucrose

An antibody assay is described based on the principle of complement mediated lysis of sheep red blood cells labelled with antigen. The technique provides a sensitive class specific assay enabling antibody in all three major immunoglobulin classes to be quantitated independently. The assay may be performed in tubes allowing precise measurement of antibody concentration, or in microtitre plates which provides a rapid estimation of antibody titre.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibody Specificity; Complement System Proteins; Dinitrobenzenes; Erythrocytes; Hemolysis; Immune Sera; Immunoglobulin M; Ovalbumin; Sheep; Swine

Oxygen dissolved in solutions is reduced at the dropping-mercury electrode of direct-current polarograph to produce two waves. The first oxygen wave displays a very pronounced maximum under some conditions. The conditions displaying or suppressing the maximum have been demonstrated experimentally. It has been found that proteins have two orders of suppressive power over other biological substances and the amount suppressed is proportional to the concentrations of proteins in lower range of concentrations. A method for microdetermination of proteins has been developed using the phenomenon. The reagent required is sodium chloride alone and oxygen dissolved naturally in a sample solution from air is utilized for the determination. The procedure required is merely to dilute a sample solution to an optimum concentration for the determination with a solution of sodium chloride. The method has been demonstrated to be specific for proteins under a certain condition and to be applicable to hematological, serological and biochemical investigations in the area of medicine.

Topics: Animals; Blood Proteins; Blood Sedimentation; Blood Stains; Cattle; Cystine; Hemolysis; Humans; Microchemistry; Ovalbumin; Oxygen; Polarography; Proteins; Solutions

Normal rats were injected with guinea pig anti-rat glomerular basement membrane antibodies of the IgG1 or IgG2 class or with their F (ab') 2 fragments, in order to study which antibody site triggers the alternate complement pathway in vivo. Both IgG classes were able to induce a heavy proteinuria and led to C3 deposition in the glomeruli in a pattern similar to their own distribution along the glomerular basement membrane, as shown by the immunofluorescence technique. The Fab(ab')2 fragment of IgG2 did not produce C3 binding or proteinuria. The F(ab')2 fragment of IgG1 was difficult to obtain devoid of Fc determinants. A F(ab')2 fragment of IgG1 still bearing Fc determinants led to C3 binding and proteinuria, whereas the true F(ab')2 fragment of IgG1 had none of these effects in two out of three animals.

Topics: Animals; Antibodies; Basement Membrane; Complement C3; Complement System Proteins; Epitopes; Fluorescent Antibody Technique; Goats; Guinea Pigs; Hemolysis; Immune Sera; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Immunoglobulin G; Kidney Glomerulus; Male; Ovalbumin; Proteinuria; Rabbits; Rats; Ultrafiltration; Zymosan

On the basis of their physicochemical and antigenic properties, and by analogy with guinea pig immunoglobulins, two antibody classes in the serum of the marsupial Setonix brachyurus have been classified as IgG1 and IgG2 isotypes. The analogy between these marsupial immunoglobulins and those of eutherian (placental) species was extended by an investigation of their biological activities. The electrophoretically slow antibody (IgG2) fixed hemolytic complement, precipitated soluble antigen and predominated early in the response mounted when quokkas were immunized with antigen emulsified in Freund's complete adjuvant (FCA). The more anodic antibody (IgG1) in this marsupial did not fix complement, was inefficient in precipitating antigen and was the predominant antibody synthesized by quokkas immunized with antigen adsorbed to alumina gel. The IgG1 isotype in this marsupial appears to possess both passive hemagglutinating (HA) and homocytotropic antibody (HCA) activities. However, the HCA and IgG1 HA activities did not develop in parallel during the course of the immune response, thus suggesting that only a functional subpopulation of the IgG1 antibodies possess homocytotropic activity.

Topics: Absorption; Aluminum; Animals; Antibodies; Antibody Specificity; Dinitrophenols; Freund's Adjuvant; Hemagglutination Tests; Hemocyanins; Hemolysis; Immune Sera; Immunity, Cellular; Immunization; Immunization, Secondary; Immunodiffusion; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; Iodine Radioisotopes; Marsupialia; Ovalbumin; Papain; Passive Cutaneous Anaphylaxis; Rabbits

Topics: Antigen-Antibody Complex; Autoradiography; Chymotrypsin; Complement System Proteins; Electrophoresis, Disc; Hemolysis; Humans; Immunodiffusion; Immunoelectrophoresis; Iodine Radioisotopes; Isotope Labeling; Microbial Collagenase; Ovalbumin; Pancreatic Elastase; Sodium Dodecyl Sulfate; Trypsin; Urea

It is shown that the paramagnetic resonance of spin labels offers a convenient technique for monitoring the complement-mediated immune lysis of erythrocyte ghosts, and sensitized phospholipid liposomes. The ghosts or liposomes are loaded with a concentrated solution of a water-soluble, membrane-impermeable spin label, so that there is a strong exchange broadening of the paramagnetic resonance signal due to labels enclosed within the liposomes or ghosts. Complement-mediated lysis releases the labels into a dilute solution, where a relatively sharp and intense paramagnetic resonance signal is detected. AN UNANTICIPATED RESULT IS ALSO REPORTED: this is the augmentation of release of spin label from loaded ghosts sensitized with egg albumin, in the presence of antibody against egg albumin, complement, and egg albumin in solution, over and above the release in the absence of egg albumin in solution. Comparison with complement fixation suggests that the augmentation of lysis is effected by crosslinking of immune complexes with the cell membrane.

Topics: Animals; Antibodies; Antigen-Antibody Complex; Antigens; Antigens, Bacterial; Binding Sites; Complement System Proteins; Electron Spin Resonance Spectroscopy; Erythrocytes; Escherichia coli; Hemolysis; Humans; Lipopolysaccharides; Liposomes; Ovalbumin; Phospholipids; Rabbits; Sheep

Topics: Ammonium Chloride; Animals; Antigens; Basophils; Blood Cells; Blood Sedimentation; Bone Marrow Cells; Cell Separation; Centrifugation, Density Gradient; Gelatin; Guinea Pigs; Hemolysis; Immunization; Iron; Leukocyte Count; Magnetics; Microscopy, Electron; Monocytes; Neutrophils; Ovalbumin; Phagocytosis; Sheep

Topics: Animals; Antigen-Antibody Reactions; Chickens; Chromium Radioisotopes; Cytotoxicity Tests, Immunologic; Erythrocytes; Hemolysis; Humans; Immunoglobulin Fab Fragments; Immunoglobulin G; In Vitro Techniques; Lymphocytes; Ovalbumin; Serum Albumin; Transferrin

Topics: Alanine; Animals; Antibodies; Antibody Formation; Antigen-Antibody Reactions; Ascitic Fluid; Cattle; Crosses, Genetic; Epitopes; Erythrocytes; Hemolysis; Immune Sera; Immunization Schedule; Immunodiffusion; Immunoglobulin G; Mice; Mice, Inbred Strains; Myeloma Proteins; Ovalbumin; Serum Albumin, Bovine; Sheep; Streptococcus

Topics: Animals; Blood Proteins; Carbon Isotopes; Cell-Free System; Centrifugation, Density Gradient; Chickens; Female; Goats; Hemolysis; Isoleucine; Leucine; Ovalbumin; Oviducts; Peptide Chain Initiation, Translational; Polyribosomes; Precipitin Tests; Rabbits; Reticulocytes; RNA; RNA, Messenger; Tritium

Topics: Acetates; Animals; Carbon Isotopes; Cell-Free System; Globins; Heme; Hemolysis; Hydrogen-Ion Concentration; Kinetics; Magnesium; Ovalbumin; Peptide Chain Elongation, Translational; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Phenylhydrazines; Polyribosomes; Protein Biosynthesis; Rabbits; Reticulocytes; RNA; RNA, Messenger; Temperature; Tritium

Topics: Animals; Antigen-Antibody Complex; Antigen-Antibody Reactions; Complement Fixation Tests; Complement System Proteins; Hemolysis; Humans; Immune Sera; Ovalbumin; Rabbits; Serum Albumin

Topics: Anaphylaxis; Animals; Antibodies; Chromatography, DEAE-Cellulose; Complement Fixation Tests; Electrophoresis; Female; Freund's Adjuvant; Guinea Pigs; Hemagglutination; Hemolysis; Immune Sera; Immunoelectrophoresis; Immunoglobulin G; Immunoglobulins; Ovalbumin; Precipitin Tests; Ultracentrifugation

Topics: Animals; Antigen-Antibody Reactions; Antigens; Binding Sites; Blood Protein Electrophoresis; Cell Membrane; Complement System Proteins; Dinitrophenols; Erythrocytes; Female; gamma-Globulins; Guinea Pigs; Haptens; Hemagglutination Tests; Hemocyanins; Hemolysis; Histamine Release; Immunization; Immunoglobulins; Mast Cells; Mice; Multiple Myeloma; Neoplasm Proteins; Nitrobenzenes; Ovalbumin; Passive Cutaneous Anaphylaxis; Rabbits; Rats; Serum Albumin, Bovine; Sheep

Topics: Animals; Antibody Formation; Antibody Specificity; Antigens; Cattle; Chemical Precipitation; Erythrocytes; Freund's Adjuvant; gamma-Globulins; Guinea Pigs; Hemagglutination; Hemolysis; Hypersensitivity, Delayed; Immunization; Ovalbumin; Passive Cutaneous Anaphylaxis; Radiation Effects; Sheep

Topics: Age Factors; Animals; Animals, Newborn; Antibody Formation; Antibody-Producing Cells; Antigens; Blood Bactericidal Activity; Dinitrophenols; Erythrocytes; Hemagglutination Tests; Hemolysis; Hemolytic Plaque Technique; Immunization; Liver; Lung; Opossums; Ovalbumin; Rabbits; Serratia marcescens; Serum Albumin, Bovine; Sheep; Species Specificity; Spleen

Topics: Animals; Antibodies; Antigens; Chickens; Chromatography, DEAE-Cellulose; Complement Fixation Tests; Cricetinae; Erythrocytes; Guinea Pigs; Hemagglutination Tests; Hemolysis; Immune Sera; Immunodiffusion; Immunoelectrophoresis; Immunoglobulin G; Ovalbumin; Passive Cutaneous Anaphylaxis; Sheep

Topics: Animals; Antigens; Chemical Precipitation; Chromatography, DEAE-Cellulose; Chromatography, Gel; Complement Fixation Tests; Edetic Acid; Guinea Pigs; Hemolysis; Humans; Hydrolysis; Immunoglobulin Fab Fragments; Immunoglobulin G; Ovalbumin; Pepsin A; Rabbits; Serum Albumin; Ultracentrifugation

Topics: Adrenalectomy; Animals; Anti-Inflammatory Agents; Arthritis; Calcium; Capillary Permeability; Carrageenan; Depression, Chemical; Dextrans; Edema; Erythema; Granuloma; Hemolysis; Hyaluronoglucosaminidase; Indicators and Reagents; Male; Malonates; Mice; Ovalbumin; Phenylbutazone; Phenylhydrazines; Prednisolone; Protein Denaturation; Radiation Injuries, Experimental; Rats; Serotonin; Ultraviolet Rays; Wound Healing

Topics: Animals; Antibodies; Antigen-Antibody Reactions; Antigens; Ascitic Fluid; Chromatography, DEAE-Cellulose; Complement Fixation Tests; Erythrocytes; Guinea Pigs; Hemagglutination Tests; Hemolysis; Immune Sera; Immunoglobulin G; Iodine Isotopes; Mice; Ovalbumin; Passive Cutaneous Anaphylaxis; Protein Binding; Species Specificity

Topics: Adjuvants, Immunologic; Aluminum; Animals; Antibody Formation; Antigens; Chromatography, DEAE-Cellulose; Dinitrophenols; gamma-Globulins; Guinea Pigs; Hemolysis; Hot Temperature; Hydroxides; Immunization; Immunoglobulin E; Mercaptoethanol; Ovalbumin; Passive Cutaneous Anaphylaxis; Rabbits; Serum Albumin; Skin Tests

Topics: Animals; Antibodies; Antigens; Cellulose; Chlorides; Chromatography; Chromium; Complement System Proteins; Erythrocytes; Guinea Pigs; Hemagglutination Tests; Hemolysis; Humans; Immune Sera; Immunoelectrophoresis; Ovalbumin; Passive Cutaneous Anaphylaxis; Rabbits; Serum Albumin, Bovine

Topics: Animals; Antibodies; Biphenyl Compounds; Cattle; Complement Fixation Tests; Fluoresceins; gamma-Globulins; Guinea Pigs; Hemagglutination; Hemolysis; Hemorrhage; Histamine; Histamine Release; Hot Temperature; Humans; Injections, Intradermal; Injections, Intravenous; Mercaptoethanol; Models, Biological; Ovalbumin; Passive Cutaneous Anaphylaxis; Pepsin A; Rabbits; Serum Albumin, Bovine; Species Specificity; Spectrophotometry

Topics: Aniline Compounds; Animals; Antibodies; Antigens; Azo Compounds; Erythrocytes; gamma-Globulins; Guinea Pigs; Hemagglutination; Hemolysis; In Vitro Techniques; Ovalbumin