Immune Rabbit Serum Research Articles

Broad vaccine coverage predicted for a bivalent recombinant factor H binding protein based vaccine to prevent serogroup B meningococcal disease

Factor H binding proteins (fHBP), are bacterial surface proteins currently undergoing human clinical trials as candidate serogroup B Neisseria meningitidis (MnB) vaccines. fHBP protein sequences segregate into two distinct subfamilies, designated A and B. Here, we report the specificity and vaccine potential of mono- or bivalent fHBP-containing vaccines. A bivalent fHBP vaccine composed of a member of each subfamily elicited substantially broader bactericidal activity against MnB strains expressing heterologous fHBP than did either of the monovalent vaccines. Bivalent rabbit immune sera tested in serum bactericidal antibody assays (SBAs) against a diverse panel of MnB clinical isolates killed 87 of the 100 isolates. Bivalent human immune sera killed 36 of 45 MnB isolates tested in SBAs. Factors such as fHBP protein variant, PorA subtype, or MLST were not predictive of whether the MnB strain could be killed by rabbit or human immune sera. Instead, the best predictor for killing in the SBA was the level of in vitro surface expression of fHBP. The bivalent fHBP vaccine candidate induced immune sera that killed MnB isolates representing the major MLST complexes, prevalent PorA subtypes, and fHBP variants that span the breadth of the fHBP phylogenetic tree. Importantly, epidemiologically prevalent fHBP variants from both subfamilies were killed.

Isolation of an Antigen Fraction from Setaria cervi Adults Having Potential for Immunodiagnosis of Human Filariasis

Crude antigenic preparations from heterologous filarial parasites gave false positive results because of complex nature of these antigens and their cross-reactivity with other helminth parasites. In the present study, efforts have been made to isolate and characterize the antigens from Setaria cervi important for diagnostic purposes. The fractionation of S. cervi somatic antigenic preparation on Sephacryl S-200 resulted in separation of three major antigenic peak fractions. Crossed immunoelectrophoretic analysis, using immune rabbit serum, revealed 13–14 antigens in SFP-I pool fraction, which showed high reactivity with filarial patients sera as compared to other two pool fractions. This SFP-I fraction was further purified by DEAE-Cellulose column chromatography. Out of the 4 antigen pool fractions, DFP-IV fraction showed high ELISA reactivity with filarial patient serum pool (Wuchereria bancrofti and Brugia malayi) as compared to other fractions. The SDS-PAGE analysis of DFP-IV fraction revealed 2 major and 1 minor protein bands (mol. wt. range 65–70 kDa). Crossed immunoelectrophoresis also showed the presence of 3 antigenic peaks in DFP-IV fraction. The purified DFP-IV fraction showed high reactivity with filarial patients sera but did not cross-react with sera from ascaris and hookworm infections thereby suggesting the filaria-specificity and potential for immunodiagnosis of human filariasis.

RNA Recognition Motif (RRM) of La/SSB: The Bridge for Interparticle Spreading of Autoimmune Response to U1-RNP

Systemic lupus erythematosus (SLE) is characterized by the production of grouped sets of autoantibodies targeting mainly the U1 ribonucleoprotein (RNP) and/or Ro/La RNP particles. Intraparticle diversification of the autoimmune response is believed to occur via epitope spreading. So far, it is not known how the autoimmune response "jumps" from one particle to another. To the extent that the majority of nuclear autoantigens in SLE are RNA binding proteins and major epitopes were previously mapped within their RRM (RNA recognition motifs), conserved sequences within RRM could be involved in the intermolecular and inter-particle diversification process of the autoimmune response. We investigated the potential of RRM of the La/SSB autoantigen to induce antibodies that cross-recognize components of the U1-RNP particle and therefore its capacity to produce interparticle epitope spreading. We immunized New Zealand white rabbits with a peptide corresponding to the epitope 145-164 of La/SSB (belonging to the RRM of La/SSB), attached in four copies on a scaffold carrier. Sera were drawn from 20 sera of patients with SLE and anti-U1-RNP antibodies and 26 sera of primary Sjögren syndrome patients with anti-La/SSB antibodies. All sera were evaluated for reactivity against the major epitope of La/SSB (pep349-364), the RNP antigen and the RRM-related epitope of La/SSB (pep145-164). Specific antibodies against pep145-164 were purified with immunoaffinity columns from selected sera. After the immunization of the animals with pep145-164, a specific IgG antibody response was detected, directed against the La/SSB autoantigen (wks 3-7), the immunizing peptide (wks 3-27), and the RNP autoantigen (wks 7-20). This response gradually decreased to low levels between postimmunization wks 27-42. Purified antibodies against pep145-164 recognized La/SSB and a 70-kD autoantigen in Western blot and exhibited significant reactivity in anti-U1-RNP ELISA. Depletion of anti-pep145-164 antibodies eliminated anti-U1-RNP reactivity from immunized rabbit sera but not from human sera. In addition, pep145-164 was recognized to a greater extent by autoimmune sera with anti-RNP reactivity compared with anti-La/SSB-positive sera, in contrast to pep349-364 of La/SSB, which was recognized almost exclusively by sera with anti-La/SSB reactivity. These data suggest that the RRM region of La/SSB can trigger interparticle B-cell diversification to U1-RNP-70 autoantigen via molecular mimicry. Identification of key sequences that trigger and perpetuate the autoimmune process is particularly important for understanding pathogenetic mechanisms in autoimmunity.

Major Urinary Protein-1 Increases Energy Expenditure and Improves Glucose Intolerance through Enhancing Mitochondrial Function in Skeletal Muscle of Diabetic Mice

Major urinary protein-1 (MUP-1) is a low molecular weight secreted protein produced predominantly from the liver. Structurally it belongs to the lipocalin family, which carries small hydrophobic ligands such as pheromones. However, the physiological functions of MUP-1 remain poorly understood. Here we provide evidence demonstrating that MUP-1 is an important player in regulating energy expenditure and metabolism in mice. Both microarray and real-time PCR analysis demonstrated that the MUP-1 mRNA abundance in the liver of db/db obese mice was reduced by approximately 30-fold compared with their lean littermates, whereas this change was partially reversed by treatment with the insulin-sensitizing drug rosiglitazone. In both dietary and genetic obese mice, the circulating concentrations of MUP-1 were markedly decreased compared with the lean controls. Chronic elevation of circulating MUP-1 in db/db mice, using an osmotic pump-based protein delivery system, increased energy expenditure and locomotor activity, raised core body temperature, and decreased glucose intolerance as well as insulin resistance. At the molecular level, MUP-1-mediated improvement in metabolic profiles was accompanied by increased expression of genes involved in mitochondrial biogenesis, elevated mitochondrial oxidative capacity, decreased triglyceride accumulation, and enhanced insulin-evoked Akt signaling in skeletal muscle but not in liver. Altogether, these findings raise the possibility that MUP-1 deficiency might contribute to the metabolic dysregulation in obese/diabetic mice, and suggest that the beneficial metabolic effects of MUP-1 are attributed in part to its ability in increasing mitochondrial function in skeletal muscle.

Antibody-Based Detection and Inhibition of Vaginolysin, the Gardnerella vaginalis Cytolysin

Bacterial vaginosis (BV) is the most common vaginal infection worldwide and is associated with significant adverse sequelae. We have recently characterized vaginolysin (VLY), the human-specific cytotoxin produced by Gardnerella vaginalis and believed to play a critical role in the pathogenesis of BV and its associated morbidities. We hypothesize that novel antibody-based strategies may be useful for detection of VLY and for inhibition of its toxic effects on human cells. Using purified toxin as an immunogen, we generated polyclonal rabbit immune serum (IS) against VLY. A western blot of G. vaginalis lysate was probed with IS and a single band (57 kD) identified. Immunofluorescence techniques using IS detected VLY production by G. vaginalis. In addition, we have developed a sandwich ELISA assay capable of VLY quantification at ng/ml concentrations in the supernatant of growing G. vaginalis. To investigate the potential inhibitory role of IS on VLY-mediated cell lysis, we exposed human erythrocytes to VLY or VLY pretreated with IS and determined the percent hemolysis. Pretreatment with IS resulted in a significant reduction in VLY-mediated lysis. Similarly, both human cervical carcinoma cells and vaginal epithelial cells exhibited reduced cytolysis following exposure to VLY with IS compared to VLY alone. These results confirm that antibody-based techniques are an effective means of VLY detection. Furthermore, VLY antiserum functions as an inhibitor of VLY–CD59 interaction, mitigating cell lysis. These strategies may have a potential role in the diagnosis and treatment of BV.

Solid‐phase tissue electrophoresis enhances sodium dodecyl sulfate‐based decellularization of xenogeneic bioscaffolds

Treatment of xenogeneic bioscaffolds with ionic detergents does not completely remove soluble protein antigens from candidate xenogeneic scaffolds for tissue engineering. The study objective was to determine if solid‐phase tissue electrophoresis enhances removal of antigenic proteins from candidate bioscaffolds.METHODSPorcine aortic valve conduit (PAV) was subjected to hypotonic lysis and then treated with 0.25% or 1% sodium dodecyl sulfate (SDS) overnight Tissues were mounted in 2% agarose gel and subjected 0 (sham), 60, or 120 V for 4 h. After treatment, soluble proteins were extracted from tissues and assayed for antigenicity by immunoblot analysis with rabbit immune serum.RESULTSSolid‐phase tissue electrophoresis enhanced removal of protein antigens from PAV compared to untreated (UT) or sham treated controls (figure).CONCLUSIONSolid‐phase tissue electrophoresis enhances SDS‐based bioscaffold decellularization. Supported by NIH HL081107.

Identification of Bacillus anthracis Spore Component Antigens Conserved across Diverse Bacillus cereus sensu lato Strains

We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals. Two high throughput approaches were used. First, an in silico screening identified 200 conserved putative B. anthracis spore components. A total of 192 of those candidate genes were expressed and purified in vitro, 75 of which reacted with the rabbit immune sera generated against B. anthracis spores. The second approach was to screen for cross-reacting antigens in the spore proteome of 10 diverse B. cereus group strains. Two-dimensional electrophoresis resolved more than 200 protein spots in each spore preparation. About 72% of the protein spots were found in all the strains. 18 of these conserved proteins reacted against anti-B. anthracis spore rabbit immune sera, two of which (alanine racemase, Dal-1 and the methionine transporter, MetN) overlapped the set of proteins identified using the in silico screen. A conserved repeat domain protein (Crd) was the most immunoreactive protein found broadly across B. cereus sensu lato strains. We have established an approach for finding conserved targets across a species using population genomics and proteomics. The results of these screens suggest the possibility of a multiepitope antigen for broad host range diagnostics or therapeutics against Bacillus spore infection.

Peptide mimics selected from immune sera using phage display technology can replace native antigens in the diagnosis of Epstein-Barr virus infection

There is an expanding area of small molecule discovery, especially in the area of peptide mimetics. Peptide sequences can be used to substitute for the entire native antigen for use in diagnostic assays. Our approach is to select peptides that mimic epitopes of the natural immune response to Epstein–Barr virus (EBV) that may be recognised by antibodies typically produced after infection with EBV. We screened a random peptide library on sera from rabbits immunised with a crude preparation of EBV and serum antibodies from a patient with a high titer of EBV antibodies. We selected four peptides (Eb1–4) with the highest relative binding affinity with immune rabbit sera and a single peptide with high affinity to human serum antibodies. The peptides were coupled to the carrier molecule BSA and the recognition of the peptides by IgM antibodies in clinical samples after infection with EBV was measured. The sensitivities were Eb1 94%, Eb2, 3, 4 88%, H1 81% and all had 100% specificity. This study illustrates that the phage display approach to select epitope mimics can be applied to polyclonal antibodies and peptides that represent several diagnostically important epitopes can be selected simultaneously. This panel of EBV peptides representing a wide coverage of immunodominant epitopes could replace crude antigen preparations currently used for capture in commercial diagnostic tests for EBV.

Reticulocyte-binding protein homologue 5 – An essential adhesin involved in invasion of human erythrocytes by Plasmodium falciparum

Invasion of erythrocytes is a prerequisite in the life history of the malaria parasite. Members of the reticulocyte-binding homologue family (PfRh) have been implicated in the invasion process and in some cases have been shown to act as adhesins, binding to specific receptors on the erythrocyte surface. We have identified a further, putatively essential, PfRh family member in the most virulent human malaria Plasmodium falciparum, called PfRh5, which binds to an unknown class of glycosylated receptors on the erythrocyte surface. This protein is an atypical PfRh family member, being much smaller than others and lacking a transmembrane and cytosolic region at the C-terminus. This suggests it may be part of a functional protein complex. PfRh5 localises to the rhoptries in merozoites and follows the tight junction during the process of erythrocyte invasion. Furthermore, rabbit immune serum raised against a portion of the ecto-domain, inhibits parasite invasion in vitro. We hypothesise an essential role for the PfRh5 adhesin in erythrocyte selection and commitment to invasion. Given its small size, we believe PfRh5 may prove to be a valuable candidate for inclusion in a multi-component anti-malarial vaccine.

IMMUNIZATION OF RABBITS AGAINST HYALOMMA ANATOLICUM ANATOLICUM USING LARVAL AND NYMPHAL EXTRACTS

Summary The protective capacity of the crude extracts from larval and nymphal stages of Hyalomma anato/icum anato/icum was examined in the New Zealand white rabbits. The rabbits were randomly divided into 3 groups of five animals. The rabbits in groups 1 and 2 were immunized with crude extracts of larval and nymphal antigens, respectively and group 3 was served as control. Following challenge of each rabbit with 2000 larvae of H. a. anato/icum, engorged nymphs were collected, weighed and then cultured in an incubator. A significant decrease in weight of engorged nymphs was only observed in group 1 that immunized with crude extracts of tick's larvae (P<0.05). Polypeptide profile of the larval and nymphal extracts was analysed by SDS-PAGE, and antigenic pattern with serum of immunized rabbits was evaluated by Dot and Western blot test. The molecular weight of the fraction of larval extract after SDS-PAGE showed six polypeptide bands as follows: 97, 84, 66, 55, 45 and 36 kDa, and in nymphal extract 13 polypeptide bands of205, 116,97,84,66,55,45,36,29,24 and 20 kDa and two bands between 116 and 205 kDa were found. In Western blotting, positive reaction was only observed with sera of group 1 in the bands with 97, 84 and 66 kDa. It seems that the larval extract of H. a. anato/icumcan be used as a source of biological material for isolation of protective antigen.

Open Sandwich FRET Immunoassay of Estrogen Receptor β in a PDMS Microfluidic Channel

Immunoassay is a fast and cost-effective protein detection method that can be applied to clinical diagnostics and biological research. Enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and protein array chips are the most widely used immunoassay techniques for the detection of a target protein. In all of these methods, however, antibodies or antigens should be immobilized on a solid substrate. As a result, they require a long assay time for complete immunoreaction between antigens and antibodies, being limited by the diffusion kinetics. Moreover, they also require several cycles of consecutive binding and washing steps to separate free reagents from binding reagents. To overcome the problem of slow immunoreactions in conventional immunoassay techniques, we recently reported a fast and sensitive one-step immunoassay of estrogen receptor β (ERβ) using quantum dot (QD)-based fluorescence resonance energy transfer (FRET). To induce a strong FRET effect, QD was used as a fluorescence donor. FRET occurs when the electronic excitation energy of a donor chromophore is transferred to a nearby acceptor molecule via a dipole-dipole interaction between the donoracceptor pair. Conventional fluorescence dyes, which cause an appreciable overlap between the emission band of the donor and the absorption band of the acceptor, have generally been used as a donor-acceptor set. However, QD provides significant advantages over conventional fluorescence dyes, including brighter fluorescence, resistance to photobleaching, and narrow emission bands, which makes it suitable as a sensitive fluorescence donor. As a result, QD reaches a longer distance to the acceptor than conventional fluorescence dyes because of their superior optical properties. Furthermore, the QD-based FRET immunoassay of ERβ was faster (30-40 min of immunoassay time including incubation) than conventional immunoassay techniques because it does not need any solid-phase carriers and multiple washing steps for free reagent separations. In our previous work, the advantages of a QD-based FRET immunoassay in an open space were fully explored. Nonetheless, a faster reaction time and a smaller sample volume are still needed to apply this QD-based FRET immunoanalysis technique to clinical diagnostics. In the present work, a lab-on-a-chip technique, combined with the QD-based FRET immunoassay method, was used to develop a more sensitive and faster immunoanalysis technique. The lab-on-a-chip technique has several advantages over conventional bench-top techniques, such as minimal sample requirement, reduced reaction time, ease of use, improved product conversion, and reduced waste generation. In particular, the reaction time can be greatly reduced to less than a few minutes if a properly designed channel to obtain highly efficient mixing is adopted. Furthermore, only a small reaction volume is required in the lab-ona-chip analysis. An alligator-teeth-shaped poly(dimethylsiloxane) (PDMS) microfluidic channel was fabricated to obtain a fast immunoreaction between antibody and antigen under a flow condition. The detailed fabrication process for this PDMS channel has been reported elsewhere. ERβ is one of the most important breast cancer markers that are closely related with cell proliferations. The antiERβ monoclonal antibody (McAb, mouse ascites, IgM isotype; Sigma, St. Louis, MO, USA) was labeled on QD 565 using an antibody conjugation kit (Invitrogen, Eugene, OR, USA). In addition, the anti-ERβ polyclonal antibody (PcAb, rabbit immune serum, IgG; Biogenex, San Ramon, CA, USA) was labeled with AF 568 protein labeling kits (Invitrogen, Eugene, OR, USA). The formation of an open sandwich immunocomplex and its energy transfer process from QD-McAb to AF-PcAb are illustrated in Figure 1(a).

GAPDH Activity and Immunogenicity of Staphylococcus aureus Recombinant GapC Protein

To characterize the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, immunogenicity and immunoprotection of Staphylococcus aureus ( S. aureus) surface protein GapC, gapC gene of S. aureus was amplified from strain BMSA/855/23-1 by PCR, and was inserted into pQE-30 vector subsequently. The recombinant plasmid, designated as pQE/ gapC, was transformed into E. coli strain M15 (pREP4). The recombinant GapC fusion proein was successfully expressed in E. coli M15 induced with IPTG and its GAPDH activity was confirmed by GAPDH activity assay. Then, the recombinant GapC protein, inactivated S. aureus whole cell and placebo (PBS) were administrated to healthy rabbits respectively. The IgG antibody titers, concentration of IFN-γ and IL-4 cytokines in immunized rabbit sera were measured with Enzyme-Linked Immunosorbent Assay (ELISA). Finally, immunized rabbits were challenged with S. aureus strain Wood46 to evaluate the immunoprotection. The IgG antibody titers against GapC and whole cell in rabbit sera reached their peaks at day 28 after boost immunization (1:64 000). The concentrations of IL-4 and IFN-γ in GapC group rabbit sera increased significantly ( P<0.05) at day 14 after boost immunization, while the concentrations of those in whole cell group did not increase ( P>0.05) compared with the placebo group. 4 rabbits in 5 of the protein immunized group were protected against challenge with 1×10 8CFU S. aureus. The results above indicate that the expressed recombinant GapC protein have high GAPDH activity and immunogenicity, can also protect against S. aureus challenge to some extent. S. aureus GapC protein could be an attractive target for further genetic engineering vaccine.

Determination of Schistosoma japonicum circulating antigens in dilution serum by piezoelectric immunosensor and S/N enhancement

A piezoelectric immunosensor assay was developed with immobilizing immunoglobulin G (IgG) as a probe to detect Schistosoma japonicum circulating antigens (SjCAg). Analytical strategy utilizes the polyclonal antibodies with broad-spectrum recognition to a complex target with high specificity. The immobilized antibodies were purified from immunized rabbit's sera ( im-S) and infected rabbit's sera ( inf-S) by S. japonicum. The detection capacities of antibodies were compared between the sera of different phenotypes and purified fractions. The sample dilution ratios were also evaluated and optimized. Additionally, the sera with a variety of infection degrees for validation could be discriminated quantitatively. The linear dose–response relationship indicates that the systematic sensitivity of this method is below 150 Hz and the lowest limit of detectable range is above 500 cercariae of S. japonicum infection for 2 weeks. The novel immunosensor technique is well potential to determine the SjCAg in serum samples for clinical diagnosis of parasitosis in early stage.

PREPARATION OF ANTIGENS FROM MIDGUT OF HYALOMMA ANATOLICUM ANATOLICUM AND DETERMINING THEIR IMMUNOPROTECTIVE EFFECTS

Partially fed (4 -5 days) H .a. anatolicum (Iranian isolate) female adult ticks were used for preparation of antigens of midgut which are, the gut supernate antigen (GS), gut membrane antigen (GM), triton extracted gut membrane antigen ( TX-GM ) and sodium dodecyl sulphate treated Triton extracted gut membrane antigen (SDS-TX-GM). The New Zealand white rabbits were immunized with GS, GM, TX- GM and SDS-TX-GM antigens. The first inoculation on day 0 was administered after emulsification with Freund`s complete adjuvant and second inoculation was given on day 14 with incomplete Freund`s adjuvant and third inoculation was given on day 28 with incomplete Freund`s adjuvant. Blood drawn was from the marginal ear vein on day 38. The humoral immune response was studied by Enzyme Linked Immunosorbent Assay (ELISA) and antibody titers in the sera of immunized rabbits on 10 days after the last booster dose of antigen was 1/32000. After electrophoresis of purified midgut antigens (GS, GM, TX-GM, SDS-TX-GM) 4µg of each were applied on a 10% acryl amide gel and blotted to nitrocellulose paper for reaction with its concern immune sera. By analysis of this Western blot, we got bands of 110, 95, 85, 66, 59, 49, 42 Kda for GSAg, 95, 85, 66, 49, 42 Kda for GM Ag, 66, 49 Kda for TX-GMAg and 66Kda for SDS-TX-GMAg responsible for induction of resistance in the host. The prepared antigens showed engorged nymphs ranged from 32-41%, 29-56%, 29-49% and 29-47% in response to GS, GM, TX-GM and SDS-TX-GM antigens respectively. Immunodominant antigenic proteins of 66 Kda were detected in the sera of rabbits immunized by all above antigens which can be candidate for single immunogen if in future studies show acceptable results. The immunized rabbits were challenged, 10 days after the last booster dose of antigens, with 700 larvae of H. a. anatolicum ticks by releasing them on one ear of rabbits and maximum protection in rabbits against H. a. anatolicum was induced with the one immunized with GSAg.

Evidence for Globally Shared, Cross-Reacting Polymorphic Epitopes in the Pregnancy-Associated Malaria Vaccine Candidate VAR2CSA

Pregnancy-associated malaria (PAM) is characterized by the placental sequestration of Plasmodium falciparum-infected erythrocytes (IEs) with the ability to bind to chondroitin sulfate A (CSA). VAR2CSA is a leading candidate for a pregnancy malaria vaccine, but its large size ( approximately 350 kDa) and extensive polymorphism may pose a challenge to vaccine development. In this study, rabbits were immunized with individual VAR2CSA Duffy binding-like (DBL) domains expressed in Pichia pastoris or var2csa plasmid DNA and sera were screened on different CSA-binding parasite lines. Rabbit antibodies to three recombinant proteins (DBL1, DBL3, and DBL6) and four plasmid DNAs (DBL1, DBL3, DBL5, and DBL6) reacted with homologous FCR3-CSA IEs. By comparison, antibodies to the DBL4 domain were unable to react with native VAR2CSA protein unless it was first partially proteolyzed with trypsin or chymotrypsin. To investigate the antigenic relationship of geographically diverse CSA-binding isolates, rabbit immune sera were screened on four heterologous CSA-binding lines from different continental origins. Antibodies did not target conserved epitopes exposed in all VAR2CSA alleles; however, antisera to several DBL domains cross-reacted on parasite isolates that had polymorphic loops in common with the homologous immunogen. This study demonstrates that VAR2CSA contains common polymorphic epitopes that are shared between geographically diverse CSA-binding lines.

Reductively Aminatedd-Xylose–Albumin Conjugate as the Immunogen for Generation of IgG and IgE Antibodies Specific tod-Xylitol, a Haptenic Allergen

Sugar alcohols are widely used as food additives and drug excipients. Xylitol, a five-carbon sugar alcohol, and a low-calorie alternative sweetener to sucrose (approx 40% fewer calories), has enjoyed an enviable record of safety, and allergic reactions to xylitol are very rare. A case of oral erosive eczema to xylitol has been reported recently [Hanakawa, Y., Hanakawa, Y., Tohyama, M., Yamasaki, K., Hashimoto, K. (2005) Xylitol as a causative agent of oral erosive eczema. Brit. J. Dermatol. 152, 821-822]. Xylitol does not contain any reactive groups; hence, it is nonimmunogenic. In order to explain the immunogenicity of xylitol, polyclonal antibodies to xylitol have been raised using the reductive aminated product of D-xylose conjugated to bovine serum albumin (BSA) as the immunogen. Rabbits immunized with xylitol-BSA conjugate (52 haptens/molecule) gave a good antibody response. Purification of antixylitol antibodies was carried out using hapten-affinity chromatography on xylitol-keyhole limpet hemocyanin-Sepharose CL-6B; the yield was approximately 40 microg/mL of rabbit immune serum. Purified xylitol-specific antibodies appeared to be homogeneous by native PAGE with a pI of approximately 7.2 by isoelectric focusing. Although the purified antibodies are specific for the xylitoyl moiety of xylitol-protein conjugates, they reacted equally well with the Schiff base conjugate of xylosyl-protein conjugates (68% cross-reactivity) indicating that carbons 2 to 5 of xylitol act as an epitope. Xylitol antibodies showed excellent specificity towards xylitol and <4.4% cross-reactivity with D-xylose and various sugar alcohols except ribitol and galactitol, which showed approximately 11% and 8% cross-reactivity, respectively. D-Xylitol-BSA conjugate was used to raise IgE antibodies in BALB/c mice by repeated intradermal administration. Passive cutaneous anaphylaxis using the immune sera confirmed the haptenic nature of xylitol.

IgG anti-GalNAc-GD1a antibody inhibits the voltage-dependent calcium channel currents in PC12 pheochromocytoma cells

We investigated the effects of IgG anti-GalNAc-GD1a antibodies, produced by immunizing rabbits with GalNAc-GD1a, on the voltage-dependent calcium channel (VDCCs) currents in nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells. VDCCs currents in NGF-differentiated PC12 cells were recorded using the whole-cell patch-clamp technique. Immunized rabbit serum that had a high titer of anti-GalNAc-GD1a antibodies inhibited the VDCCs currents in the NGF-differentiated PC12 cells (36.0 ± 9.6% reduction). The inhibitory effect of this serum was reversed to some degree within 3–4 min by washing with bath solution. Similarly, application of purified IgG from rabbit serum immunized with GalNAc-GD1a significantly inhibited the VDCCs currents in PC12 cells (30.6 ± 2.5% reduction), and this inhibition was recovered by washing with bath solution. Furthermore, the inhibitory effect was also observed in the GalNAc-GD1a affinity column binding fraction (reduction of 31.1 ± 9.85%), while the GalNAc-GD1a affinity column pass-through fraction attenuated the inhibitory effect on VDCCs currents. Normal rabbit serum and normal rabbit IgG did not affect the VDCCs currents in the PC12 cells. In an immunocytochemical study using fluorescence staining, the PC12 cells were stained using GalNAc-GD1a binding fraction. These results indicate that anti-GalNAc-GD1a antibodies inhibit the VDCCs currents in NGF-differentiated PC12 cells.

An analysis of the calcium-binding protein 1 of Fasciola gigantica with a comparison to its homologs in the phylum Platyhelminthes

A full-length cDNA encoding the Fasciola gigantica calcium-binding protein 1 (FgCaBP1) was cloned from an adult stage cDNA expression library in an immunoscreen using rabbit immune serum against the parasite's excretion/secretion antigens. The deduced amino acid sequence showed 96.3% identity to Fh22CBP of Fasciola hepatica. During development in the mammalian host FgCaBP1 RNA was detected in metacercariae, juveniles and adults and was exclusively localized to the tegumental cell bodies. Immune serum of a rabbit infected with F. gigantica detected recombinant FgCaBP1 starting from the sixth week of infection. Immune sera of mice infected with Schistosoma mansoni and Schistosoma mekongi cross-reacted with recombinant FgCaBP1 in immunoblots. Recombinant FgCaBP1 showed calcium and magnesium-binding activity by a mobility shift during non-denaturing PAGE in the presence of Ca 2+ or Mg 2+, respectively. A polyclonal mouse anti-rFgCaBP1 antiserum detected the native protein as a major component of the parasite's tegumental antigens in immunoblots and as a strictly tegumental antigen in tissue cross-sections of adult and juvenile parasites. Comparative sequence analysis of homologs from Fasciola and Schistosoma present in the GenBank database revealed sequence signatures specific to these trematode proteins and thereby indicates their origin from a single ancestor. FgCaBP1 contains two adjacent, N-terminal located EF-hands and a C-terminal located domain similar to dynein light chain type 1. Independent structure predictions of the two domains suggest that they will fold according to the already determined structures of the EF-hand motif and the dynein light chain type 1 proteins.

Quantitative Assessment of Protection in Experimental Syphilis

Protection in experimental rabbit syphilis has been previously assessed by lesion development following intradermal challenge with Treponema pallidum. We have recently reported that passive immunization using monoclonal antibody M131 conveys partial protection as evidenced by significant lesion delays following intradermal challenge. To determine whether such delays in time to lesion appearance corresponded to decreases in the numbers of spirochetes, we used real-time PCR to quantitate T. pallidum genomic DNA copy numbers in lesion biopsies taken throughout the course of lesion development. Three groups of animals were given one prechallenge passive immunization with immune rabbit serum (IRS), M131, or control monoclonal antibody (CMAb) and then challenged with treponemal admixtures of IRS or monoclonal antibody in normal rabbit serum (NRS). As compared to the CMAb NRS controls, delays in the mean time to lesion appearance of 5.8 days for IRS and 8.8 days for M131 were observed. At the earliest time point (10 days postchallenge), real-time PCR showed a mean T. pallidum DNA copy number per mug of rabbit DNA in the CMAb NRS group of 7.65 x 10(3) copies, while no T. pallidum DNA could be detected in the M131 group. At approximately the mean time to lesion appearance in the IRS and M131 groups (17 and 20 days, respectively), the numbers of T. pallidum DNA copies were still 5- and 30-fold less, respectively, than those in the control group at these times. By 30 days postchallenge, the T. pallidum DNA copy numbers were similar in all three groups. These findings indicate that the delays in appearance of syphilitic lesions conferred by IRS and M131 corresponded to a marked decrease in treponemal numbers during the course of lesion development.

Use of the skin protection assay in experimental syphilis to assess protective immunity against a specific Treponema pallidum surface epitope

We have recently shown that a monoclonal antibody, designated M131, that binds a surface phosphorylcholine epitope on Treponema pallidum possesses complement-dependent killing activity and confers partial protection in rabbits following passive immunization (Blanco et al., 2005, Infect. Immun. 73:3083–3095). In this study, the protective potential of M131 was further tested using the rabbit skin protection assay of Titus and Weiser. Both M131 and infection-derived immune rabbit serum resulted in significant lesion delays corresponding to at least a 90% reduction of the treponemal challenge inoculum. The skin protection assay provides a way to assess the protective potential of specific immunogens while using far less antibody than in passive immunization protocols.