Fraction Of Antibodies Research Articles

Abstract 3856: Dimerized single chain TRAIL-receptor agonists do not depend on Fc-gamma-receptor cross-linking for anti-tumor efficacy in vivo

Abstract APG350 is a TRAIL-receptor (TRAIL-R) agonist comprising two single-chain TRAIL-R binding domains (scTRAIL-RBD) that are dimerized via the Fc-part of a human IgG1-mutein thereby creating six receptor binding sites per molecule. This improved ability to form clusters on target cells composed of six TRAIL-Rs each, distinguishes APG350 from current clinical development candidates. In vitro, comparison of APG350 with recombinant APO2L/TRAIL and a TRAIL-R2 specific agonistic antibody revealed superior apoptosis induction for APG350 on primary and established human tumor cell-lines. Treatment of mice bearing Colo205 xenograft tumors with APG350, APO2L/TRAIL or an agonistic TRAIL-R2 specific antibody showed superior anti-tumor efficacy for APG350. Pronounced anti tumor efficacy was also shown on colon cancer stem cell (CSC) derived xenografts and successful APG350 re-treatment of relapsed CSC derived tumors demonstrated that tumors did not develop drug resistance. For most agonistic TRAIL-R antibodies effective apoptosis induction is achieved only upon additional cross-linking. SEC-based fractionation of a TRAIL-R2 specific agonistic antibody indicates a small amount of multimerized antibody that showed efficient apoptosis induction in vitro. However, the respective monomeric antibody showed poor apoptosis induction in vitro that could be enhanced upon cross-linking. In contrast apoptosis induction by APG350 was only marginally enhanced by cross-linking. Although monomeric agonistic TRAIL-R antibodies are poor apoptosis inducers in vitro, they show efficient apoptosis induction on xenograft tumors in vivo. A likely explanation for this difference is given by a recent publication showing that anti-tumor efficacy of an agonistic TRAIL-R2 specific antibody (Drozitumab) depends on cross-linking by Fcα-receptors (FcαR) in vivo. These data suggest that FcαR cross-linking might be a common requirement for the anti-tumor efficacy of agonistic TRAIL-R antibodies. To exclude that in vivo efficacy of APG350 depends on cross-linking by FcαRs we designed APG350-muteins with strongly reduced (APG808) or lacking FcαR binding (APG780). Side by side comparison of APG808, APG780 and APG350 in mice bearing Colo205-derived xenograft tumors showed identical anti-tumor efficacy for all respective proteins. Given that APG780 cannot bind to FcαRs these results suggest that the anti-tumor efficacy of APG808, APG780 and APG350 is solely based on the unique construction principle of the dimerized scTRAIL-RBD. APG350 induces superior clustering of TRAIL-Rs that in contrast to agonistic TRAIL-R antibodies does not require cross-linking via FcαRs for its potent anti-tumor efficacy. APG350 based dimerized scTRAIL-RBD formats may therefore have the capacity to bridge the current gap seen between preclinical and clinical efficacy for agonistic TRAIL-R specific antibodies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3856. doi:1538-7445.AM2012-3856

A proteomics approach for the identification and cloning of monoclonal antibodies from serum

We describe a proteomics approach that identifies antigen-specific antibody sequences directly from circulating polyclonal antibodies in the serum of an immunized animal. The approach involves affinity purification of antibodies with high specific activity and then analyzing digested antibody fractions by nano-flow liquid chromatography coupled to tandem mass spectrometry. High-confidence peptide spectral matches of antibody variable regions are obtained by searching a reference database created by next-generation DNA sequencing of the B-cell immunoglobulin repertoire of the immunized animal. Finally, heavy and light chain sequences are paired and expressed as recombinant monoclonal antibodies. Using this technology, we isolated monoclonal antibodies for five antigens from the sera of immunized rabbits and mice. The antigen-specific activities of the monoclonal antibodies recapitulate or surpass those of the original affinity-purified polyclonal antibodies. This technology may aid the discovery and development of vaccines and antibody therapeutics, and help us gain a deeper understanding of the humoral response.

Rapid Identification of Monospecific Monoclonal Antibodies Using a Human Proteome Microarray

To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His6 fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.

An engineered mutant of HIV-1 gp120 formulated with adjuvant Quil A promotes elicitation of antibody responses overlapping the CD4-binding site.

A major priority in HIV vaccine research is the development of an immunogen to elicit broadly neutralizing antibodies (NAbs). Monoclonal antibody (mAb) b12 is one of now several broadly neutralizing mAbs that bind epitopes overlapping the CD4-binding site (CD4bs) on HIV-1 gp120 and that serve as templates to engineer effective immunogens. We are exploring a strategy whereby extra glycans are incorporated onto gp120 to occlude the epitopes of non-neutralizing mAbs while maintaining exposure of the b12 site. Immunizing with these so-called hyperglycosylated gp120s is hypothesized to preferentially elicit b12-like NAbs. Here, the effects of two adjuvants, monophosphoryl lipid A (MPL) and Quil A, on eliciting b12-like responses when formulated with a new hyperglycosylated mutant, ΔN2mCHO(Q105N), is presented. Sera from ΔN2mCHO(Q105N)_MPL immunized animals bound the homologous antigen ΔN2mCHO(Q105N) with greater preference than sera from ΔN2mCHO(Q105N)_QuilA immunized animals, demonstrating the modulation of antibody fine specificity by these two adjuvants. We also found that sera from ΔN2mCHO(Q105N)_QuilA immunized animals bound best to a resurfaced HIV gp120 core protein on which non-CD4bs epitopes are substituted with non-HIV residues, suggesting that these sera contain a relatively larger fraction of CD4bs-specific antibodies. Consistent with these data, inhibition assays revealed epitope overlap with the binding sites of the CD4bs-specific antibodies b12, b13 and VRC03. Unexpectedly, these sera did not exhibit significant neutralizing activity against a set of HIV-1 primary strains. Our results show that although formulating mutant ΔN2mCHO(Q105N) with Quil A promotes the elicitation of CD4bs-directed antibodies relative to wild-type gp120, tweaking of the immunization regimen is needed to yield robust, CD4bs-focused NAbs.

Determination of Anti-Factor VIIII Antibody Titers, Isotypes and Epitopes by SPR Analysis of Human Plasma

Abstract 202Development of neutralizing anti-factor VIII (FVIII) allo-antibodies (“inhibitors”) occurs in a significant proportion of congenital Hemophilia A (HA) patients receiving exogenous FVIII, and autoimmune responses to FVIII can also occur. Accurate early detection of these immune responses, including low-titer or non-neutralizing antibodies, could have prognostic value and thus assist clinicians in evaluating therapeutic options. Rapid, sensitive, high-information-content surface plasmon resonance (SPR) assays have been developed to quantify the titer, IgG subtype distribution and domain/epitope specificity of anti-FVIII antibodies by direct analysis of pre-treated plasma samples. Samples were injected directly onto the biosensor of a Biacore T-100 instrument, on which recombinant FVIII was captured via a covalently immobilized anti-FVIII monoclonal antibody (mAb), and the increase in resonance units (RU) was converted to antibody titers. The lower limit of detection was 100ng/mL (∼1nM ∼0.01 Bethesda units/mL), allowing characterization of anti-FVIII antibodies that could not be detected by the Bethesda assay. Subsequent serial injections of isotype-specific anti-human mAbs yielded quantitative measurements of the fractions of anti-FVIII antibodies with specific subtypes. Plasma samples from 200 HA subjects age 2 and above (with and without a measurable inhibitor) as well as samples from 3 autoimmune adults were analyzed. Distinct IgG subtype distributions were observed, with most inhibitor subjects exhibiting mixed IgG1 + IgG4, or IgG1 + IgG2 + IgG4 responses. Serial samples from 1 subject following initial FVIII infusions showed transient class switching with the appearance of anti-FVIII-IgG3.Epitope mapping was next carried out via competition studies utilizing (a) a panel of 11 monoclonal antibodies (mAbs) whose epitopes on the FVIII-C2 domain have been mapped at high resolution and (b) recombinant FVIII-C2 proteins. The mAb-competition assays allowed the (FVIII-C2) epitope specificity of anti-FVIII antibodies in plasma to be mapped at a similar resolution, identifying specific clusters of surface-exposed FVIII residues that comprise or overlap immunodominant epitope(s) recognized by human antibodies. Complementary solution-phase competition SPR assays utilized wild type FVIII-C2 protein and selected proteins from a panel of 60 FVIII-C2 point mutants (muteins) that were identified during epitope mapping of the mAbs by SPR. This second approach alleviated several technical issues associated with the earlier blocking studies that utilized mAbs to compete the polyclonal plasma IgG (e.g. dissociation of the blocking mAb and steric effects) and provided reproducible quantitative results. Because saturating concentrations of FVIII-C2 proteins were used, changes in the number of RUs measured before and after addition of WT-FVIII-C2 indicated the fraction of polyclonal anti-FVIII antibodies that recognized this domain. Notably, the IgG subtype distribution of all FVIII-C2 specific antibodies reflected the total IgG subtype distribution, indicating that affinity maturation of B-cell epitopes preceded antibody class switching. Interestingly, saturating amounts of either FVIII-C2 or the high-affinity anti-FVIII-C2 mAb BO2C11 completely competed binding of antibodies from 1 subject (with HA genotype A2201P) to FVIII captured on the biosensor surface, indicating that his antibody response was to a single B-cell epitope or else to closely overlapping epitopes. However, although rare subjects had a completely FVIII-C2 specific response or no binding to FVIII-C2, between 30–50% of the anti-FVIII antibodies in the vast majority of inhibitor-positive samples targeted the FVIII-C2 domain. This was true for inhibitor subjects with severe HA due to the intron 22 inversion, for subjects with other HA genotypes resulting in severe HA and for the 3 autoimmune subjects.Results of these competition SPR assays are directly translatable to our laboratory's efforts to design FVIII muteins that retain adequate pro-coagulant activity but can avoid neutralization by anti-FVIII antibodies in inhibitor patients' blood. FVIII muteins having amino acid substitutions that eliminate immunodominant B-cell epitopes are expected to have potential for development as less immunogenic/antigenic bypass therapies for patients who develop a high-titer inhibitor. Disclosures:Howard:Haplomics: Equity Ownership; Baxter, Bayer, Biogen: Research Funding; Haplomics: Patents & Royalties. Pratt:Bayer, CSL Behring, Pfizer: Research Funding; Puget Sound Blood Center: Patents & Royalties.

Aspects immunologiques du syndrome des antiphospholipides

Antiphospholipid antibodies constitute a group of heterogeneous antibodies, which mainly recognize complexes made of proteins and anionic phospholipids. The nature of these complexes is currently better defined, as well as known, the structure of the antiphospholipid antibodies owing to the analysis of the monoclonal forms of these antibodies which were also studied both in terms of their precise specificities and cross-reactivity. However, the origin of these autoantibodies is not clearly understood, as well as the possible link between antiphospholipid antibodies present in healthy individuals, and those observed in autoimmune diseases. Only a fraction of antiphospholipid antibodies are pathogenic and directly responsible for the clinical manifestations of the antiphospholipid syndrome, but there is, to date, no biological test able to accurately detect pathogenic antiphospholipid antibodies. The diverse mechanisms which link these autoantibodies to the occurrence of symptoms, mainly during obstetrical complications, are better understood, and suggest new therapeutic avenues.

Consensus Substrate Sequence for Protein-tyrosine Phosphatase Receptor Type Z

Protein-tyrosine phosphatase receptor type Z (Ptprz) has multiple substrate proteins, including G protein-coupled receptor kinase-interactor 1 (Git1), membrane-associated guanylate kinase, WW and PDZ domain-containing 1 (Magi1), and GTPase-activating protein for Rho GTPase (p190RhoGAP). We have identified a dephosphorylation site at Tyr-1105 of p190RhoGAP; however, the structural determinants employed for substrate recognition of Ptprz have not been fully defined. In the present study, we revealed that Ptprz selectively dephosphorylates Git1 at Tyr-554, and Magi1 at Tyr-373 and Tyr-858 by in vitro and cell-based assays. Of note, the dephosphorylation of the Magi1 Tyr-858 site required PDZ domain-mediated interaction between Magi1 and Ptprz in the cellular context. Alignment of the primary sequences surrounding the target phosphotyrosine residue in these three substrates showed considerable similarity, suggesting a consensus motif for recognition by Ptprz. We then estimated the contribution of surrounding individual amino acid side chains to the catalytic efficiency by using fluorescent peptides based on the Git1 Tyr-554 sequence in vitro. The typical substrate motif for the catalytic domain of Ptprz was deduced to be Glu/Asp-Glu/Asp-Glu/Asp-Xaa-Ile/Val-Tyr(P)-Xaa (Xaa is not an acidic residue). Intriguingly, a G854D substitution of the Magi1 Tyr-858 site matching better to the motif sequence turned this site to be susceptible to dephosphorylation by Ptprz independent of the PDZ domain-mediated interaction in cells. Furthermore, we found by database screening that the substrate motif is present in several proteins, including paxillin at Tyr-118, its major phosphorylation site. Expectedly, we verified that Ptprz efficiently dephosphorylates paxillin at this site in cells. Our study thus provides key insights into the molecular basis for the substrate recognition of Ptprz.

Protective immunity to ricin toxin conferred by antibodies against the toxin's binding subunit (RTB)

The B subunit (RTB) of ricin toxin is a galactose-/N-acetyl galactosamine-specific lectin that promotes attachment and entry of ricin into host cells. RTB is also the archetype of the so-called R-type lectin family, whose members include haemagglutinins of botulinum neurotoxin (BoNT) progenitor toxins, as well as the binding subunits of cytolethal distending toxins. Although RTB is an appealing subunit vaccine candidate, as well as a potential target for immunotherapeutics, the degree to which RTB immunization elicits protective antibodies against ricin toxin remains unresolved. To address this issue, groups of mice were immunized with RTB and then challenged with 5×LD(50)s of ricin administered intraperitoneally. Despite high RTB-specific serum antibody titers, groups of RTB immunized mice were only partially immune to ricin challenge. Analysis of a collection of RTB-specific B cell hybridomas suggested that only a small fraction of antibodies against RTB have demonstrable neutralizing activity. Two RTB-specific neutralizing monoclonal IgG(1) antibodies, 24B11 and SylH3, when passively administered to mice, were sufficient to protect the animals against a 5×LD(50) dose of ricin. Both 24B11 and SylH3 blocked ricin attachment to terminal galactose residues and prevented toxin binding to the surfaces of bone marrow-derived macrophages (BMM), suggesting that they function by steric hindrance and recognize epitopes located on RTB's carbohydrate recognition sub-domains (1α or 2γ). These data raise the possibility of using specific RTB sub-domains, rather than RTB itself, as antigens to more efficiently elicit neutralizing antibodies and protective immunity against ricin.

Use of affinity‐directed liquid chromatography‐mass spectrometry to map the epitopes of a factor VIII inhibitor antibody fraction

Neutralizing factor (F) VIII antibodies develop in approximately 30% of individuals with hemophilia A and show specificity to multiple sites in the FVIII protein. Reactive epitopes to an immobilized IgG fraction prepared from a high-titer, FVIII inhibitor plasma were determined after immuno-precipitation (IP) of tryptic and chymotryptic peptides derived from digests of the A1 and A2 subunits of FVIIIa and FVIII light chain. Peptides were detected and identified using highly sensitive liquid chromatography-mass spectrometry (LC-MS). Coverage maps of the A1 subunit, A2 subunit and light chain represented 79%, 69% and 90%, respectively, of the protein sequences. Dot blots indicated that the inhibitor IgG reacted with epitopes contained within each subunit of FVIIIa. IP coupled with LC-MS identified 19 peptides representing epitopes from all FVIII A and C domains. The majority of peptides (10) were derived from the A2 domain. Three peptides mapped to the C2 domain, while two mapped to the A1 and A3 domains, and single peptides mapped to the a1 segment and C1 domain. Epitopes were typically defined by peptide sequences of < 12 residues. IP coupled with LC-MS identified extensive antibody reactivity at high resolution over the entire functional FVIII molecule and yielded sequence lengths of < 15 residues. A number of the peptides identified mapped to known sequences involved in functionally important protein-protein and protein-membrane interactions.

Myelin Basic Protein as a Novel Genetic Risk Factor in Rheumatoid Arthritis—A Genome-Wide Study Combined with Immunological Analyses

Rheumatoid arthritis (RA) is a major cause of adult chronic inflammatory arthritis and a typical complex trait. Although several genetic determinants have been identified, they account for only a part of the genetic susceptibility. We conducted a genome-wide association study of RA in Japanese using 225,079 SNPs genotyped in 990 cases and 1,236 controls from two independent collections (658 cases and 934 controls in collection1; 332 cases and 302 controls in collection2), followed by replication studies in two additional collections (874 cases and 855 controls in collection3; 1,264 cases and 948 controls in collection4). SNPs showing p<0.005 in the first two collections and p<10−4 by meta-analysis were further genotyped in the latter two collections. A novel risk variant, rs2000811, in intron2 of the myelin basic protein (MBP) at chromosome 18q23 showed strong association with RA (p = 2.7×10−8, OR 1.23, 95% CI: 1.14–1.32). The transcription of MBP was significantly elevated with the risk allele compared to the alternative allele (p<0.001). We also established by immunohistochemistry that MBP was expressed in the synovial lining layer of RA patients, the main target of inflammation in the disease. Circulating autoantibody against MBP derived from human brain was quantified by ELISA between patients with RA, other connective tissue diseases and healthy controls. As a result, the titer of anti-MBP antibody was markedly higher in plasma of RA patients compared to healthy controls (p<0.001) and patients with other connective tissue disorders (p<0.001). ELISA experiment using citrullinated recombinant MBP revealed that a large fraction of anti-MBP antibody in RA patients recognized citrullinated MBP. This is the first report of a genetic study in RA implicating MBP as a potential autoantigen and its involvement in pathogenesis of the disease.

Intrathecal EBV antibodies are part of the polyspecific immune response in multiple sclerosis

One mechanism underlying the link between multiple sclerosis (MS) and Epstein-Barr virus (EBV) might be a direct CNS infection. Viral CNS infections cause elevated antibody indices (AIs). Elevated EBV AIs were found in MS; however, patients with MS frequently show a polyspecific intrathecal immune response with elevated antiviral AIs. To discriminate whether elevated EBV AIs indicate a virus-driven or a polyspecific intrathecal immune response, we determined the intrathecal fraction of anti-EBV antibodies. The fraction of intrathecally synthesized EBV-specific immunoglobulin G (IgG) of the total intrathecally synthesized IgG (F(S) anti-EBV) was determined in 24 patients with a clinically isolated syndrome (CIS) or MS and 3 patients with cerebral posttransplantation lymphoproliferative disorder (PTLD), all of whom had elevated EBV AIs. F(S) anti-measles and AIs for measles, rubella, varicella zoster, and herpes simplex virus were measured as well. The prevalence of an elevated EBV AI was analyzed in another 36 patients with CIS. Median F(S) anti-EBV in patients with CIS/MS was low (0.65%) and did not differ from F(S) anti-measles (0.9%). Median F(S) anti-EBV was about 40-fold higher in patients with cerebral PTLD than in patients with CIS/MS. All 24 patients with CIS/MS with an elevated EBV AI had at least one further elevated antiviral AI. Only 2 of 36 (5.6%) patients with CIS showed an intrathecal synthesis of anti-EBV antibodies. Intrathecally produced anti-EBV antibodies are part of the polyspecific intrathecal immune response in CIS/MS and only rarely detectable in patients with CIS, both arguing against a direct CNS infection with EBV in patients with CIS/MS.

Validation of an Analytical Method for Assay of Immunoreactivity of 99mTechnetium‐radiolabelled Monoclonal Antibodies

A validation is described of the popular Lindmo assay which is widely used for the determination of immunoreactive fraction of radiolabeled monoclonal antibodies used in tumour targeting. 99mTechnetium-labelled PR1A3 anti-carcinoembryonic antigen (CEA) antibody was incubated with tumour cells bearing the appropriate CEA epitope over a range of concentrations and using optimized assay conditions. Cell-bound radioactivity was measured following centrifugation and the immunoreactive fraction determined by extrapolation of a Burke-Lineweaver plot to the y-axis. The specificity, repeatability, reproducibility, accuracy, sensitivity and linearity of the assay were explored and found to lie within acceptable limits.

Phosphorylation Regulates OLIG2 Cofactor Choice and the Motor Neuron-Oligodendrocyte Fate Switch

SummaryA fundamental feature of central nervous system development is that neurons are generated before glia. In the embryonic spinal cord, for example, a group of neuroepithelial stem cells (NSCs) generates motor neurons (MNs), before switching abruptly to oligodendrocyte precursors (OLPs). We asked how transcription factor OLIG2 participates in this MN-OLP fate switch. We found that Serine 147 in the helix-loop-helix domain of OLIG2 was phosphorylated during MN production and dephosphorylated at the onset of OLP genesis. Mutating Serine 147 to Alanine (S147A) abolished MN production without preventing OLP production in transgenic mice, chicks, or cultured P19 cells. We conclude that S147 phosphorylation, possibly by protein kinase A, is required for MN but not OLP genesis and propose that dephosphorylation triggers the MN-OLP switch. Wild-type OLIG2 forms stable homodimers, whereas mutant (unphosphorylated) OLIG2S147A prefers to form heterodimers with Neurogenin 2 or other bHLH partners, suggesting a molecular basis for the switch.

Hepatitis C patient-derived glycoproteins exhibit marked differences in susceptibility to serum neutralizing antibodies: genetic subtype defines antigenic but not neutralization serotype.

Neutralizing antibodies have a role in controlling hepatitis C virus (HCV) infection. A successful vaccine will need to elicit potently neutralizing antibodies that are capable of preventing the infection of genetically diverse viral isolates. However, the specificity of the neutralizing antibody response in natural HCV infection still is poorly understood. To address this, we examined the reactivity of polyclonal antibodies isolated from chronic HCV infection to the diverse patient-isolated HCV envelope glycoproteins E1 and E2 (E1E2), and we also examined the potential to neutralize the entry of pseudoparticles bearing these diverse E1E2 proteins. The genetic type of the infection was found to determine the pattern of the antibody recognition of these E1E2 proteins, with the greatest reactivity to homologous E1E2 proteins. This relationship was strongest when the component of the antibody response directed only to linear epitopes was analyzed. In contrast, the neutralization serotype did not correlate with genotype. Instead, serum-derived antibodies displayed a range of neutralization breadth and potency, while different E1E2 glycoproteins displayed different sensitivities to neutralization, such that these could be divided broadly into neutralization-sensitive and -resistant phenotypes. An important additional observation was that entry mediated by some E1E2 proteins was enhanced in the presence of some of the polyclonal antibody fractions isolated during chronic infection. These data highlight the need to use diverse E1E2 isolates, which represent extremes of neutralization sensitivity, when screening antibodies for therapeutic potential and for testing antibodies generated following immunization as part of vaccine development.

Immunization with different Pf AMA1 alleles in sequence induces clonal imprint humoral responses that are similar to responses induced by the same alleles as a vaccine cocktail in rabbits

BackgroundAntibodies to key Plasmodium falciparum surface antigens have been shown to be important effectors that mediate clinical immunity to malaria. The cross-strain fraction of anti-malarial antibodies may however be required to achievestrain-transcending immunity. Such antibody responses against Plasmodium falciparum apical membrane antigen 1 (PfAMA1), a vaccine target molecule that is expressed in both liver and blood stages of the parasite, can be elicited through immunization with a mixture of allelic variants of the parasite molecule. Cross-strain antibodies are most likely elicited against epitopes that are shared by the allelic antigens in the vaccine cocktail.MethodsA standard competition ELISA was used to address whether the antibody response can be further focused on shared epitopes by exclusively boosting these common determinants through immunization of rabbits with different PfAMA1 alleles in sequence. The in vitro parasite growth inhibition assay was used to further evaluate the functional effects of the broadened antibody response that is characteristic of multi-allele vaccine strategies.ResultsA mixed antigen immunization protocol elicited humoral responses that were functionally similar to those elicited by a sequential immunization protocol (p > 0.05). Sequential exposure to the different PfAMA1 allelic variants induced immunological recall of responses to previous alleles and yielded functional cross-strain antibodies that would be capable of optimal growth inhibition of variant parasites at high enough concentrations.ConclusionsThese findings may have implications for the current understanding of the natural acquisition of clinical immunity to malaria as well as for rational vaccine design.

Purification of Monoclonal Antibodies from Cell-Culture Supernatant by Use of Anion-Exchange Convective Interaction Media (CIM) Monolithic Columns

Purified monoclonal antibodies (mAb) have been used in therapeutics and some analytical procedures. Purification of mAb by use of high-throughput anion-exchange methacrylate monolithic systems has been attempted in this work. Monolithic macroporous convective interaction media (CIM) with diethylaminoethyl (DEAE) and ethylene diamine (EDA) as anion-exchange ligands were used and evaluated for purification of anti-glycophorin-A IgG1 mouse mAbs from cell culture supernatant (CCS) after precipitation with 50% ammonium sulfate. The adsorption and elution of mAb from the CCS on CIM-DEAE and CIM-EDA disks were studied with three different buffer systems, acetate, MOPS (3-(N-morpholino)propanesulfonic acid), and Tris, to study the effect of the nature of buffer ions and to find the optimum buffer conditions for purification of mAb. The optimum buffers for purification of mAb using CIM-DEAE and CIM-EDA were 50 mM acetate buffer, pH 5.1 and 20 mM Tris buffer, pH 8.0, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA) showed the antibody fractions obtained were highly pure, with high antigen-binding efficiency. High specific activity with purification factors of 130 ± 34 (unretained fraction with acetate buffer) and 74 ± 13 (fraction eluted with Tris buffer containing 0.6 M NaCl) was obtained for IgG1 using the CIM-DEAE and CIM-EDA disks, respectively. The results indicate that rapid separation and efficient recovery of high-purity anti-glycophorin-A mAbs could be achieved by use of anion-exchange CIM disks.

Tandem Affinity Depletion: A Combination of Affinity Fractionation and Immunoaffinity Depletion Allows the Detection of Low-Abundance Components in the Complex Proteomes of Body Fluids

Protein biomarker discovery in the low concentration range of human body fluids requires the enrichment of the proteins of interest. Here we report on a tandem affinity strategy: In the first step, we isolated a human plasma glyco-subproteome of healthy individuals by wheat germ agglutinin (WGA) lectin affinity chromatography. In the second step, the proteins of this subproteome were used to raise antibodies in llama (Lama glama). The heavy-chain fraction of the llama antibodies was used to deplete from the WGA lectin binding fraction all proteins normally found in human plasma. In this way, we selectively enriched the glycoprotein, CEA, a known cancer marker which had been spiked into normal plasma. As a proof of concept, we applied this method to the analysis of plasma sample from colon cancer patients. We could demonstrate the selective enrichment of CEA by a factor of 600-800.

Characterization of Anti- Salmonella enterica Serotype Typhi Antibody Responses in Bacteremic Bangladeshi Patients by an Immunoaffinity Proteomics-Based Technology

Salmonella enterica serotype Typhi is the cause of typhoid fever and a human-restricted pathogen. Currently available typhoid vaccines provide 50 to 90% protection for 2 to 5 years, and available practical diagnostic assays to identify individuals with typhoid fever lack sensitivity and/or specificity. Identifying immunogenic S. Typhi antigens expressed during human infection could lead to improved diagnostic assays and vaccines. Here we describe a platform immunoaffinity proteomics-based technology (IPT) that involves the use of columns charged with IgG, IgM, or IgA antibody fractions recovered from humans bacteremic with S. Typhi to capture S. Typhi proteins that were subsequently identified by mass spectrometry. This screening tool identifies immunogenic proteins recognized by antibodies from infected hosts. Using this technology and the plasma of patients with S. Typhi bacteremia in Bangladesh, we identified 57 proteins of S. Typhi, including proteins known to be immunogenic (PagC, HlyE, OmpA, and GroEL) and a number of proteins present in the human-restricted serotypes S. Typhi and S. Paratyphi A but rarely found in broader-host-range Salmonella spp. (HlyE, CdtB, PltA, and STY1364). We categorized identified proteins into a number of major groupings, including those involved in energy metabolism, protein synthesis, iron homeostasis, and biosynthetic and metabolic functions and those predicted to localize to the outer membrane. We assessed systemic and mucosal anti-HlyE responses in S. Typhi-infected patients and detected anti-HlyE responses at the time of clinical presentation in patients but not in controls. These findings could assist in the development of improved diagnostic assays.

Anti‐idiotypic antibody mimicking a T‐antigen‐specific lectin inhibits human epithelial tumor cell proliferation

Cancer-associated mucins show frequent alterations of oligosaccharide chain profile. Terminal structures may be deleted, thereby exposing normally 'cryptic' structures such as Tn (GalNAcα-O-Ser/Thr) and T antigen (Galβ1-3GalNAcα-O-Ser/Thr). Overexpression of these commonly hidden glycoforms, and reduced level of naturally occurring anti-T or anti-Tn antibodies, is associated with epithelial tumor progression and aggressiveness. The lectin from the common edible mushroom Agaricus bisporus (ABL) shows high affinity binding to T antigen, and reversible noncytotoxic inhibitory effect on epithelial tumor cell proliferation. The aim of this study was to induce immune response with tumor-associated glycan specificity and biological activity similar to those of ABL. An anti-idiotypic (Id) antibody strategy was developed using ABL as first template. ABL was purified by affinity chromatography and assayed as immunogen in rabbit. Rabbit IgG was purified from anti-ABL serum using a protein G column, and specific anti-ABL IgG was obtained by affinity chromatography using immobilized ABL. Affinity-purified anti-ABL IgG contained an antibody fraction that recognizes the carbohydrate-binding site of ABL. This IgG was used as immunogen in mouse to yield anti-Id antibody recognizing tumor-associated glycans such as Tn and T antigen. Competitive assays showed that α-anomeric GalNAc is the main binding subsite of anti-Id antibody in glycan recognition. Anti-Id antibody bound human epithelial tumor cells, as shown by cell enzyme-linked immunosorbent assay and immunofluorescence. Anti-Id antibody raised by immunization with affinity-purified anti-ABL IgG had antiproliferative effect on human epithelial tumor cells through apoptosis induction similar to that of ABL. The anti-Id immune response developed here has potential application in cancer therapy.

Polyclonal antibodies against kallikrein-related peptidase 4 (KLK4): immunohistochemical assessment of KLK4 expression in healthy tissues and prostate cancer

KLK4 is a member of the human kallikrein-related peptidase family of (chymo)trypsin-like serine proteases. The aim of the present study was to generate polyclonal antibodies (pAb) directed against KLK4 for the analysis of KLK4 by immunohistochemistry in human tissues. Recombinantly expressed human mature KLK4 was used for immunization of chickens. pAb 617A is an affinity-purified monospecific pAb fraction reacting with a linear epitope within a flexible surface-exposed loop of KLK4. pAb 617C is the KLK-directed pAb fraction completely depleted from pAb 617A. In healthy adult tissues, KLK4 was immunodetected by both antibody fractions in kidney, liver, and prostate, but not in other organs such as colon and lung. To evaluate protein expression of KLK4 in prostate cancer, samples of tumor tissue plus corresponding tumor-free areas of 44 prostate cancer patients, represented on a tissue microarray, were investigated. Distinct KLK4 immunostaining was observed with both antibodies in cancerous glandular epithelial cells, but not in surrounding stromal cells. KLK4 expression was lower in stage pT3+4 than in pT1+2 tumors, which was highly significant when employing pAb 617A. Thus, our results indicate that KLK4, which is expressed in the healthy prostate, is upregulated in early-stage but not late-stage prostate cancer.