Human Immunoglobulin G Antibodies Research Articles

Characterisation of two snake toxin-targeting human monoclonal immunoglobulin G antibodies expressed in tobacco plants

Current snakebite antivenoms are based on polyclonal animal-derived antibodies, which can neutralize snake venom toxins in envenomed victims, but which are also associated with adverse reactions. Therefore, several efforts within antivenom research aim to explore the utility of recombinant monoclonal antibodies, such as human immunoglobulin G (IgG) antibodies, which are routinely used in the clinic for other indications. In this study, the feasibility of using tobacco plants as bioreactors for expressing full-length human monoclonal IgG antibodies against snake toxins was investigated. We show that the plant-produced antibodies perform similarly to their mammalian cell-expressed equivalents in terms of in vitro antigen binding. Complete neutralization was achieved by both the plant and mammalian cell-produced anti-α-cobratoxin antibody. The feasibility of using plant-based expression systems may potentially make it easier for laboratories in resource-poor settings to work with human monoclonal IgG antibodies.

Selective and high-capacity binding of immunoglobulin G to zirconia nanoparticles modified with phosphate groups

High-performance and cost-effective purification is necessary for the development of antibody drugs. This study found that nanoparticles of zirconia modified with phosphate groups selectively adsorb immunoglobulin G (IgG) antibodies against serum proteins with high adsorption capacity. The IgG antibodies collected from the zirconia nanoparticle surfaces retain their molecular conformation. Importantly, zirconia nanoparticles have the highest affinity for human IgG antibodies among tested mammalian IgG antibodies. The affinity for human IgG subclasses is in the order IgG3 > IgG1 > IgG2, which contrasts with a conventional ligand (Protein A) that has a lower affinity for IgG3. Because zirconia nanoparticles are chemically and mechanically stable, they can be utilized for the purification of antibody drugs not only in batch methods but also in chromatography as a process upstream or downstream of Protein A chromatography and even as an alternative process.

Seroprevalences of antibodies against pertussis, diphtheria, tetanus, measles, mumps and rubella: A cross-sectional study in children following vaccination procedure in Guangzhou, China

This study investigated the concentrations and seroprevalence of immunoglobulin G (IgG) antibodies against pertussis, diphtheria, tetanus, measles, mumps and rubella among children in Guangzhou, China. We conducted a cross-sectional study focusing on the post-vaccination immune statuses of children on scheduled immunisation. Human IgG antibody against six diseases were measured using commercial enzyme-linked immunosorbent assay kits. Of 620 subjects, the male-to-female ratio was 2.04 (416/204). Seroprevalence (81.97% vs 90.20%) and IgG concentrations (686.55 IU/mL vs 884.26 IU/mL, P < 0.05) for measles, tetanus (0.94 IU/mL vs 1.21 IU/mL) and rubella (34.33 IU/mL vs 47.37 IU/mL) were all higher in females. No differences based on sex were observed in the seroprevalence and IgG concentrations for anti-pertussis antibodies, anti-diphtheria antibodies and anti-mumps. Slight increase in seroprevalence and IgG concentration occurred with anti-pertussis antibodies after primary and booster vaccinations (from 0.00% [1 m], 5.45% [6 m], to 17.14% [1.5 yr]; and from 8.57% [5 yr] to 15.79% [6 yr]). Although no booster vaccination was given after age 6 yr, the seroprevalence and IgG concentration for anti-pertussis antibodies remained relatively stable. For diphtheria, tetanus, measles and rubella, seroprevalence reached their peaks after the primary and first booster vaccination. A plateau occurred after age 1.5 yr with a declining trend in subjects >8–10 yr. The IgG concentrations of these 4 pathogens showed a dramatic increase after primary vaccination, with steadily declining trends thereafter. For mumps, subjects showed increased seroprevalence and IgG concentration after the primary mumps-containing vaccination in 1.5-yr-olds (from 7.14% to 57.14%; 52.13 IU/mL to 214.18 IU/mL); however, following that low seroprevalence levels (from 42.86% to 80.00%) were observed. The post-vaccination immune statuses against diphtheria, tetanus, measles and rubella were relatively satisfactory, compared to those against pertussis and mumps. Booster vaccination against pertussis and mumps at appropriate time should be considered.

The enzymatic removal of immunoglobulin variable domain glycans by different glycosidases

About 15% of immunoglobulin G (IgG) molecules contain glycans linked to the antigen-binding fragments (Fab arms) in addition to the glycans linked to the crystallizable fragment (Fc tail) of all IgGs. Fab glycosylation appears to be an important feature of antibodies, for example by influencing antigen binding and antibody stability. The reliable generation of antibodies that either have or lack Fab glycans would be very helpful to study the role of Fab glycans in more detail. In this study, we set out to remove Fab glycans by treating polyclonal and monoclonal human IgG antibodies with two commonly used glycosidases and an improved version of one of the two (Endo F3, PNGase F, and Rapid™ PNGase F). Fc glycans can be removed using PNGase F and Rapid™ PNGase F, but not with Endo F3. For most antibody clones, Endo F3 partially cleaved off the Fab glycans. In contrast, PNGase F left the Fab glycans of most clones unaffected, but could remove glycans of some clones. Rapid™ PNGase F showed a higher glycosidase efficacy than PNGase F, and more clones could be deglycosylated using this enzyme. In summary, not all Fab glycans can be cleaved off by the tested glycosidases (under non-denaturing conditions), suggesting that Fab glycans are exposed to different degrees.

In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies

The black mamba (Dendroaspis polylepis) is one of the most feared snake species of the African savanna. It has a potent, fast-acting neurotoxic venom comprised of dendrotoxins and α-neurotoxins associated with high fatality in untreated victims. Current antivenoms are both scarce on the African continent and present a number of drawbacks as they are derived from the plasma of hyper-immunized large mammals. Here, we describe the development of an experimental recombinant antivenom by a combined toxicovenomics and phage display approach. The recombinant antivenom is based on a cocktail of fully human immunoglobulin G (IgG) monoclonal antibodies capable of neutralizing dendrotoxin-mediated neurotoxicity of black mamba whole venom in a rodent model. Our results show the potential use of fully human monoclonal IgGs against animal toxins and the first use of oligoclonal human IgG mixtures against experimental snakebite envenoming.

Patterns of Interindividual Variability in the Antibody Repertoire Targeting Proteins Across the Epstein-Barr Virus Proteome.

Little is known about variation in antibody responses targeting the full spectrum of Epstein-Barr virus (EBV) proteins and how such patterns inform disease risk. We used a microarray to measure immunoglobulin G (IgG) and immunoglobulin A (IgA) antibody responses against 199 EBV protein sequences from 5 EBV strains recovered from 289 healthy adults from Taiwan. We described positivity patterns, estimated the correlation between antibodies, and investigated the associations between environmental and genetic risk factors and variations in antibody responses. Healthy adults were more likely to mount IgG antibody responses to EBV proteins (median positivity frequency, 46.5% for IgG and 17.3% for IgA; P = 1.6 × 10-46, by the Wilcoxon rank sum test). Responses against glycoproteins were particularly prevalent. The correlations between antibody responses of the same class were higher than correlations across classes. The mucosal exposure to proteins involved in EBV reactivation (as determined by the IgA response) was associated with smoking (P = .002, by the sequence kernel association test-combined), and approximately one quarter of adults displayed antibody responses associated with EBV-related cancer risk. These data comprehensively define the variability in human IgG and IgA antibody responses to the EBV proteome. Patterns observed can serve as the foundation for elucidating which individuals are at highest risk of EBV-associated clinical conditions and for identifying targets for effective immunodiagnostic tests.

Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability.

Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion-mobility mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins.

A label-free voltammetric immunoassay based on 3D-structured rGO–MWCNT–Pd for detection of human immunoglobulin G

In this work, a 3D-structured reduced graphene oxide–multiwalled carbon nanotube–palladium nanoparticles (NPs) nanocomposite (rGO–MWCNT–Pd) was prepared by a facile and green way and used as substrate material for the development of a label-free electrochemical immunoassay for human immunoglobulin G (HIgG) detection. The network structure of rGO–MWCNT nanocomposite provides more active sites for Pd nucleation. And the synergistic effect of rGO, MWCNTs and Pd NPs significantly improved the electrochemical performance of the modified electrode. Pd particles can combine HIgG antibodies and enhance the conductivity of electrode. The structure of rGO–MWCNT–Pd nanocomposite was characterized by scanning electron microscopy (SEM) and high resolution transmission electron microscopy (HRTEM). The HRTEM of Pd NPs and energy dispersive X-ray spectroscopy (EDS) of rGO–MWCNT–Pd nanocomposite confirmed the composition of the as-synthesized nanocomposite. K3[Fe(CN)6] was used as a redox probe. HIgG antibodies are immobilized onto the nanocomposite via PdNH2 bonds. Cyclic voltammetry and square wave voltammetry were used to characterize the recognizability of HIgG. Under optimum experimental conditions, the proposed immunoassay exhibited a low detection limit (3.3pg/mL), a wide linear range (from 0.01 to 25ng/mL) as well as good stability, reproducibility and selectivity.

Questioning the added value of Luminex single antigen beads to detect C1q binding donor HLA-specific antibodies.

Luminex single antigen bead (SAB) technology enables highly sensitive and rapid characterization of immunoglobulin G (IgG) human leukocyte antigen (HLA)-specific antibodies. It is widely used to determine antibody compatibility for renal transplantation and to aid the diagnosis of antibody-mediated rejection and response to therapy. HLA-specific antibodies may contribute to allograft rejection through a variety of mechanisms, including activation of the classical complement pathway, endothelial cell activation, and recruitment of Fc-dependent effector cells. Of these, complement-mediated cytotoxicity has long been associated with hyperacute rejection and, more recently, antibody-mediated rejection for which the deposition of the complement component C4d on peritubular capillaries is a diagnostic marker. In the standard SAB assay, patient serum is incubated with microbeads coated with purified HLA proteins. Human leukocyte antigenYspecific IgG antibody binding is then detected using a fluorescent anti-IgG detection antibody. Increasing levels of donor HLA-specific antibodies detected by this assay predict inferior long-term graft outcome, but not all patients with IgG donor-specific HLA antibodies (DSA) have a poor graft outcome (1, 2). This observation is consistent with the notion that HLA antibodies with the same specificity may differ in their ability to cause graft injury, and this variability may be related to differences in complement fixing activity. In an effort to improve the clinical utility of the IgG

The effect of sugar removal on the structure of the Fc region of an IgG antibody as observed with single molecule Förster Resonance Energy Transfer

The deglycosylation of immunoglobulin G (IgG) antibodies leads to a diminished immune response. This reduction in immune response is thought to arise from weakened binding of IgG antibodies to effector molecules as a result of a conformational change in the antibody. The nature of this structural alteration is uncertain due to the conflicting results obtained from different experimental methods. We have examined the impact of deglycosylation by the endoglycosidase PNGase F on the structure of the Fc region of a human IgG antibody using single molecule Förster Resonance Energy Transfer (FRET). The FRET efficiency histograms obtained indicate that the structure of the Fc region becomes more flexible upon deglycosylation. This is demonstrated by a change in the width of the energy transfer efficiency peak, which increases from 0.19±0.02 to 0.6±0.1 upon deglycosylation.

Evaluation of the Human IgG Antibody Response to Aedes albopictus Saliva as a New Specific Biomarker of Exposure to Vector Bites

BackgroundThe spread of Aedes albopictus, a vector for re-emergent arbovirus diseases like chikungunya and dengue, points up the need for better control strategies and new tools to evaluate transmission risk. Human antibody (Ab) responses to mosquito salivary proteins could represent a reliable biomarker for evaluating human-vector contact and the efficacy of control programs.Methodology/Principal FindingsWe used ELISA tests to evaluate specific immunoglobulin G (IgG) responses to salivary gland extracts (SGE) in adults exposed to Aedes albopictus in Reunion Island. The percentage of immune responders (88%) and levels of anti-SGE IgG Abs were high in exposed individuals. At an individual level, our results indicate heterogeneity of the exposure to Aedes albopictus bites. In addition, low-level immune cross-reactivity between Aedes albopictus and Aedes aegypti SGEs was observed, mainly in the highest responders.Conclusion/SignificanceAb responses to saliva could be used as an immuno-epidemiological tool for evaluating exposure to Aedes albopictus bites. Combined with entomological and epidemiological methods, a “salivary” biomarker of exposure to Aedes albopictus could enhance surveillance of its spread and the risk of arbovirus transmission, and could be used as a direct tool for the evaluation of Aedes albopictus control strategies.

Can IgE-mediated allergic diseases be prevented by using allergen-specific IgG antibodies?

The prevalence of allergic disease has increased significantly over the past few decades, especially in children. Prenatal period may represent an ideal time for the implementation of interventions to prevent allergy and allergic diseases. In this hypothesis, allergen-specific immunoglobulin G (IgG) antibodies may combine with maternal allergens as blocking antibodies with immunoglobulin E (IgE) or mask antigenic determinants in their binding to prevent the sensitization of the fetus. It may also modify fetal immune response to prevent the production of allergen-specific IgE antibodies and the further development of allergic diseases. Recent epidemiological, immunological and intervention studies supporting the hypothesis were summarized. Anti-mite human IgG Fabs (antigen binding fragments) were prepared from two immunoglobulin genes libraries and they were confirmed to have a specific binding blocking property by an in vitro mastocyte degranulation inhibition test. Allergen-specific human IgG antibodies may have a potential use in the prenatal immunoprophylaxis or the prevention of allergic diseases after birth.

Detection of human cytomegalovirus IgG antibody using automated immunoassay with recombinant antigens gives uniform results to established assays using whole virus antigens

Serological testing for human cytomegalovirus (CMV) immunoglobulin G (IgG) continues to play a major role in the evaluation of tissue and organ transplant donors and recipients, screening blood donors and in estimating the risk of infection in pregnant women. In this study we evaluated the performance and technical characteristics of two semi-automated commercial CMV IgG immunoassays. One hundred consecutive sera submitted to a referral serology laboratory for CMV IgG antibody testing were assayed using two automated CMV IgG immunoassays: the Architect (Abbott Diagnostics) and Elecsys (Roche Diagnostics) immunoassays. Because this patient population has a high rate (∼87%) of seropostivity, we took a further 50 sera that were CMV IgG negative on Architect and tested on the Elecsys assay, in order to assess the specificity of the assays. The assays showed an excellent qualitative agreement with two (2%) discrepant results in the 100 patients tested initially, with all of the 50 additional sera negative on Architect also negative on the Elecsys. However, there was no correlation between the optical density of results generated by the instruments for the set of samples. Both Elecsys and Architect immunoassays are convenient for detection of CMV IgG antibody, with near identical qualitative results.

Label-free amperometric immunobiosensor based on a gold colloid and Prussian blue nanocomposite film modified carbon ionic liquid electrode

A novel experimental methodology based on a Prussian blue (PB) and gold nanoparticles (AuNPs) modified carbon ionic liquid electrode (CILE) was developed for use in a label-free amperometric immunosensor for the sensitive detection of human immunoglobulin G (HIgG) as a model protein. The CILE was fabricated by using the ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate as binder. Controllable electrodeposition of PB on the surface of the CILE and coating with 3-aminopropyl triethylene silane (APS) formed a film with high electronic catalytic activity and large surface area for the assembly of AuNPs and further immobilization of HIgG antibody. The electrochemistry of the formed nanocomposite biofilm was investigated by electrochemical techniques including cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy. The HIgG concentration was measured through the decrease of amperometric responses in the corresponding specific binding of antigen and antibody. The decreased differential pulse voltammetric values were proportional to the HIgG concentration in two ranges, 0.05-1.25 ng mL(-1) and 1.25-40 ng mL(-1), with a detection limit of 0.001 ng mL(-1) (S/N = 3). This electrochemical immunoassay combined the specificity of the immunological reaction with the sensitivity of the AuNPs, ionic liquid, and PB amplified electrochemical detection and would therefore be valuable for clinical immunoassays.

Integrated microfluidic magnetic immunosensor for quantification of human serum IgG antibodies to Helicobacter pylori

In this paper, we have developed and characterized a microfluidic magnetic immunosensor coupled to a gold electrode for the rapid and sensitive quantification of human serum IgG antibodies to Helicobacter pylori. This microorganism cause peptic ulcers and chronic gastritis, affecting around the 10% of the world population. The sensor was completely automated and the antibodies detection in serum samples was carried out using a non-competitive immunoassay based on the use of purified H. pylori antigens that are immobilized on magnetic microspheres 3-aminopropyl-modified. The magnetic microbeads were injected into microchannel devices and manipulated for an external removable magnet. The IgG antibodies in human serum sample are allowed to react immunologically with the immobilized antigens, and the bounded antibodies are quantified by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. The p-aminophenyl phosphate ( p-APP) was converted to p-aminophenol ( p-AP) by AP and an electroactive product was detected on gold layer electrode at 0.250 V. The response current obtained from the product of enzymatic reaction is directly proportional to the activity of the enzyme and, consequently, to the amount of IgG antibodies to H. pylori in serum samples. The electrochemical detection can be done within 1 min and total assay time was 25 min. The calculated detection limits for electrochemical detection and the ELISA procedure were 0.37 and 2.1 U mL −1, respectively, and the within- and between-assay coefficients of variation were below 5%. Our results indicate the potential usefulness of our fabricated microbiochip for the early assessment of human serum immunoglobulin G (IgG) antibodies to H. pylori.

Serologic Assay To Quantify Human Immunoglobulin G Antibodies to the Staphylococcus aureus Iron Surface Determinant B Antigen

A direct binding Luminex assay has been developed and validated for the detection of human immunoglobulin G (IgG) antibodies to the Staphylococcus aureus iron surface determinant B protein (IsdB) in serum following natural infection or immunization with investigational Saccharomyces cerevisiae-derived IsdB-based vaccines. To ensure that IsdB-specific IgG antibodies are measured following immunization with S. cerevisiae-derived IsdB, an Escherichia coli-produced IsdB antigen is used in the assay. The IsdB antigen is covalently conjugated to maleimide microspheres via an engineered carboxy-terminal cysteine residue. Antibody titers are determined in a direct binding format, where the phycoerythrin-labeled monoclonal antibody (HP6043) specific for IgG1 to IgG4 binds to human serum IgG antibodies. Fluorescent signal emitted from bound HP6043 is directly proportional to an individual's antibody levels. A pooled human reference serum from vaccinees with high titers to IsdB is used to generate a 12-point standard curve. The correlation of mean fluorescent intensity (MFI) units to microg/ml of IsdB-specific IgG is made by interpolating the MFI data through a four-parameter curve-fitting algorithm. The assay is sensitive to 1.06 microg/ml with a dynamic range of 2.1 to 10,625 microg/ml. The overall specificity of the assay is >96% and the linearity (parallelism) of the assay is -4% per 10-fold dilution. The total precision of the assay was 16.6% relative standard deviation across three different IsdB antigen lots, three different microsphere lots, two secondary antibody lots, and three different operators. The assay has proven useful for evaluating the immune response following the administration of different dosages and formulations of investigational IsdB-based vaccines.

Detection of Human Immunoglobulin G at Physiological Conditions with Chemically Functionalizated Carbon Nanotube Field Effect Transistors

In this paper we report a label-free biosensor able to detect 10 mg/L of human immunoglobulin G (HIgG) at physiological conditions. It is based on a field effect transistor in which a network of carbon nanotubes (CNTs) acts as the conductor channel. HIgG antibodies are linked to the CNTs in three steps. First, the polymer polyethyleneimine (PEI) covers the CNTssurface preventing the nonspecific binding of proteins. Second, the HIgG antibodies are linked to the CNTs using glutaraldehyde as a cross-linker. Finally, glycine is used to block the unreacted aldehyde groups and minimize unspecific adsorption effects. The selectivity of the sensor has been tested against 10 mg/L of serum albumin, the most abundant protein in plasma. Keywords: Carbon nanotube field effect transistor, human immunoglobulin G, CNT functionalization, immunosensor, antigen, –, antibody interaction, physiological conditions

Immuno-column for on-line quantification of human serum IgG antibodies to Helicobacter pylori in human serum samples

This study report an human serum IgG antibodies to Helicobacter pylori quantitation procedure based on the multiple use of an immobilized H. pylori antigen on an immuno-column incorporated into an a flow-injection (FI) analytical system. The immuno-adsorbent column was prepared by packing 3-aminopropyl-modified controlled-pore glass (APCPG) covalently linking H. pylori antigens in a 3-cm of Teflon tubing (0.5 i.d.). Antibodies in the serum sample are allowed to react immunologically with the immobilized H. pylori antigen, and the bound antibodies are quantified by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. p-Aminophenyl phosphate ( pAPP) was converted to p-aminophenol ( pAP) by AP and an electroactive product was quantified on glassy carbon electrode (GCE) modified with multiwall carbon nanotubes (MWCNT) (GCE-CNTs) at 0.30 V. The total assay time was 25 min. The calculated detection limits for amperometric detection and the ELISA procedure are 0.62 and 1.8 U mL −1, respectively. Reproducibility assays were made using repetitive standards of H. pylori-specific antibody and the intra- and inter-assay coefficients of variation were below 5%. The immuno-affinity method showed higher sensitivity and lower time-consumed, demonstrate its potential usefulness for early assessment of human serum immunoglobulin G (IgG) antibodies to H. pylori.

Carbon nanotube field effect transistors for the fast and selective detection of human immunoglobulin G

We report a field effect transistor (FET) based on a network of single-walled carbon nanotubes (SWCNTs) which can selectively detect human immunoglobulin G (HIgG). HIgG antibodies, which are strongly adsorbed onto the walls of the SWCNTs, are the basic elements of the recognition layer. The non-specific binding of proteins and the effects of other interferences are avoided by covering the non-adsorbed areas of the SWCNTs with Tween 20. The selectivity of the sensor has been tested against bovine serum albumin (BSA), the most abundant protein in plasma. HIgG in aqueous solution with concentrations from 1.25 mg L(-1) (8 nM) can be readily detected with response times of about 10 min. The SWCNT networks that form the basis of the sensor are easily grown by chemical vapour deposition. Silver screen-printed electrodes make the sensor quick to build. The sensitivity obtained with this sensor is similar to other FET devices based on SWCNTs built using much more complicated lithography processes. Moreover, the sensor is a reagentless device that does not need labels to detect HIgG.

Screen-printed immunosensor for quantification of human serum IgG antibodies to Helicobacter pylori

Abstract This paper describes the development of a screen-printed immunosensor for the rapid and sensitive quantification of human serum immunoglobulin G (IgG) antibodies to Helicobacter pylori . This microorganism cause peptic ulcers and chronic gastritis, affecting around the 10% of the world population. Antibodies in the serum sample are allowed to react immunologically with the purified H. pylori antigens that are immobilized on graphite screen-printed electrodes (GSPE). The bound antibodies are quantified by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. p -Aminophenyl phosphate ( p -APP) was converted to p -aminophenol ( p -AP) by AP, and an electroactive product was quantified using Osteryoung square wave voltammetry (OSWV). The electrochemical detection can be done within 1 min and total assay time was 25 min. The calculated detection limits for electrochemical detection and the ELISA procedure are 0.5 and 1.8 U ml −1 , respectively. Reproducibility assays were made using repetitive standards of H. pylori specific antibody (measured as the activity of the correspondent anti-serum's enzyme conjugated) and the intra- and inter-assay coefficients of variation were below 5%. The electrochemical immunosensor showed higher sensitivity and lower time consumed than the standard spectrophotometric detection ELISA method, demonstrate its potential usefulness for early assessment of human serum immunoglobulin G (IgG) antibodies to H. pylori .