Plasmodium falciparum: sequence diversity and antibody recognition of the Merozoite surface protein-2 (MSP-2) in Brazilian Amazonia

The merozoite surface protein-2 (MSP-2) of Plasmodium falciparum comprises repeats flanked by dimorphic domains defining the allelic families FC27 and IC1. Here, we examined sequence diversity at the msp-2 locus in Brazil and its impact on MSP-2 antibody recognition by local patients. Only 25 unique partial sequences of msp-2 were found in 61 isolates examined. The finding of identical msp-2 sequences in unrelated parasites, collected 6-13 years apart, suggests that no major directional selection is exerted by variant-specific immunity in this malaria-endemic area. To examine antibody cross-reactivity, recombinant polypeptides derived from locally prevalent and foreign MSP-2 variants were used in ELISA. Foreign IC1-type variants, such as 3D7 (currently tested for human vaccination), were less frequently recognized than FC27-type and local IC1-type variants. Antibodies discriminated between local and foreign IC1-type variants, but cross-recognized structurally different local IC1-type variants. The use of evolutionary models of MSP-2 is suggested to design vaccines that minimize differences between local parasites and vaccine antigens. (C) 2004 Elsevier B.V. All rights reserved.

Novel monoclonal antibody recognition of oxidative DNA damage adduct, deoxycytidine-glyoxal

Glyoxal, a reactive aldehyde, is a decomposition product of lipid hydroperoxides, oxidative deoxyribose breakdown, or autoxidation of sugars, such as glucose. It readily forms DNA adducts, generating potential carcinogens such as glyoxalated deoxycytidine (gdC). A major drawback in assessing gdC formation in cellular DNA has been methodologic sensitivity. We have developed an mAb that specifically recognizes gdC. Balb/c mice were immunized with DNA, oxidatively modified by UVC/hydrogen peroxide in the presence of endogenous metal ions. Although UVC is not normally considered an oxidizing agent, a UVC/hydrogen peroxide combination may lead to glyoxalated bases arising from hydroxyl radical damage to deoxyribose. This damaging system was used to induce numerous oxidative lesions including glyoxal DNA modifications, from which resulted a number of clones. Clone F3/9/H2/G5 showed increased reactivity toward glyoxal-modified DNA greater than that of the immunizing antigen. ELISA unequivocally showed Ab recognition toward gdC, which was confirmed by gas chromatography-mass spectrometry of the derivatized adduct after formic acid hydrolysis to the modified base. Binding of Ab F3/9 with glyoxalated and untreated oligomers containing deoxycytidine, deoxyguanosine, thymidine, and deoxyadenosine assessed by ELISA produced significant recognition (p 0.0001) of glyoxal-modified deoxycytidine greater than that of untreated oligomer. Additionally, inhibition ELISA studies using the glyoxalated and native deoxycytidine oligomer showed increased recognition for gdC with more than a 5-fold difference in IC50 values. DNA modified with increasing levels of iron (II)/EDTA produced a dose-dependent increase in Ab F3/9 binding. This was reduced in the presence of catalase or aminoguanidine. We have validated the potential of gdC as a marker of oxidative DNA damage and showed negligible cross-reactivity with 8-oxo-2'-deoxyguanosine or malondialdehyde-modified DNA as well as its utility in immunocytochemistry. Formation of the gdC adduct may involve intermediate structures; however, our results strongly suggest Ab F3/9 has major specificity for the predominant product, 5-hydroxyacetyl-dC.

Application of impedance spectroscopy for monitoring colloid Au-enhanced antibody immobilization and antibody-antigen reactions

We used colloidal An to enhance the amount of antibody immobilized on a gold electrode and ultimately monitored the interaction of antigen-antibody by impedance measurement. Self-assembly of 6 nm (diameter) colloidal An onto the self-assembled monolayers (SAMs) of 4-aminothiophenol modified gold electrode resulted in an easier attachment of antibody. The redox reactions of [Fe(CN)(6)](4-)/[Fe(CN)(6)](3-) on the gold surface were blocked due to the procedures of self-assembly of 4-aminothiophenol and antibody immobilization, which were investigated by cyclic voltammetry and impedance spectroscopy. The interaction of antigen with grafted antibody recognition layers was carried out by soaking the modified electrode into a phosphate buffer at pH 7.4 with various concentrations of antigen at 37 degreesC for 30 min. The antibody recognition layers and their interactions with various concentrations of antigen could be detected by measurements of the impedance change. The results show that this method has good correlation for detection of Hepatitis B virus surface antigen in the range of 0.5-200 mug/l and a detection limit of about 50 ng/l.

Amplification of antigen-antibody interactions based on biotin labeled protein-streptavidin network complex using impedance spectroscopy

Antibody was covalently immobilized by amine coupling method to gold surfaces modified with a self-assembled monolayer of thioctic acid. The electrochemical measurements of cyclic voltammetry and impedance spectroscopy showed that the hexacyanoferrate redox reactions on the gold surface were blocked due to the procedures of self-assembly of thioctic acid and antibody immobilization. The binding of a specific antigen to antibody recognition layer could be detected by measurements of the impedance change. A new amplification strategy was introduced for improving the sensitivity of impedance measurements using biotin labeled protein- streptavidin network complex. This amplification strategy is based on the construction of a molecular complex between streptavidin and biotin labeled protein. This complex can be formed in a cross-linking network of molecules so that the amplification of response signal will be realized due to the big molecular size of complex. The results show that this amplification strategy causes dramatic improvement of the detection sensitivity of hIgG and has good correlation for detection of hIgG in the range of 2-10 mug/ml. (C) 2001 Elsevier Science B.V. All rights reserved.

Ultra-trace detection of diagnostically important biomarkers using functionalised-Surface Enhanced Raman Spectroscopy (SERS)

Here we report an ultrasensitive method for detecting bio-active compounds in biological samples by means of functionalised nanoparticles interrogated by surface enhanced Raman spectroscopy (SERS). This method is applicable to the recovery and detection of many diagnostically important peptidyl analytes such as insulin, human growth hormone, growth factors (IGFs) and erythropoietin (EPO), as well as many small molecule analytes and metabolites. Our method, developed to detect EPO, demonstrates its utility in a complex yet well defined biological system. Recombinant human EPO (rhEPO) and EPO analogues have successfully been used to treat anaemia in end-stage renal failure, chronic disorders and infections, cancer and AIDS. Current methods for EPO testing are lengthy, laborious and relatively insensitive to low concentrations. In our rapid screening methodology, gold nanoparticles were functionalised with anti-EPO antibodies to provide very high selectivity towards the EPO protein in urine. These “smart sensor” nanoparticles interact with and trap EPO. Subsequent SERS screening allows for the detection and quantisation of ultra trace amounts (<<10-15 M) of EPO in urine samples with minimal sample preparation. We present data showing that the SERS spectrum differentiates between human endogenous EPO and rhEPO in unpurified urine, and potentially distinguishes between purified EPO isoforms. The elimination of sample preparation and direct screening in biological fluids significantly reduces the time required by current methods. Antibody recognition against a variety of biological targets and the availability of portable commercial SERS analysers for rapid onsite testing suggest broad diagnostic applicability in a flexible analytical platform.

IgE cross-reactivity between Pas n 1 of Bahia grass pollen and other group 1 grass pollen allergens

Background The subtropical Bahia grass (Paspalum notatum) is an important source of pollen allergens with an extended season of pollination and wide distribution in warmer climates. The immunological relationship between pollen allergens of Bahia grass and temperate grasses is unresolved. Methods Serum IgE reactivity of grass pollen-allergic patients with Bahia, Ryegrass and Bermuda grass pollen extracts and their purified group 1 allergens, Pas n 1, Lol p 1 and Cyn d 1, were compared by immunoblotting, ELISA, inhibition ELISA, basophil activation by flow cytometry and molecular modeling. Results Differences in antibody recognition of allergenic components between Bahia grass and Ryegrass pollen were observed by immunoblotting. Eight grass pollen-allergic patients from a temperate region showed greater serum IgE reactivity with Ryegrass pollen than Bahia grass by ELISA. For seven of these sera, Ryegrass pollen inhibited IgE reactivity with Bahia grass pollen but not the converse. For 51 sera from grass pollen-allergic patients in this temperate region, IgE reactivity with Lol p 1 was greater than Pas n 1 or Cyn d 1. IgE reactivity with Lol p 1 was not inhibited by Pas n 1 or Cyn d 1, but Pas n 1 IgE reactivity was inhibited by Lol p 1. Two group 1 grass pollen allergen-specific mAb distinguished between temperate and subtropical grass pollens. Basophil activation for three patients tested was greater by Ryegrass pollen than Bahia or Bermuda grass, and by Lol p 1 than Pas n 1 or Cyn d 1. In contrast, two patients from a subtropical region had higher serum IgE reactivity with Bahia grass pollen than Ryegrass and Bahia grass pollen inhibited IgE reactivity with Ryegrass. A structural model of Pas n 1 showed amino acids implicated in IgE epitopes of other group 1 allergens were juxtaposed on the surface. Conclusion Allergens from subtropical Bahia grass pollen, including Pas n 1, share antigenic determinants with temperate grass pollen allergens, but patients exhibit higher serum IgE reactivity to their locally predominant grass pollen. Basophil activation by Bahia grass pollen and Pas n 1 in patients from a temperate climate indicates clinically relevant cross-sensitization between temperate and subtropical grass pollens.

Mapping the common antigenic determinants in avidin and streptavidin

An epitope scan analysis of the whole sequence of avidin and core streptavidin using polyclonal antibodies to these two antigens reveal the presence of multiple common epitopes in both the proteins. These antigenic determinants consist mostly of either identical or similar residues. The antibody recognition sites in both antigens are shown to be localized to homologous regions.

Immunochemical analysis of prolamins in gluten-free foods

People with coeliac disease have to maintain a gluten-free diet, which means excluding wheat, barley and rye prolamin proteins from their diet. Immunochemical methods are used to analyse the harmful proteins and to control the purity of gluten-free foods. In this thesis, the behaviour of prolamins in immunological gluten assays and with different prolamin-specific antibodies was examined. The immunoassays were also used to detect residual rye prolamins in sourdough systems after enzymatic hydrolysis and wheat prolamins after deamidation. The aim was to characterize the ability of the gluten analysis assays to quantify different prolamins in varying matrices in order to improve the accuracy of the assays. Prolamin groups of cereals consist of a complex mixture of proteins that vary in their size and amino acid sequences. Two common characteristics distinguish prolamins from other cereal proteins. Firstly, they are soluble in aqueous alcohols, and secondly, most of the prolamins are mainly formed from repetitive amino acid sequences containing high amounts of proline and glutamine. The diversity among prolamin proteins sets high requirements for their quantification. In the present study, prolamin contents were evaluated using enzyme-linked immunosorbent assays based on ω- and R5 antibodies. In addition, assays based on A1 and G12 antibodies were used to examine the effect of deamidation on prolamin proteins. The prolamin compositions and the cross-reactivity of antibodies with prolamin groups were evaluated with electrophoretic separation and Western blotting. The results of this thesis research demonstrate that the currently used gluten analysis methods are not able to accurately quantify barley prolamins, especially when hydrolysed or mixed in oats. However, more precise results can be obtained when the standard more closely matches the sample proteins, as demonstrated with barley prolamin standards. The study also revealed that all of the harmful prolamins, i.e. wheat, barley and rye prolamins, are most efficiently extracted with 40% 1-propanol containing 1% dithiothreitol at 50 °C. The extractability of barley and rye prolamins was considerably higher with 40% 1-propanol than with 60% ethanol, which is typically used for prolamin extraction. The prolamin levels of rye were lowered by 99.5% from the original levels when an enzyme-active rye-malt sourdough system was used for prolamin degradation. Such extensive degradation of rye prolamins suggest the use of sourdough as a part of gluten-free baking. Deamidation increases the diversity of prolamins and improves their solubility and ability to form structures such as emulsions and foams. Deamidation changes the protein structure, which has consequences for antibody recognition in gluten analysis. According to the resuts of the present work, the analysis methods were not able to quantify wheat gluten after deamidation except at very high concentrations. Consequently, deamidated gluten peptides can exist in food products and remain undetected, and thus cause a risk for people with gluten intolerance. The results of this thesis demonstrate that current gluten analysis methods cannot accurately quantify prolamins in all food matrices. New information on the prolamins of rye and barley in addition to wheat prolamins is also provided in this thesis, which is essential for improving gluten analysis methods so that they can more accurately quantify prolamins from harmful cereals.

Immunosensors based on layer-by-layer self-assembled Au colloidal electrode for the electrochemical detection of antigen

Colloidal Au particles have been deposited on the gold electrode through layer-by-layer self-assembly using cysteamine as cross-linkers. Self-assembly of colloidal Au on the gold electrode resulted in ail easier attachment of antibody, larger electrode surface and ideal electrode behavior. The redox reactions of [Fe(CN)(6)]-/[Fe(CN)(6)](3-) on the gold surface were blocked due to antibody immobilization, which were investigated by cyclic voltammetry and impedance spectroscopy. The interaction of antigen with grafted antibody recognition layers was carried out by soaking the modified electrode into a phosphate buffer at pH 7.0 with various concentrations of antigen at 37degreesC for 30 min. Further, an amplification strategy to use biotin conjugated antibody was introduced for improving the sensitivity of impedance measurements. Thus, the sensor based oil this immobilization method exhibits a large linear dynamic range, from 5 - 400 mug/L for detection of Human IgG. The detection limit is about 0.5 mug/L.

The ultrasensitive detection of prostate cancer biomarker based on resonant Rayleigh light-scattering response of single Au nanoparticle

A proof-of-concept study was reported on analysis of antigen-antibody recognition based on resonant Rayleigh scattering response of single Au nanoparticles on a microimaging chamber. As benefited by a traditional dark-field microscope and a spectrograph, tiny 30 nm Au nanoparticles were effectively used as nanosensors to monitor changes in refractive index induced by every single binding of the adsorbates. The individual Au nanoparticles were observed with very high signal-to-noise ratio, and a LSPR ?max shift of about 2.5 nm accounting for the detection of PSA antigen with concentration as low as 0.1 pg ml-1 was recorded. This resulted in the successful demonstration of a non-labelling detection system for proteins as well as thousands of different chemical or biological species with possibility of miniaturization and multiplexing scheme.

Naturally Acquired Antibodies to Plasmodium vivax Duffy Binding Protein (DBP) in Rural Brazilian Amazon

Duffy binding protein (DBP), a leading malaria vaccine candidate, plays a critical role ill Plasmodium vivax erythrocyte invasion. Sixty-eight of 366 (18.6%) subjects had IgG anti-DBP antibodies by enzyme-linked immunosorbent assay (ELISA) in a community-based cross-sectional survey ill the Brazilian Amazon Basin. Despite Continuous exposure to low-level malaria transmission, the overall seroprevalence decreased to 9.0% when the Population was reexamined 12 months later. Antibodies from 16 of 50 (360%) Subjects who were ELISA-positive at the baseline were able to inhibit erythrocyte binding to at least one of two DBP variants tested. Most (13 of 16) of these subjects still had inhibitory antibodies when reevaluated 12 months later. Cumulative exposure to malaria was the strongest predictor of DBP seropositivity identified by Multiple logistic regression models in this population. The poor antibody recognition of DBP elicited by natural exposure to P. vivax in Amazonian populations represents a challenge to be addressed by vaccine development strategies.

Population dynamics of genetically diverse Plasmodium falciparum lineages: community-based prospective study in rural Amazonia

Temporal changes in the prevalence of antigenic variants in Plasmodium falciparum populations have been interpreted as evidence of immune-mediated frequency-dependent selection, but evolutively neutral processes may generate similar patterns of serotype replacement. Over 4 years, we investigated the population dynamics of P. falciparum polymorphisms the community level by using 11 putatively neutral microsatellite markers. Plasmodium falciparum Populations were less diverse than sympatric P. vivax isolates, with less multiple-clone infections, lower number of alleles per locus and lower Virtual heterozygosity, but both species showed significant multilocus linkage disequilibrium. Evolutively neutral P. falciparum polymorphisms showed a high turnover rate, with few lineages persisting for several months in the population. Similar results had previously been obtained, in the same community, for sympatric P. vivax isolates. In contrast, the prevalence of the 2 dimorphic types of a major antigen, MSP-2, remained remarkably stable throughout the Study period. We Suggest that the relatively fast turnover of parasite lineages represents the typical population dynamics of neutral polymorphisms in small populations, with clear implications for the detection of frequency-dependent selection of polymorphisms.

Sequence diversity and evolutionary dynamics of the dimorphic antigen merozoite surface protein-6 and other Msp genes of Plasmodium falciparum

Immune evasion by Plasmodium falciparum is favored by extensive allelic diversity of surface antigens. Some of them, most notably the vaccine-candidate merozoite surface protein (MSP)-1, exhibit a poorly understood pattern of allelic dimorphism, in which all observed alleles group into two highly diverged allelic families with few or no inter-family recombinants. Here we describe contrasting levels and patterns of sequence diversity in genes encoding three MSP-1-associated surface antigens of P. falciparum, ranging from an ancient allelic dimorphism in the Msp-6 gene to a near lack of allelic divergence in Msp-9 to a more classical multi-allele polymorphism in Msp-7 Other members of the Msp-7 gene family exhibit very little polymorphism in non-repetitive regions. A comparison of P. falciparum Msp-6 sequences to an orthologous sequence from P. reichenowi provided evidence for distinct evolutionary histories of the 5` and 3` segments of the dimorphic region in PfMsp-6, consistent with one dimorphic lineage having arisen from recombination between now-extinct ancestral alleles. In addition. we uncovered two surprising patterns of evolution in repetitive sequence. Firsts in Msp-6, large deletions are associated with (nearly) identical sequence motifs at their borders. Second, a comparison of PfMsp-9 with the P. reichenowi ortholog indicated retention of a significant inter-unit diversity within an 18-base pair repeat within the coding region of P. falciparum, but homogenization in P. reichenowi. (C) 2009 Elsevier B.V. All rights reserved.

Nano- und mikrostrukturierte organische Erkennungsstrukturen in Lipidmembranen

Im Rahmen dieser Arbeit wurden zunächst die ausgewählten Lipide anhand ihrer p-ArnIsothermen charakterisiert. Dabei zeigte sich, dass die flüssiganaloge Phase umso ausgeprägter ist, je größer die hydrophile Kopfgruppe des jeweiligen Lipids ist, so dass diese schon bei sehr großen Flächen miteinander wechselwirken. Außerdem wurde das Mischungsverhalten und die Domänenbildung binärer und ternärer Mischungen untersucht.rnDabei konnte im Fall der binären Mischungen der polymerisierbaren Lipide 1 und 2 mit dem perfluorierten Lipid 3 eine Phasenseparation beobachtet werden. Hierbei zeigte sich, dass sowohl die Form als auch die Größe der Domänen durch zwei Faktoren kontrolliert werden konnte, nämlich zum einen durch den Lateraldruck und zum anderen durch die molare Zusammensetzung der binären Mischung. Bei niedrigen Lateraldrücken (10 mN/m) warenrnfluorierte und nichtfluorierte Lipide in der homogenen flüssiganalogen Phase vollständig mischbar. Bei sehr hohen Lateraldrücken (30 mN/m) lag eine nahezu vollständige Entmischung vor, da das Lipid 1 hier schon vollständig auskristallisiert war. Im Fall der binären Mischung 2/3 konnte ein solches Verhalten nicht beobachtet werden, was an dem Phasenverhalten des Lipids 2 liegt. Aber auch hier lies sich die Domänenbildung sowohl überrnden Lateraldruck als auch über die Komposition der binären Mischung kontrollieren. Des Weiteren konnte gezeigt werden, dass die Monoschichten dieser beiden binären Mischungen durch photochemische Polymerisation stabilisiert werden können.rnDer Einfluss des Fluorierungsgrades auf das Phasenseparationsverhalten wurde anhand der binären Mischungen 1/6 und 1/8 untersucht. Es zeigte sich hier, dass sich auch hier in beiden Fällen die Domänenbildung sowohl über den Lateraldruck als auch über die Komposiion der Mischung steuern lies. Ein Vergleich der gefundenen Strukturen belegte darüber hinaus klare Unterschiede zwischen dem fluorierten und teilfluorierten Lipid. Denn in der binären Mischung 1/8 (perfluoriertes Lipid) konnte die hexagonale Grundstruktur, die zuvor schon inrnder binären Mischung 1/3 gefunden wurde, beobachtet werden. Demgegenüber hatten die kristallinen Domänen, die in der binären Mischung 1/6 (teilfluoriertes Lipid) beobachtet wurden, die hexagonale Grundform verloren. Hier wurden rundliche unförmige Domänen gefunden, die zudem deutlich kleiner waren als die bei der Mischung mit dem perfluorierten Lipid gefundenen Domänen. In den ternären Mischungen zeigte sich, dass diese ternären Mischungen eine ähnliche Phasenseparation zeigten wie die binäre Mischung 1/3. Die hier auftretenden kristallanalogen Domänen bestanden aus Lipid 1, wobei die umgebendernflüssiganaloge Matrix die beiden fluorierten Lipide enthielt. Deswegen war es von besonderer Bedeutung wie sich die beiden fluorierten Verbindungen untereinander verhalten würden.rnDazu wurden die p-A Isothermen der binären Mischung 3/5 ausgewertet und dabei zeigte sich, dass sich das Vakzinlipid, sofern der Anteil nicht höher ist als der des fluorierten Matrixlipides, in idealer Weise mit diesem mischt. Eine Untersuchung der Antigen-Antikörper Erkennung der binären Mischungen 3/5 zeigte, dass die Reaktion mit dem Antikörper umso effektiver war, je größer der Anteil des Vakzinlipids 5 war. Allerdings konnte hier kein linearer Zusammenhang gefunden werden. Stattdessen handelte es sich umrneine Sättigungskurve, da bereits ein Anteil an Lipid 5 von 42,2 mol% eine nur geringfügig schwächere Antikörperanbindung zeigte, als das reine Vakzinlipid.

Biomolecular interaction analysis of gestrinone-anti-gestrinone using arrays of high aspect ratio SU-8 nanopillars

In this paper, label-free biosensing for antibody screening by periodic lattices of high-aspect ratio SU-8 nano-pillars (BICELLs) is presented. As a demonstration, the determination of anti-gestrinone antibodies from whole rabbit serum is carried out, and for the first time, the dissociation constant (KD = 6 nM) of antigen-antibody recognition process is calculated using this sensing system. After gestrinone antigen immobilization on the BICELLs, the immunorecognition was performed. The cells were interrogated vertically by using micron spot size Fourier transform visible and IR spectrometry (FT-VIS-IR), and the dip wavenumber shift was monitored. The biosensing assay exhibited good reproducibility and sensitivity (LOD = 0.75 ng/mL).