Pistachio vicilin, Pis v 3, is immunoglobulin E-reactive and cross-reacts with the homologous cashew allergen, Ana o 1.

BACKGROUND: Patients allergic to cashew nuts often report allergy to pistachio, which could be a result of cross-reactivity between the two as both are members of the Anacardiaceae family. OBJECTIVE: Because cashew 7S globulin (vicilin, Ana o 1) is a recognized major allergen, we cloned the pistachio homologue and assayed it for IgE reactivity and cross-reactivity with Ana o 1. METHODS: Degenerate primers for 7S globulin were used in PCR to amplify DNA from a pistachio cDNA library. An isolate was sequenced, cloned and expressed in Escherichia coli. Reactivity to the allergen was screened by dot blot using 19 pistachio and/or cashew-allergic patients' sera. Cross-reactivity was investigated by inhibition dot- and Western immunoblot assays using pistachio/cashew-allergic patients' sera, and monoclonal antibodies (MAbs) raised against recombinant Ana o 1 (rAna o 1). RESULTS: An isolate was found that coded for a 7S vicilin-like protein, designated Pis v 3. IgE reactivity to Pis v 3 was found in the serum of seven of the 19 (37%) patients with histories of allergy to both pistachio and cashew or who were allergic to cashew but had never eaten pistachio. The seven patients with IgE that recognized rPis v 3 also recognized rAna o 1. Six of nine anti-rAna o 1 MAbs also showed reactivity to rPis v 3 on dot blots. CONCLUSION: Of the 37% of pistachio/cashew-allergic patients' sera that recognized the pistachio allergen, rPis v 3, all showed complete cross-reactivity with rAna o 1. The data does not identify the primary sensitizing agent but suggests that IgE reactivity to rPis v 3 and rAna o 1 is focused on the most conserved regions of the proteins. Clinical histories suggest that in some cases, cashew was the sensitizing agent. rPis v 3 is a likely contributor to the observed co-sensitivity to pistachio and cashew in some patients.

Electron microscopic and immunochemical analysis of the broadly neutralizing HIV-1-specific, anti-carbohydrate antibody, 2G12.

2G12 is one of only a few cloned antibodies with broadly neutralizing specificity to HIV-1 envelope proteins. Crystallographic and electron microscopic (EM) data showed that the Fab arms are locked together via a novel VH domain exchange. Both the conventional and the unprecedented additional VH-VH antigen binding sites show specificity for high mannose oligosaccharides on the silent face of gp120. We have now extended the EM and biochemical analysis of 2G12. Unligated 2G12 IgG1 molecules clearly show paired (parallel attached) Fab arms in the "doughnut" configuration attached to the Fc both in individual and computationally averaged images. A minority of the IgG molecules in the 2G12 prep showed the open "Y" configuration of conventional IgG. The averaged EM image compares well to the atomic structure model of 2G12. Papain digests of 2G12 yielded paired Fab arms (Fab dimer), as observed by EM, which dissociated into Fab-sized fragments in non-reducing SDS-PAGE. Purified 2G12 reduced and alkylated H and L chains can reassociate to form IgG molecules with the Fab dimer configuration and can combine with L and H chains from conventional human IgG to form hybrid molecules. 2G12 is heavily aggregated following brief acid exposure possibly as a result of its unique structure. A model of the aggregation process is proposed. An anti-Id MAb was shown by EM to react with neither the conventional nor additional antigen binding sites, but bound to the lateral faces of the Fab arms of intact, reduced and alkylated, and reconstructed 2G12 molecules. Efforts to identify IgG molecules with a similar intertwined Fab dimer structure in a large IgG pool were unsuccessful.

Effects of roasting, blanching, autoclaving, and microwave heating on antigenicity of almond (Prunus dulcis L.) proteins.

Whole, unprocessed Nonpareil almonds were subjected to a variety of heat processing methods that included roasting (280, 300, and 320 degrees F for 20 and 30 min each; and 335 and 350 degrees F for 8, 10, and 12 min each), autoclaving (121 degrees C, 15 psi, for 5, 10, 15, 20, 25, and 30 min), blanching (100 degrees C for 1, 2, 3, 4, 5, and 10 min), and microwave heating (1, 2, and 3 min). Proteins were extracted from defatted almond flour in borate saline buffer, and immunoreactivity of the soluble proteins (normalized to 1 mg protein/mL for all samples) was determined using enzyme linked immunosorbent assay (ELISA). Antigenic stability of the almond major protein (amandin) in the heat-processed samples was determined by competitive inhibition ELISA using rabbit polyclonal antibodies raised against amandin. Processed samples were also assessed for heat stability of total antigenic proteins by sandwich ELISA using goat and rabbit polyclonal antibodies raised against unprocessed Nonpareil almond total protein extract. ELISA assays and Western blotting experiments that used both rabbit polyclonal antibodies and human IgE from pooled sera indicated antigenic stability of almond proteins when compared with that of the unprocessed counterpart.

A molecular model of type I allergy: identification and characterization of a nonanaphylactic anti-human IgE antibody fragment that blocks the IgE-FcepsilonRI interaction and reacts with receptor-bound IgE.

BACKGROUND: The IgE-mediated activation of effector cells and antigen-presenting cells through the high-affinity receptor for IgE (FcepsilonRI) represents a key pathomechanism in type I allergy and many forms of asthma. OBJECTIVE: We sought to establish an in vitro molecular model for the interaction of human FcepsilonRI, IgE, and the corresponding allergen and to identify monoclonal anti-human IgE antibodies with a therapeutic profile different from previously established anti-IgE antibodies. METHODS: Human FcepsilonRI alpha chain, a human monoclonal allergen-specific IgE antibody (chimeric Bip 1), and the corresponding allergen, the major birch pollen allergen Bet v 1, were produced as recombinant proteins and analyzed by means of circular dichroism and native overlays, respectively. Using this molecular model, as well as negative stain immunoelectron microscopic analysis, and in vitro cultivated human basophils, we characterized mouse anti-human IgE antibodies. RESULTS: We established a molecular model for the interaction of human IgE with FcepsilonRI. Using this molecular model, we identified a nonanaphylactic anti-human IgE antibody fragment (Fab12), which blocked the IgE-FcepsilonRI interaction and reacted with effector cell-bound IgE. CONCLUSION: Fab12 represents a candidate molecule for therapy of atopy and asthma because it can be used for the depletion of circulating IgE antibodies, as well as for the depletion of IgE-bearing cells.

Structural flexibility and functional valence of CD4-IgG2 (PRO 542): potential for cross-linking human immunodeficiency virus type 1 envelope spikes.

CD4-immunoglobulin G2 (CD4-IgG2) incorporates four copies of the D1D2 domains of CD4 into an antibody-like molecule that potently neutralizes primary human immunodeficiency virus type 1. Here electron microscopy was used to explore the structure and functional valence of CD4-IgG2 in complex with gp120. CD4-gamma2, a divalent CD4-immunoglobulin fusion protein, was evaluated in parallel. Whereas CD4-gamma2-gp120 complexes adopted a simple Y-shaped structure, CD4-IgG2-gp120 complexes consisted of four gp120s arrayed about a central CD4-IgG2 molecule, a structure more reminiscent of complement C1q. Molecular modeling corroborated the electron microscopy data and further indicated that CD4-IgG2 but not CD4-gamma2 has significant potential to cross-link gp120-gp41 trimers on the virion surface, suggesting a mechanism for the heightened antiviral activity of CD4-IgG2.

Detection and stability of the major almond allergen in foods.

Almond major protein (AMP or amandin), the primary storage protein in almonds, is the major allergen recognized by almond-allergic patients. A rabbit antibody-based inhibition ELISA assay for detecting and quantifying AMP in commercial foods has been developed, and this assay, in conjunction with Western blotting analyses, has been applied to the investigation of the antigenic stability of AMP to harsh food-processing conditions. The ELISA assay detects purified AMP at levels as low as 87 +/-16 ng/mL and can detect almond at between 5 and 37 ppm in the tested foods. The assay was used to quantify AMP in aqueous extracts of various foods that were defatted and spiked with known amounts of purified AMP or almond flour. In addition, AMP was quantified in commercially prepared and processed almond-containing foods. Neither blanching, roasting, nor autoclaving of almonds markedly decreased the detectability of AMP in subsequent aqueous extracts of almonds. Western blots using both rabbit antisera and sera from human almond-allergic patients confirm a general stability of the various peptides that comprise this complex molecule and show that the rabbit antibody-based assay recognizes substantially the same set of peptides as does the IgE in sera from almond-allergic patients.

Electrophoretic and immunological analyses of almond (Prunusdulcis l.) genotypes and hybrids.

Aqueous extracts from sixty almond samples representing various genotypes and interspecies hybrids of almond, including almond-peach, were analyzed for protein and peptide content using electrophoresis, Western immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Nondenaturing nondissociating polyacrylamide gel electrophoresis (NDND-PAGE) of the aqueous extracts indicated that a single major storage protein (almond major protein -- AMP or amandin) dominated the total soluble protein composition. Denaturing SDS--PAGE analyses of the aqueous extracts revealed that the AMP was mainly composed of two sets of polypeptides with estimated molecular masses in the ranges of 38--41 kDa and 20--22 kDa, regardless of the source; however, distinct variations in the intensity and electrophoretic mobility of some bands were noted between samples. In addition to AMP, several minor polypeptides were also present in all the genotypes, and variations were seen in these as well. Regardless of the genotype, AMP was recognized in Western blots by rabbit polyclonal anti-AMP antibodies, mouse monoclonal anti-AMP antibodies (mAbs), and serum IgE from patients displaying strong serum anti-almond IgE reactivity. As with protein staining results, antibody reactivity also revealed common patterns but displayed some variation between samples. An anti-AMP inhibition ELISA was used to quantify and compare aqueous extracts for various samples. All samples (n = 60) reacted in this assay with a mean +/- standard deviation (sigma n) = 0.82 +/- 0.18 when compared to reference aqueous extract from Nonpareil designated as 1.0.

Parameters influencing measurement of the Gag antigen-specific T-proliferative response to HIV type 1 infection.

We have analyzed factors that might influence the in vitro quantitation of the T-proliferative response to HIV-1 Gag antigens, a common and increasingly used clinical measurement of helper T cell function in the context of HIV-1 infection. We have compared the rate and extent of T cell proliferation in freshly prepared and previously frozen PBMC samples, and have concluded that frozen cells can be used successfully; we have assessed whether the suppression of any HIV-1 replication in the PBMC cultures affects the extent of T cell proliferation; we have studied which forms of the Gag antigens are the most efficient at inducing T cell proliferation. From the latter experiments, we conclude that Gag proteins that include p17, and perhaps also p7, sequences flanking the central p24 capsid protein, are better stimulants than proteins that comprise only p24 sequences.

Production and characterization of rabbit polyclonal antibodies to almond (Prunus dulcis L.) major storage protein.

Rabbits were immunized with purified almond major protein (AMP), the primary storage protein in almonds. Rabbit anti-AMP polyclonal antibodies (PA) could detect AMP when as little as 1-10 ng/mL were used to coat microtiter plates in a noncompetitive enzyme linked-immunosorbent assay (ELISA). Competitive inhibition ELISA assays detected the AMP down to 300 ng/mL. PA recognized the AMP in protein extracts from all U.S. major marketing cultivars of almonds (Mission, Neplus, Peerless, Carmel, and Nonpareil) with specific reactivity of 52.6-75% as compared to that of the AMP alone. Immunoreactivity of protein extracts prepared from commercial samples of blanched almonds, roasted almonds, and almond paste was respectively reduced by 50.0%, 56.6%, and 68.4% (noncompetitive ELISA) when compared to the immunoreactivity of the AMP. Moist heat (121 degrees C, 15 min) pretreatment of the AMP reduced the PA reactivity by 87% (noncompetitive ELISA). Exposing AMP to pH extremes (12.5 and 1.5-2.5) caused a 53% and 57% reduction in PA reactivity, respectively (noncompetitive ELISA). PA showed some cross-reactivity with the cashew major globulin, and to a lesser extent, the Tepary and Great Northern bean major storage protein (7S or phaseolin). The presence of almonds in a commercial food was detected using PA in a competitive ELISA.

Immunoglobulin structure and function as revealed by electron microscopy.

Electron-microscopic (EM) analysis preceded crystallographic analysis [1,2] of Igs by over a decade and was for a time the only direct way of analyzing their 3-D molecular structure. Once the X-ray structures were deduced, the role of EM gradually shifted from gross structural analysis to the addressing of more sophisticated structural and functional questions. EM remains a vital adjunct to the many physicochemical, biochemical, and serological tools brought to bear on these remarkable molecules as we try to relate form to function. In this review I will highlight some of the many contributions that have been made possible by virtue of being able to 'see' Ig molecules and immune complexes.

Comparisons of the ability of human IgG3 hinge mutants, IgM, IgE, and IgA2, to form small immune complexes: a role for flexibility and geometry.

Various native and hinge-modified forms of Ig with identical Ids were reacted with an anti-Id mAb, and the resultant immune complexes were analyzed by negative stain immunoelectron microscopy. Complexes were scored for their geometry (linear versus ring complexes) and size (dimer, trimer, etc.). Ring dimers are the thermodynamically most favorable configuration, unless inhibited by steric and/or flexibility constraints. We found ring dimerization to correlate with the length of the upper, but not middle or lower, hinge. In contrast, the geometry and size of complexes of those molecules lacking formal hinges were unpredictable. A hingeless IgG mutant and native IgE readily formed ring dimers. Remarkably, monomeric IgM formed more ring dimers than any of the other Igs tested, including IgG3. We also tagged the Fab arms and measured the mean Fab-Fab angles and the degree of angular variation for each type of Ig. Surprisingly, IgM proved the most flexible by this assay. In hinged Igs, there was a correlation between length of the upper hinge and Fab-Fab flexibility. In contrast, we found no correlation between the mean Fab-Fab angle in uncomplexed Igs and their ability to dimerize with anti-Id mAb. These data suggest that the physicochemical methods typically used to evaluate molecular flexibility are often of low predictive value when tested in a functional assay.

Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins.

We recently have identified an antigen receptor in sharks called NAR (new or nurse shark antigen receptor) that is secreted by splenocytes but does not associate with Ig light (L) chains. The NAR variable (V) region undergoes high levels of somatic mutation and is equally divergent from both Ig and T cell receptors (TCR). Here we show by electron microscopy that NAR V regions, unlike those of conventional Ig and TCR, do not form dimers but rather are independent, flexible domains. This unusual feature is analogous to bona fide camelid IgG in which modifications of Ig heavy chain V (VH) sequences prevent dimer formation with L chains. NAR also displays a uniquely flexible constant (C) region. Sequence analysis and modeling show that there are only two types of expressed NAR genes, each having different combinations of noncanonical cysteine (Cys) residues in the V domains that likely form disulfide bonds to stabilize the single antigen-recognition unit. In one NAR class, rearrangement events result in mature genes encoding an even number of Cys (two or four) in complementarity-determining region 3 (CDR3), which is analogous to Cys codon expression in an unusual human diversity (D) segment family. The NAR CDR3 Cys generally are encoded by preferred reading frames of rearranging D segments, providing a clear design for use of preferred reading frame in antigen receptor D regions. These unusual characteristics shared by NAR and unconventional mammalian Ig are most likely the result of convergent evolution at the molecular level.

Flexibility of human IgG subclasses.

A variable region (Id)-matched set of genetically engineered human IgG1, -2, -3, and -4 subclass molecules was analyzed by electron microscopy for hinge-mediated differences in flexibility. The hinge-mediated bending was studied, as was the ability of the subclasses to form immune complexes with two anti-Id mAbs. The data show that the rank order (most to least flexible) of the IgG subclasses for hinge-folding mode of flexibility between Fab arms is IgG3 > IgG1 > IgG4 > IgG2. The mean Fab-Fab angles for the subclasses are IgG3, 136 degrees; IgG4, 128 degrees; IgG2, 127 degrees; and IgG1, 117 degrees. Fab-Fc angles were similarly analyzed. By sampling of equimolar mixtures of Id-bearing IgGs and each of two anti-Id mAb after incubation over time (1.5 min to 3.5 h), different kinetic profiles of immune complex formation of defined geometry were documented. Both anti-Id mAbs displayed unique kinetic profiles when complexed with the four IgG subclass molecules but also shared important features. Most notable was the higher propensity to form closed bivalent ring Id-anti-Id dimers with IgG3 than with IgG2 and IgG4. IgG1 was intermediate in its ability to form such dimers.

Rabbit RAG-2 UTRs: characterization of an unusually large 3' untranslated region.

We previously characterized the rabbit recombination activating gene-2 (RAG-2) coding region and a portion of the cDNA. Rabbit RAG-2 mRNA, however, was shown to be approximately twice as large as the predominant form expressed in other vertebrate species, suggesting that it contained additional coding and/or untranslated regions (UTR). In this report, we map and sequence the complete 5' and 3' UTRs of the rabbit RAG-2 transcript and identify and sequence the genomic regions from which they are transcribed. The data show that, with the exception of a 300 nucleotide 5' UTR, almost all of the additional sequence belongs to the 3' UTR and that the 3' UTR sequence is transcribed from a single large exon that encodes most of the coding region and all of the 3' UTR. The 3' UTR contains four poly A signal sites, the last of which is closely followed by a GU-rich region. The rabbit 3' UTR has a high level of identity with the homologous region downstream of the human RAG-2 gene but not with the mouse RAG-2 gene. The region of identity extends several hundred nucleotides beyond the transcribed region and terminates in a series of dinucleotide (TG) repeats. The data are discussed in terms of RAG gene and 3' UTR function, regulation, and evolution.

Cross-reactivity of human IgG anti-F(ab')2 antibody with DNA and other nuclear antigens.

OBJECTIVE: To characterize immunologic specificity and possible antiidiotype activity of IgG anti-F(ab')2 in normal subjects as well as in patients with active and inactive systemic lupus erythematosus (SLE). METHODS: IgG anti-F(ab')2 and anti-double-stranded DNA (anti-dsDNA) were affinity isolated from immunoadsorption columns of F(ab')2 and dsDNA linked to Sepharose 4B. Affinity-purified IgG anti-F(ab')2 (APAF) and affinity-isolated IgG anti-dsDNA (APAD) were tested by enzyme-linked immunosorbent assay (ELISA) for other cross-reacting specificities including anti-Sm, anti-Sm/RNP, and anti- Crithidia binding. Anti-DNA specificity of APAF and APAD was assayed by S1 nuclease treatment of heat-denatured DNA. Rabbit antiidiotypic antisera were prepared by immunization with APAF and APAD from normal subjects and SLE patients and absorption with insolubilized human Cohn fraction II (Fr II). VL and VH regions of 5 monoclonal IgM antibodies with anti-F(ab')2/anti-DNA specificity generated by Epstein-Barr virus B cell stimulation were sequenced by polymerase chain reaction and characterized for VH and VL subgroup. APAF and APAD were also examined by high-resolution electron microscopy for possible ring forms indicative of antiidiotypic V-region interactions. RESULTS: APAF from normal subjects, representing 0.08-0.18% of serum IgG, showed striking relative concentrations of both anti-F(ab')2 and anti-DNA, as well as anti-Sm and anti-Sm/RNP ELISA reactivity. Both APAF and APAD reacting with F(ab')2 or dsDNA on the ELISA plate could be cross-inhibited by F(ab')2 or DNA in solution. Anti-DNA reactivity in normal APAF and APAD was much more sensitive to S1 nuclease treatment than similar fractions from SLE patients. Neither APAF nor APAD from controls produced positive antinuclear immunofluorescence or positive Crithidia staining, whereas these were strongly positive using SLE APAF and APAD. Absorbed rabbit antisera against normal or SLE APAF and APAD showed strong ELISA reactivity against both APAF and APAD, but no residual reactivity with normal Fr II. VL and VH sequencing of monoclonal human IgM antibodies showing both anti-F(ab')2 and anti-DNA reactivity showed relative VH3, V kappa 1 or VH1, V kappa 3 restriction. No evidence of ring forms or V-region "kissing" dimers was obtained when normal or SLE APAD or APAF was examined by high-resolution electron microscopy. CONCLUSION: IgG anti-F(ab')2 in both normal subjects and SLE patients represents a polyreactive Ig subfraction with concomitant anti-DNA, anti-Sm, and anti-Sm/RNP specificities. Anti-DNA reactivity in SLE is qualitatively different from that in normal APAD and APAF since normal APAD and APAF anti-DNA is much more sensitive to S1 nuclease digestion of denatured dsDNA. APAF and APAD share distinct V-region antigens which may be related to prominent VH3 or VH1 antigenic components. No evidence for in vivo complexing of anti-DNA and anti-F(ab')2 as ring forms or antiidiotype-IgG complexes was observed during ultrastructural studies. In both normal individuals and SLE patients, APAF may represent a small polyreactive IgG subfraction which also contains antinuclear and anti-DNA specificities.

High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR.

Touchdown (TD) PCR represents a versatile one-step procedure for optimizing PCRs even if the degree of primer-template complementarity is not fully known. The protocol relies on incremental annealing temperature decreases in progressive cycles designed to bracket the melting temperature (Tm) of the reaction. Here we investigate the characteristics of TD PCR that serve to minimize the need to optimize annealing temperature or buffer conditions and yet produce single strong target amplicons. We demonstrate that priming initiates above the optimum annealing temperature; this helps to ensure a competitive advantage for the target amplicon. On the other hand, as the cycling program progresses, annealing temperatures well below the Tm can serve to significantly increase yields in reactions that would otherwise be marginal due to suboptimal buffer composition and yet do not promote spurious amplification. Modified forms of TD PCR, termed stepdown PCR, consisting of fewer but steeper incremental declines in annealing temperature, are also shown to be effective and can simplify thermal cycler programming.

Characterization of biosynthetic IgG oligomers resulting from light chain variable domain duplication.

We have characterized a human IgG1 monoclonal antibody composed of altered light chains. Each light chain consists of two identical variable domains and a kappa constant domain, in association with a normal gamma chain. This antibody assembled biosynthetically into a mixture of stable oligomers and monomers. Employing gel filtration, PAGE, and electron microscopy, we examined the antibody and the nature of the associations involved in oligomer formation. By engineering a protease factor Xa site between the duplicated light chain variable domains and examining the fragments produced following factor Xa cleavage, we demonstrated the association of the IgG monomers occurred through their duplicated VL domains. Electron microscopy showed the oligomeric antibody to be predominantly dimers and trimers in which the monomeric units were associated through the tips of the Fab portion of the antibody, presumably through the protruding N-terminal VL domains. Similar examination of monomers demonstrated several molecular forms, including individual molecules with self-crosslinked Fab arms and others displaying the open Y and T shapes typically observed for IgG antibodies. The monomers also displayed distally protruding domain-like structures. The oligomers produced by this cell line therefore occurred through the noncovalent interaction between the extra light chain variable domains.