Serological tests using Sporothrix species antigens for the accurate diagnosis of sporotrichosis: a meta-analysis.

Some species of the fungus Sporothrix cause a chronic granulomatous infection in humans and animals called sporotrichosis. In the last decades, some research into serological tests has been carried out by different groups for the rapid detection of this infection. We performed a systematic review of the literature with meta-analysis to evaluate studies using Sporothrix spp. antigens and to evaluate their accuracy for sporotrichosis diagnostic. We searched Scopus, MEDLINE, Web of Science, GALE, Technology Research Database, DOA, Elsevier, SciELO, and Google Scholar Databases. The united results of sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio with their corresponding 95% confidence intervals (CI) were assessed. A total of 15 assays from 8 studies using 7 different serological methods and 8 different antigens were analyzed. The studies were performed in the USA, Brazil, and Venezuela from 1973 until 2015 and presented good quality. A high heterogeneity for sensitivity [I2 = 90.7%; 87% CI = (84-89), P < 0.001] and specificity [I2 = 89.2%; 93% CI = (92-95), P < 0.001] was observed. The performance of diagnostic tests was 0.93. Enzyme-linked immunosorbent assay was the main tool used, and the ConA-binding fraction antigen of the strain 1099-18 appears as a promising diagnostic biomarker candidate.

Toxoplasma gondii Histone 4 Affects Some Functions of Murine Ana-1 Macrophages In Vitro.

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan that can infect almost all nucleated cells. Histone proteins and DNA form the nucleosomes, which are the fundamental building blocks of eukaryotic chromatin. Histone 4 is an essential component of a histone octamer. In the present study, T. gondii histone 4 (TgH4) was cloned and the regulatory effect of TgH4 on murine macrophages was characterized. Bioinformatics analysis revealed that TgH4 was highly conserved in structure. Recombinant TgH4 (rTgH4) protein was identified by sera from rats experimentally infected with T. gondii and native TgH4 in the total soluble protein of T. gondii tachyzoites was recognized by polyclonal antibodies against rTgH4, as indicated by immunoblotting analysis. Immunofluorescence assay showed that TgH4 binds to macrophages. Following incubation with rTgH4, the toll-like receptor 4 (TLR4) level of the macrophages was downregulated. Meanwhile, chemotaxis and the proliferation of macrophages were inhibited. However, rTgH4 can promote phagocytosis, apoptosis, and the secretion of nitric oxide, interleukin-6, and tumor necrosis factor-α from macrophages. Just 80 µg/ml rTgH4 can significantly elevate the secretion of interleukin-10 and interleukin-1ß (p < 0.05 and p < 0.01). Viewed together, these outcomes indicated that rTgH4 can affect the functions of murine macrophages in vitro.

Characterization of Epitope-Specific Anti-Respiratory Syncytial Virus (Anti-RSV) Antibody Responses after Natural Infection and after Vaccination with Formalin-Inactivated RSV.

UNLABELLED: Antibodies against the fusion (F) protein of respiratory syncytial virus (RSV) play an important role in the protective immune response to this important respiratory virus. Little is known, however, about antibody levels against multiple F-specific epitopes induced by infection or after vaccination against RSV, while this is important to guide the evaluation of (novel) vaccines. In this study, we analyzed antibody levels against RSV proteins and F-specific epitopes in human sera and in sera of vaccinated and experimentally infected cotton rats and the correlation thereof with virus neutralization. Analysis of human sera revealed substantial diversity in antibody levels against F-, G (attachment)-, and F-specific epitopes between individuals. The highest correlation with virus neutralization was observed for antibodies recognizing prefusion-specific antigenic site Ø. Nevertheless, our results indicate that high levels of antibodies targeting other parts of the F protein can also mediate a potent antiviral antibody response. In agreement, sera of experimentally infected cotton rats contained high neutralizing activity despite lacking antigenic site Ø-specific antibodies. Strikingly, vaccination with formalin-inactivated RSV (FI-RSV) exclusively resulted in the induction of poorly neutralizing antibodies against postfusion-specific antigenic site I, although antigenic sites I, II, and IV were efficiently displayed in FI-RSV. The apparent immunodominance of antigenic site I in FI-RSV likely explains the low levels of neutralizing antibodies upon vaccination and challenge and may play a role in the vaccination-induced enhancement of disease observed with such preparations. IMPORTANCE: RSV is an importance cause of hospitalization of infants. The development of a vaccine against RSV has been hampered by the disastrous results obtained with FI-RSV vaccine preparations in the 1960s that resulted in vaccination-induced enhancement of disease. To get a better understanding of the antibody repertoire induced after infection or after vaccination against RSV, we investigated antibody levels against fusion (F) protein, attachment (G) protein, and F-specific epitopes in human and animal sera. The results indicate the importance of prefusion-specific antigenic site Ø antibodies as well as of antibodies targeting other epitopes in virus neutralization. However, vaccination of cotton rats with FI-RSV specifically resulted in the induction of weakly neutralizing, antigenic site I-specific antibodies, which may play a role in the enhancement of disease observed after vaccination with such preparations.

Prophylactic Injection of Recombinant Alpha-Enolase Reduces Arthritis Severity in the Collagen-Induced Arthritis Mice Model.

OBJECTIVE: To evaluate the ability of the glycolytic enzyme alpha-enolase (ENO1) or its immunodominant peptide (pEP1) to reduce the severity of CIA in DBA/1 mice when injected in a prophylactic way. METHODS: Mice were treated with mouse ENO1 or pEP1 one day prior to collagen II immunization. Clinical assessment was evaluated using 4 parameters (global and articular scores, ankle thickness and weight). Titers of serum anti-ENO1, anti-cyclic citrullinated peptides (anti-CCP) and anti-CII (total IgG and IgG1/IgG2a isotypes) antibodies were measured by ELISA at different time-points. Disease activity was assessed by histological analysis of both anterior and hind paws at the end of experimentation. RESULTS: Prophylactic injection of 100 µg of ENO1 reduced severity of CIA. Serum levels of anti-CII antibodies were reduced in ENO1-treated mice. Concordantly, ENO1-treated mice joints presented less severe histological signs of arthritis. ENO1 did not induce a shift toward a Th2 response since IgG1/IgG2a ratio of anti-CII antibodies remained unchanged and IL-4 serum levels were similar to those measured in the control group. CONCLUSIONS: Pre-immunization with ENO1 or its immunodominant peptide pEP1 reduces CIA severity at the clinical, immunological and histological levels. Effects of pEP1 were less pronounced. This immunomodulatory effect is associated with a reduction in anti-CII antibodies production but is not due to a Th1/Th2 shift.

Novel immunodominant epitopes derived from MAGE-A3 and its significance in serological diagnosis of gastric cancer.

PURPOSE: To evaluate the significance of MAGE-A3 novel immunodominant epitopes in serological diagnosis of gastric cancer. METHODS: B cell, CTL, and Th epitopes of MAGE-A3 were analyzed using computer-assisted techniques. Three possible immunodominant epitope peptides located at 5aa-23aa (QRSQHCKPEEGLEARGEAL), 112aa-131aa (KVAELVHFLLLKYRAREPVT), and 232aa-246aa (EGREDSILGDPKKLL) with potential B cell-dominant epitope, high-score HLA-A2 and A24 restriction CTL epitope, and HLA-DRB restriction Th epitope were selected. After optimized by prokaryotic codon, these genes were expressed as Trx-His-tag recombinant proteins in Escherichia coli and purified by Ni-NTA agarose beads. Three recombinant proteins were identified by Western blotting using His-tag monoclonal antibody and the serum antibodies from the patient of gastric cancer. The level of specific antibodies in the sera from 210 patients with gastric cancer, 56 patients with chronic gastritis, and 116 healthy controls was further analyzed by indirect ELISA. RESULTS: Three MAGE-A3 epitope recombinant proteins about 20 kDa molecular weight were specifically recognized by His-tag monoclonal antibody and the serum of gastric cancer patients. ELISA based on the epitope recombinant protein indicated that gastric cancer patients had significantly higher reactivity to these immunodominant epitope proteins compared with chronic gastritis and healthy individuals (P < 0.05). Furthermore, the serum antibody positive rate in the gastric cancer group was also significantly higher than that in the chronic gastritis patients and healthy controls (P < 0.05), while there was no significant difference in gastritis group and the healthy control group (P > 0.05). CONCLUSIONS: These study results demonstrated that these three predictive epitopes may be potential targets for applications in the design of serological diagnosis tools for gastric cancer.

Serologic markers of effective tumor immunity against chronic lymphocytic leukemia include nonmutated B-cell antigens.

Patients with chronic lymphocytic leukemia (CLL) who relapse after allogeneic transplant may achieve durable remission following donor lymphocyte infusion (DLI), showing the potency of donor-derived immunity in eradicating tumors. We sought to elucidate the antigenic basis of the effective graft-versus-leukemia (GvL) responses associated with DLI for the treatment of CLL by analyzing the specificity of plasma antibody responses developing in two DLI-treated patients who achieved long-term remission without graft-versus-host disease. By probing high-density protein microarrays with patient plasma, we discovered 35 predominantly intracellular antigens that elicited high-titer antibody reactivity greater in post-DLI than in pre-DLI plasma. Three antigens-C6orf130, MDS032, and ZFYVE19-were identified by both patients. Along with additional candidate antigens DAPK3, SERBP1, and OGFOD1, these proteins showed higher transcript and protein expression in B cells and CLL cells compared with normal peripheral blood mononuclear cells. DAPK3 and the shared antigens do not represent minor histocompatibility antigens, as their sequences are identical in both donor and tumor. Although ZFYVE19, DAPK3, and OGFOD1 elicited minimal antibody reactivity in 12 normal subjects and 12 chemotherapy-treated CLL patients, 5 of 12 CLL patients with clinical GvL responses were serologically reactive to these antigens. Moreover, antibody reactivity against these antigens was temporally correlated with clinical disease regression. These B-cell antigens represent promising biomarkers of effective anti-CLL immunity.

Pancreatic duodenal homeobox 1 protein is a novel beta-cell-specific autoantigen for type I diabetes.

Pancreatic duodenal homeobox 1 (Pdx1) protein is a key transcription factor involved in the regulation of insulin gene expression that is expressed at high levels in the beta-cells of the pancreatic islets. We asked whether Pdx1 is a target of anti-islet autoimmunity in type I diabetes (T1D). Pdx1 autoantibodies (PAAs) were detected in non-obese diabetic (NOD) mice using ELISA, western blotting, and radioimmunoprecipitation of [(35)S]-labeled insulinoma cell line-derived Pdx1 protein. PAAs were detected as early as at 5 weeks of age, and generally peaked before the onset of clinically overt diabetes in diabetes-prone female NOD mice. Levels declined substantially after the onset of diabetes. PAAs were not detected in the sera of NOD-scid, C57BL/6, or BALB/c mice. The titers of PAAs in NOD mouse sera were as high as 1/93 750 by ELISA. The fine specificity of PAAs was determined by western blotting using a series of truncated recombinant Pdx1 proteins. The immunodominant epitopes were located to the C-terminus of the Pdx1 (p200-283) in NOD mice. PAAs also were detected in sera from human T1D patients, but the major epitopes were localized to amino acids 159-200 as well as the same region (p200-283) recognized by PAAs from NOD mice. Using [(3)H]thymidine incorporation, the p83 fragment of Pdx1 specifically stimulated proliferation of splenic T cells from recent-onset diabetic NOD mice. The presence of PAAs in prediabetic NOD mice and human T1D patients, and Pdx1-specific T-cell proliferation in NOD mice provide a strong rationale for further investigation of the pathogenic role of immune responses against Pdx1 in T1D.

The kinetics of Theileria parva infection and lymphocyte transformation in vitro.

Theileriaparva is an intracellular protozoan parasite that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. The parasite infects host lymphocytes causing their transformation and uncontrolled proliferation. Infiltration of major organs with parasitized lymphoblasts results in most cases in death within 3 weeks. Although both T and B lymphocytes are susceptible to infection, the majority of cell lines arising from infection of peripheral blood mononuclear cells in vitro are of T cell lineage. To explore the basis of this phenotypic bias we have followed the very early stages of parasite development in vitro at the single cell level. Peripheral blood mononuclear cells were infected and stained for both surface phenotype and intracellular parasite antigen and analysed by flow cytometry. Although the parasite antigen was detected intracellularly as early as 6h p.i., our data indicate that parasite infection does not lead to cell transformation in all instances. Rather, specific cell types appear to undergo selection very early after infection and expansion of particular cell subsets results in survival and growth of only a small proportion of the cells originally parasitized.

One NY-ESO-1-derived epitope that promiscuously binds to multiple HLA-DR and HLA-DP4 molecules and stimulates autologous CD4+ T cells from patients with NY-ESO-1-expressing melanoma.

NY-ESO-1 is expressed by a broad range of human tumors and is often recognized by Abs in the sera of cancer patients with NY-ESO-1-expressing tumors. The NY-ESO-1 gene also encodes several MHC class I- and class II-restricted tumor epitopes recognized by T lymphocytes. In this study we report one novel pan-MHC class II-restricted peptide sequence, NY-ESO-1 87-111, that is capable of binding to multiple HLA-DR and HLA-DP4 molecules, including HLA-DRB1*0101, 0401, 0701, and 1101 and HLA-DPB1*0401 and 0402 molecules. We also demonstrate that peptide NY-ESO-1 87-111 stimulates Th1-type and Th-2/Th0-type CD4(+) T cells and clones when presented in the context of these HLA-DR and HLA-DP4 molecules. Both bulk CD4(+) T cells and CD4(+) T cell clones were capable of recognizing not only peptide-pulsed APCs, but also autologous dendritic cells, either loaded with the NY-ESO-1 protein or transfected with NY-ESO-1 cDNAs. Using IFN-gamma and IL-5 ELISPOT assays and PBL from patients with NY-ESO-1-expressing tumors, we observed the existence of Th1-type circulating CD4(+) T cells recognizing peptide NY-ESO-1 87-111 in the context of HLA-DP4 molecules. Taken together, these data represent the first report of an HLA-DR- and HLA-DP-restricted epitope from a tumor Ag. They also support the relevance of cancer vaccine trials with peptides NY-ESO-1 87-111 in the large number of cancer patients with NY-ESO-1-expressing tumors.

Production of recombinant protein Pap31 and its application for the diagnosis of Bartonella bacilliformis infection.

Tropical bartonellosis is a highly fatal epidemic and endemic infectious disease that occurs throughout the communities of the Andes Mountains in South America. The disease is caused by the facultative intracellular bacteria, Bartonella bacilliformis. The emergence of bartonellosis in new geographic areas and an increase in the number of reported cases suggest the need for a rapid test for epidemiologic study and investigation of the disease burden. The objective of this research is to develop a rapid serologic diagnostic test using recombinant antigens to overcome the limitations of the current standard IFA technique for laboratory diagnosis. Western blot analysis with patient sera of whole cell lysate separated on a 2D gel identified Pap31 as a dominant antigen. PCR primers were designed according to the sequence of ATCC strain 35685 to amplify the gene coding for Pap31 from a local isolate (HOSP 800-09, Peru). The amplicon was subsequently cloned into pET24a, adding the T7 tag, and expressed in E. coli. Patient sera with different IFA titers confirmed the diagnostic band of 31 kDa on a Western blot of SDS-PAGE. The performance of affinity-purified recombinant Pap31 (rPap31) was also evaluated in an ELISA format with 137 patient sera of known IFA titers. The range of ELISA reading from positive sera did not overlap with the range of those from negative sera, suggesting the potential application of rPap31 in both ELISA for high throughput regional hospital settings and in the construction of handheld rapid tests for rural clinical sites.

Analysis of immunodominance among minor histocompatibility antigens in allogeneic hematopoietic stem cell transplantation.

In major histocompatibility complex (MHC)-matched allogeneic hematopoietic stem cell transplantation (HSCT), donor responses are directed against multiple host minor histocompatibility antigens (mHAgs), producing graft-versus-host disease (GVHD) and graft-versus-tumor (GVT) effects. We studied MHC-matched, mHAg-mismatched C3H.SW>C57BL/6 HSCT in which three mHAg are molecularly defined (B6dom1, H3, H13) to determine if there is a hierarchy of immunodominance among the mHAgs and to learn the contribution of each to GVHD. We found that B6dom1 was the immunodominant mHAg. B6dom1 did not block responses to the subdominant mHAgs H3 and H13. The mechanism of immunodominance was not mHAg avidity or affinity for class I. B6dom1 elicited a broader variety of Vbeta clonotypes than either H3 or H13. Severe GVHD could occur in the absence of a strong B6dom1 response. Alloreactivity to isolated B6dom1, H3 or H13 differences did not produce severe GVHD. We concluded that immunodominance is explained by both mHAg density on host cells and the repertoire of donor T cells capable of responding to the mHAgs. Clinically significant GVHD requires donor responses to multiple mHAgs. Modulation of responses to a single immunodominant mHAg is insufficient for the prevention of GVHD, while immunotherapies directed against isolated mHAgs may not provoke severe GVHD.

Monoclonal antibodies to Rh D--development and uses.

Monoclonal anti-D has proved impossible to make in rodent systems. Human monoclonal anti-D has been produced using EBV transformed peripheral B cells, coupled with fusions to myeloma cell lines. More recently molecular biology techniques have been used to produce monoclonal anti-D. The range of monoclonal anti-D produced is considered. The selection of monoclonal anti-D for use as blood grouping reagents for typing donors and recipients is reviewed--all types of D positive should be typed as positive when donors are considered. However, DVI patients should be typed as D negative. Considerations for the development of monoclonal anti-D for prophylactic use are reviewed.

Characterization of a dodecapeptide containing a dominant epitope of Par j 1 and Par o 1, the major allergens of P. judaica and P. officinalis pollen.

The pollen of Parietaria, a weed of the Urticaceae family, is a major cause of respiratory allergy in Europe, where the most common species are P. judaica and P. officinalis. Previously, we reported that a beta-galactosidase fusion protein (6a-BG) expressing a 26-bp cDNA fragment (6a cDNA) contained a dominant IgE-binding epitope (6a epitope) of the major allergens Par o 1 and Par j 1. The present study aimed to define the amino-acid sequence containing the 6a epitope. We analyzed the reactivity of anti-Par o 1 antibodies affinity purified from allergic patient sera with: 1) a panel of synthetic peptides deduced from the 6a nucleotide sequence using different reading frames 2) glutathione S-transferase (GST) fusion proteins containing selected peptides. The peptide NSARARADSCRI (p102) specifically bound anti-Par o 1 antibodies affinity purified from allergic patient sera or from rabbit anti-Par o 1 antiserum (ELISA). The related peptide NSARAGTSSCRI (p101) reacted to human but not to rabbit, anti-Par o 1 antibodies. GST fusion proteins containing p101 (GST 3.5) or p102 (GST 3.2) extensively inhibited the binding between Par o 1 and IgE or IgG antibodies from an allergic patient serum pool according to a dose-response curve. Percent inhibition of IgE antibodies binding obtained by absorbing a solution (50 microl) of affinity-purified antibodies with 5 microg of GST 3.2 or with 1.2 mg of GST 3.5 was 69% and 66%, respectively. In conclusion, the results of the present study indicate that the amino-acid sequences NSARARADSCRI (p102) and NSARAGTSSCRI (p101) contain the dominant epitope of Par o 1 and Par j 1 for human IgE and IgG antibodies indicated as 6a epitope. Moreover, the study shows that the epitope is conserved in recombinant molecules containing these peptides, irrespective of the fused polypeptide (beta-galactosidase or GST). The knowledge of the amino-acid sequence of this dominant epitope is important in therapeutic approaches to the development of allergen-derived haptens.

Identification of epitopes on a mutant form of Pseudomonas exotoxin using serum from humans treated with Pseudomonas exotoxin containing immunotoxins.

PE38 is a 38-kDa derivative of the 66-kDa Pseudomonas exotoxin (PE) in which the cell binding domain of PE (domain Ia, amino acids 1-252) and a portion of domain Ib (amino acids 365-380) are deleted. The immunotoxins LMB-1 and LMB-7 contain PE38 and kill cancer cells by exploiting the cytotoxic action of PE38. The major human B cell epitopes of PE38 were mapped by measuring the reactivity of 45 serum samples from patients treated with the PE38-containing immunotoxins LMB-1 or LMB-7 to two panels of overlapping synthetic peptides representing the sequence of PE38. One panel of peptides is ten amino acids long and overlap by seven amino acids, and the second panel of peptides is twenty amino acids long and overlap by ten. Five major epitopes were identified: amino acids 274-283, 470-492, 531-540, 555-564, and the C-terminal amino acids 596-609. Two minor epitopes were identified as well: amino acids 501-510 and 582-589. These epitopes are predominantly located on the surface of the protein. The amino acids believed to be critical for binding are highly solvent-accessible residues. The results of the human antibody response to peptides are compared to the pattern of reactivity previously identified with serum samples obtained from monkeys administered LMB-1 and LMB-7. The epitopes between monkey and human are almost identical, demonstrating similarity in the response of antibody repertoires between the two species and providing further support that these are the immunodominant epitopes. This information is critical for genetically engineering less immunogenic immunotoxins and provides a foundation for the development of a vaccine against pseudomonal infections which plague immunocompromised individuals and individuals with cystic fibrosis.

Identification of HCV core mimotopes: improved methods for the selection and use of disease-related phage-displayed peptides.

Disease-specific epitope discovery from random peptide libraries displayed on phage using sera from patients involves a number of screening steps with many immune and non-immune sera. To rapidly identify mimotopes of the human hepatitis C virus (HCV) core protein, we have used an anti-core human monoclonal antibody (mAb; B12.F8) as a probe in screening phage that were affinity-selected using a serum from an HCV infected patient. Three different positive phage were isolated displaying low or no homology with the natural antigen, but which still efficiently bound to the antigen binding site of the B12.F8 antibody. Testing the reactivity of these phage with forty-five sera from HCV infected patients showed that antibodies recognizing them are present in more than 80% of this population. These antibodies showed distinct fine specificity, as they bound the selected phage in a mutually exclusive fashion. Co-expression of two mimotopes in the same cells led to chimeric particles which were recognized by antibodies of different specificity. These data provide novel information on the potential use of the phage display technology for the characterization of antibody specificity as well as disease diagnosis and prevention.

[The use of synthetic peptides for detection of antibodies against foot-and-mouth disease virus in the blood from convalescent animals]. / Ispol'zovanie sinteticheskikh peptidov dlia vyiavleniia antitel k virusu iashchura v syvorotkakh krovi perebolevshikh zhivotnykh.

Peptides were synthesized, which, according to theoretical analysis of the antigenic structure of protein VP1 of foot-and-mouth disease (FMD) virus types A, 0, and Asia 1, corresponded to potential immunodominant protein sites. Activities of the peptides were studied by solid-phase indirect radioimmunoassay on polyethylene film with purified immunoglobulins against intact FMD virus. Virtually no cross reactions were observed. Blood sera of cattle convalescent after FMD were tested with the FMD virus and peptides containing VP1 fragments 141-160 (A22 No. 550), 140-160 (O1 No. 194), and 140-153 (Asia 1 No. 48). The specificity of interactions between the sera and peptides and the virus was uniform, this permitting the identification of the virus type which caused the disease.

Absence of continuous epitopes in the house dust mite major allergens Der p I from Dermatophagoides pteronyssinus and Der f I from Dermatophagoides farinae.

BACKGROUND: The house dust mite has been shown to be an important source of domestic allergens associated with immediate hypersensitivities. The Group I mite allergens Der p I from Dermatophagoides pteronyssinus and Der f I from D. farinae display extensive amino acid sequence homology and have similarities with cysteine protease enzymes. OBJECTIVE: The availability of the complete amino acid sequences for these allergens allowed us to search for the allergic determinants within these molecules. The aim of the present investigation was to identify any continuous IgE-binding epitopes within these amino acid sequences. We also sought to test the validity of previously reported Der p I peptide epitope sequences. METHODS: In order to identify any continuous IgE epitopes, the amino acid sequences of Der p I and Der f I were synthesized as decapeptides overlapping in sequence and coupled to plastic pins. The specific IgE-binding capacity of these peptides was assayed using an enzyme-linked biotin-streptavidin procedure and sera from patients known to be sensitive to these allergens. Previously reported Der p I peptide epitopes were synthesized as free peptides and tested for their ability to inhibit specific IgE binding to allergen extract discs. RESULTS: None of the pin-coupled Der p I or Der f I peptides was found by the continuous epitope mapping procedure to bind significantly to specific IgE in the sera of hypersensitive patients. The previously reported Der p I peptide epitopes did not inhibit specific IgE binding to mite extract discs. CONCLUSION: The specific IgE binding epitopes of the house dust mite allergens Der p I and Der f I are discontinuous in nature.