Well-known Allergens Research Articles

Neglected but Clinically Relevant Allergens in Korea.

Allergy diagnostics and immunotherapeutics in Asia heavily rely on imported products from Western countries, raising concerns about the accuracy and efficacy of these products for the management of Asian allergy patients. Recent advancements in allergen research have led to the identification and characterization of novel allergens from indigenous Korean species. While some allergens share homology with well-known allergens, others lack counterparts in imported allergen extracts. Classifying regional allergens in Asia into three categories based on their cross-reactivity with imported allergens offers valuable insights. Highly cross-reactive allergens, such as oak allergens Que m 1 from Quercus mongolica and Que ac 1 from Q. acutissima, can be effectively substituted with the imported allergens. Allergens with partial cross-reactivity, like the Asian needle ant allergen Pac c 3 (Antigen 5), permit limited diagnostic value by the currently available products. Unique allergens, including the Japanese hop allergen Hum j 6 (pectin methylesterase inhibitor) and the silkworm pupa allergen Bomb m 4 (30kDa hemolymph lipoprotein) lack alternatives in the available product list. Greater attention is needed, particularly for species listed as ecologically invasive in Western regions. Additionally, allergens from domestic fruits and vegetables causing pollen food allergy syndrome require characterization for the development of improved diagnostics.

Vegan diet - alternative protein sources as potential allergy risk

ZusammenfassungBackground: As a vegan diet is per definition a plant-based diet, consumers rely on plant protein sources in order to substitute animal proteins. Plant protein sources commonly used in this context are the following: cereals, like wheat (Triticum aestivum, Triticum durum), oat flakes; pseudo cereals like quinoa; nuts and oil seeds like cashew (Anacardium occidentale), hazelnut (Corylus avellana), walnut (Juglans regia); seeds like psyllium seeds (Plantago ovata), sesame (Sesamum indicum), and chia seed (Salvia hispanica).Methods: In order to assess the allergy risk posed by vegan diet, a literature search focusing on the composition of this particular diet and whether the respective foods are potentially allergenic was performed.Results: At first glance, it is evident for allergologists that these protein sources are well-known allergen sources. Particularly nuts and legumes harbour storage proteins, oleosins, and lipid trans- fer proteins that as such are associated with severe allergic reactions to food. In addition, there is increasing evidence that the simultaneous consumption of several of these foods may produce a summation effect where many single allergens of high allergenic potential sum up, thereby inducing anaphylaxis. Furthermore, food processing - or the lack of it - puts patients with pollen-associated food allergy at risk to react to some of the plant foods used in vegan diets.Conclusion: Therefore, individuals with a history of atopy should be educated regarding the allergy risk of a vegan diet.Cite this as Jappe U. Vegan diet - alternative protein sources as potential allergy risk. Allergo J Int 2023;32:251-7https://doi.org/10.1007/s40629-023-00248-7

Vegan diet—alternative protein sources as potential allergy risk

Abstract Background As a vegan diet is per definition a plant-based diet, consumers rely on plant protein sources in order to substitute animal proteins. Plant protein sources commonly used in this context are the following: cereals, like wheat (Triticum aestivum, Triticum durum), oat flakes; pseudo cereals like quinoa; nuts and oil seeds like cashew (Anacardium occidentale), hazelnut (Corylus avellana), walnut (Juglans regia); seeds like psyllium seeds (Plantago ovata), sesame (Sesamum indicum), and chia seed (Salvia hispanica). Methods In order to assess the allergy risk posed by vegan diet, a literature search focusing on the composition of this particular diet and whether the respective foods are potentially allergenic was performed. Results At first glance, it is evident for allergologists that these protein sources are well-known allergen sources. Particularly nuts and legumes harbour storage proteins, oleosins, and lipid transfer proteins that as such are associated with severe allergic reactions to food. In addition, there is increasing evidence that the simultaneous consumption of several of these foods may produce a summation effect where many single allergens of high allergenic potential sum up, thereby inducing anaphylaxis. Furthermore, food processing—or the lack of it—puts patients with pollen-associated food allergy at risk to react to some of the plant foods used in vegan diets. Conclusion Therefore, individuals with a history of atopy should be educated regarding the allergy risk of a vegan diet.

Gibberellin-regulated proteins: Emergent allergens.

About 10 years ago, a protein family was shown for the first time to contain allergenic members, gibberellin-regulated protein (GRP). The first reported member was from peach, Pru p 7. One can hypothesize that it was not detected before because its physicochemical characteristics overlap with those of lipid transfer protein (LTP), a well-known allergen, or because the exposure to GRP increased due to an increase in the gibberellin phythormone level in plant food, either exogenous or endogenous. Like LTPs, GRPs are small cationic proteins with disulfide bridges, are resistant to heat and proteolytic cleavage, and are involved in the defense of the plant. Besides peach, GRP allergens have been described in Japanese apricot (Pru m 7), sweet cherry (Pru av 7), orange (Cit s 7), pomegranate (Pun g 7), bell pepper (Cap a 7), strawberry (Fra a GRP), and also in pollen with a restriction to Cupressaceae tree family (Cup s 7, Cry j 7, and Jun a 7). IgE cross-reactivities were described between GRPs, and the reported peach/cypress and citrus/cypress syndromes may therefore be explained because of these GRP cross-reactivities. GRPs are clinically relevant, and severe adverse reactions may sometimes occur in association with cofactors. More than 60% and up to 95% sequence identities are calculated between various allergenic GRPs, and three-dimensional models show a cleft in the molecule and predict at least three epitopic regions. The structure of the protein and its properties and the matrix effect in the original allergenic source should be unraveled to understand why, despite the ubiquity of the protein family in plants, only a few members are able to sensitize patients.

Role of ventilation and cleaning for controlling house dust mite allergen infestation: A study on associations of house dust mite allergen concentrations with home environment and life styles in Tianjin area, China.

House dust mites produce well-known allergens for asthma and allergy among children. To study house dust mite allergen exposure level in northeast China and characterize its association with indoor environmental factors and cleaning habits, we inspected 399 homes in Tianjin area and collected dust from mattresses. Dermatophagoides farinae (Der f) and Dermatophagoides pteronyssinus (Der p) were detected by the enzyme-linked immunosorbent assay (ELISA) method. The medians of total allergen concentrations for spring, summer, autumn, and winter were 524 ng/g, 351 ng/g, 1022 ng/g, and 1010 ng/g. High indoor air relative humidity (RH), low air change rate, indoor dampness, and frequent changing of quilt cover/bedsheet/pillow case were significantly associated with high house dust mite allergen concentration (relative risk [RR]: RH, 1.18-1.34; air change rate, 0.97-1.00; dampness, 2.92-3.83; changing quilt cover/bedsheet/pillow case, 0.66-0.75). The decrease in the absolute humidity gradient between indoors and outdoors that occurs with increased air change rate may explain why a high ventilation reduces house dust mite allergen concentration. The findings of this study show the importance of ventilation and cleaning for controlling house dust mite allergens. We found that the decrease in additional absolute humidity (e.g., humidity indoor -humidity outdoor ) with increased air change rate may be the main reason that a high ventilation rate reduces house dust mite allergen concentration. Ventilation and cleaning should be both considered for creating a healthy home environment.

Candida-induced asthma steps up to the plate-lets

Fungal proteases are well-known allergens. In this issue of Immunity, Wu etal. (2021) observe that allergic airway responses to Candida albicans are mediated by the peptide toxin candidalysin rather than proteases. Candidalysin promotes these responses by stimulating platelets to release the Wnt antagonist Dickkopf-1.

Airborne Bacterial and Eukaryotic Community Structure across the United Kingdom Revealed by High-Throughput Sequencing

Primary biological aerosols often include allergenic and pathogenic microorganisms posing potential risks to human health. Moreover, there are airborne plant and animal pathogens that may have ecological and economic impact. In this study, we used high-throughput sequencing techniques (Illumina, MiSeq) targeting the 16S rRNA genes of bacteria and the 18S rRNA genes of eukaryotes, to characterize airborne primary biological aerosols. We used a filtration system on the UK Facility for Airborne Atmospheric Measurements (FAAM) research aircraft to sample a range of primary biological aerosols across southern England overflying surface measurement sites from Chilbolton to Weybourne. We identified 30 to 60 bacterial operational taxonomic units (OTUs) and 108 to 224 eukaryotic OTUs per sample. Moreover, 16S rRNA gene sequencing identified significant numbers of genera that have not been found in atmospheric samples previously or only been described in limited number of atmospheric field studies, which are rather old or published in local journals. This includes the genera Gordonia, Lautropia, and Psychroglaciecola. Some of the bacterial genera found in this study include potential human pathogens, for example, Gordonia, Sphingomonas, Chryseobacterium, Morganella, Fusobacterium, and Streptococcus. 18S rRNA gene sequencing showed Cladosporium to be the major genus in all of the samples, which is a well-known allergen and often found in the atmosphere. There were also genetic signatures of potentially allergenic taxa; for example, Pleosporales, Phoma, and Brassicales. Although there was no significant clustering of bacterial and eukaryotic communities depending on the sampling location, we found meteorological factors explaining significant variations in the community composition. The findings in this study support the application of DNA-based sequencing technologies for atmospheric science studies in combination with complementary spectroscopic and microscopic techniques for improved identification of primary biological aerosols.

Walnut antigens can trigger autoantibody development in patients with pemphigus vulgaris through a “hit-and-run” mechanism

Environmental factors, as well as genetic predisposition, are known to be critical for the development of autoimmunity. However, the environmental agents that trigger autoimmune responses have remained elusive. One possible explanation is the "hit-and-run" mechanism in which the inciting antigens that initiate autoimmune responses are not present at the time of overt autoimmune disease. After our previous findings that some allergens can incite autoimmune responses, we investigated the potential role of environmental allergens in triggering autoantibody development in patients with an autoimmune skin disease, pemphigus vulgaris (PV). Revertant/germline mAbs (with mutations on variable regions of heavy and light chains reverted to germline forms) of 8 anti-desmoglein (Dsg) 3 pathogenic mAbs from patients with PV were tested for reactivity against a panel of possible allergens, including insects, pollens, epithelia, fungi, and food antigens. All the PV germline mAbs were reactive to antigens from walnut, including the well-known allergen Jug r 2 and an uncharacterized 85-kDa protein component. Sera from patients with PV contained significantly greater levels of anti-Dsg3 autoantibodies than walnut-specific antibodies, suggesting that the autoreactive B-cell response in patients with PV might be initially triggered by walnut antigens but is subsequently driven by Dsg3. Our findings suggest that walnut antigens/allergens can initiate autoantibody development in patients with PV through a "hit-and-run" mechanism. The revertant/germline mAb approach might provide a paradigm for the etiological study of other allergic and autoimmune diseases.

Fungal Contamination of Airconditioner Units in Five Hospitals of Erbil Province- Kurdistan Region /Iraq

During April 2018, 75samples were collected from five hospitals in Erbil city. Samples were taken by sterile cotton swabs from the air dispenser window of air conditioner units (ACU) . They were cultured directly on Sabouraud's dextrose agar incubated at 25oC ±2. The samples were collected from five departments in each hospital include out-patient ward (OP),in-patient ward (IP), emergency room (ER), intensive care unit(ICU), and operation theater (OT). A total of (410) fungal colonies were counted, they belong to13 genera include ten hyphomycetes and only one of zygomycetes ,basidiomycetes , and hemiascomycetes. Penicillium represented in the highest total occurrence (40%) followed by Aspergillus(38.66%) and Alternaria (21.33%). The total frequency showed that Candidawas the highest (30%) followed by Penicillium(27.56%) and Aspergillus(13.17%). The highest density of filamentous fungi (no. of colony/sample) was detected in OP=16.75, and the lowest in OT=1.5. The diversity of isolates showed that ( 9) genera were recorded from ER(60%) and only one in OP (6%). All recorded genera in current study were regarded as indoor air pollutants. The predominant genera Alternaria, Aspergillus, Penicillium ,Cladosporiumand Candida are well-known allergens and may cause several pulmonary disorders as well as fetal infections in particular cases.

Microbial diversity in biodeteriorated Greek historical documents dating back to the 19th and 20th century: A case study.

Paper documents in archives, libraries, and museums often undergo biodeterioration by microorganisms. Fungi and less often bacteria have been described to advance paper staining, so called “foxing” and degradation of paper substrates. In this study, for the first time, the fungal and bacterial diversity in biodeteriorated paper documents of Hellenic General State Archives dating back to the 19th and 20th century has been assessed by culture‐dependent and independent methods. The internally transcribed spacer (ITS) region and 16S rRNA gene were amplified by PCR from fungal and bacterial isolates and amplicons were sequenced. Sequence analysis and phylogeny revealed fungal phylotypes like Penicillium sp., Cladosporium sp., Penicillium citrinum, Alternaria infectoria, Alternaria alternata, Epicoccum nigrum, and Penicillium chrysogenum which are often implicated in paper deterioration. Bacterial phylotypes closely related to known biodeteriogenic bacteria such as Bacillus spp., Micrococcus spp., Kocuria sp. in accordance with previous studies were characterized. Among the fungal phylotypes described in this study are included well‐known allergens such as Penicillium spp., Alternaria spp., and Cladosporium spp. that impose a serious health threat on staff members and scholars. Furthermore, fungal isolates such as Chalastospora gossypii and Trametes ochracea have been identified and implicated in biodeterioration of historical paper manuscripts in this study for the first time. Certain new or less known fungi and bacteria implicated in paper degradation were retrieved, indicating that particular ambient conditions, substrate chemistry, or even location might influence the composition of colonizing microbiota.

Thermal treatment reduces gliadin recognition by IgE, but a subsequent digestion and epithelial crossing permits recovery

Wheat is one of the most important crops in the world in terms of human nutrition. With regards to health, some individuals exhibit wheat-related disorders such as food allergy to wheat (FAW). In this disorder, gluten is involved, particularly the gliadins which are among the main proteins responsible for FAW. Food processing, as well as digestibility and intestinal transport are key factors to consider since they may affect the allergenic potential of food allergens.Wheat is always consumed after heat processing and this step may impact epitope accessibility by inducing aggregation and may irreversibly destroy conformational epitopes. Our aim was to investigate the effects of heating and digestion on the structure of well-known allergens (total gliadins and α-gliadins) and their capacity to maintain their allergenic potential after crossing an intestinal barrier.The sizes of the processed (heated and heated/digested) proteins were characterized by laser light scattering and chromatographic reverse phase. The IgE-binding capacities of native and processed proteins were checked using a dot blot with sera from wheat allergenic patients. Furthermore, the abilities of these samples to cross the intestinal barrier and to induce mast cell degranulation were investigated by combining two in vitro cellular models, Caco-2 and RBL-SX38.The heat treatment of total gliadins and α-gliadins induced the production of large aggregates that were hardly recognized by IgE of patients in dot-blot. However, after limited pepsin hydrolysis, the epitopes were unmasked, and they were able to bind IgE again. Native proteins (gliadins and α-type) and processed forms were able to cross the Caco-2 cells in small amount. Permeability studies revealed the capacity of α-gliadins to increase paracellular permeability. In the RBL assay, the total native gliadins were able to trigger cell degranulation, but none of their processed forms. However after crossing the CaCo-2 monolayer, processed gliadins recovered their degranulation capacity to a certain extent. Total native gliadins remained the best allergenic form compared to α-type.

In vitro detection of chemical allergens: an optimized assay using mouse bone marrow-derived dendritic cells.

Identification of the allergenic potency of chemicals is a key step in the safety assessment process. Predictive assays that require no or few animals are needed. To develop an alternative in vitro mouse bone marrow-derived dendritic cell (BMDC) assay to determine the allergenic potential of chemicals. BMDCs were exposed to well-known allergens and to non-allergenic chemicals. Surface marker expression and cytokine release of BMDCs were analysed after treatment. Eleven tested chemicals showed a significant stimulation index (SI) of >1.5 (accuracy, 75%; sensitivity, 69%). The four non-allergens all showed a SI of <1.5. Eight contact allergens tested showed a significant SI of >1.5 (accuracy, 92%; sensitivity, 89%), whereas only two respiratory allergens showed a significant SI of >1.5 (accuracy, 60%; sensitivity, 33%). The results indicate that the BMDC assay could become a reliable test for assessment of the allergenic potential of chemicals. The next step should include the testing of further chemicals, with the aim of integrating this assay into the toolbox of in vitro methods for the evaluation of the allergenic potential of chemicals.

Taxonomy of Allergenic Fungi

The Kingdom Fungi contains diverse eukaryotic organisms including yeasts, molds, mushrooms, bracket fungi, plant rusts, smuts, and puffballs. Fungi have a complex metabolism that differs from animals and plants. They secrete enzymes into their surroundings and absorb the breakdown products of enzyme action. Some of these enzymes are well-known allergens. The phylogenetic relationships among fungi were unclear until recently because classification was based on the sexual state morphology. Fungi lacking an obvious sexual stage were assigned to the artificial, now-obsolete category, "Deuteromycetes" or "Fungi Imperfecti." During the last 20 years, DNA sequencing has resolved 8 fungal phyla, 3 of which contain most genera associated with important aeroallergens: Zygomycota, Ascomycota, and Basidiomycota. Advances in fungal classification have required name changes for some familiar taxa. Because of regulatory constraints, many fungal allergen extracts retain obsolete names. A major benefit from this reorganization is that specific immunoglobulin E (IgE) levels in individuals sensitized to fungi appear to closely match fungal phylogenetic relationships. This close relationship between molecular fungal systematics and IgE sensitization provides an opportunity to systematically look at cross-reactivity and permits representatives from each taxon to serve as a proxy for IgE to the group.

Phenylephrine is a frequent cause of periorbital allergic contact dermatitis.

Phenylephrine is a selective α1-adrenergic receptor agonist used in eye drops, in order to dilate the pupil to facilitate visualization of the retina, and also to treat nasal congestion and increased sinus pressure resulting from rhinitis. However, in Denmark it is only marketed as mydriatic eye drops. Phenylephrine hydrochloride (HCl) is a well-known contact allergen. The Allergen Bank is a service established in 1992 for dermatologists in practice in Denmark, where, on a subscription basis, they can gain access to extra contact allergens, for example phenylephrine HCl, as supplements to the baseline series for case-by-case testing of individual patients (1). Phenylephrine HCl has been available from the Allergen Bank since 2000. We performed a retrospective analysis of patients patch tested with phenylephrine HCl from 2000 to 2014 both in private dermatological practices and in a department of dermatology. The vast majority of patients tested at the dermatology department were referred by the ophthalmologist because of acute periorbital dermatitis and suspected allergic contact dermatitis caused by eye drops. We assume that the same referral mechanism was in effect in dermatological practice; however, detailed information is not available.

The Impact of Atopic Dermatitis on Quality of Life

Approximately 5-20% of children worldwide suffer from atopic dermatitis (AD), a kind of dermatitis characterized as an inflammatory, relapsing, noncontagious and itchy skin disorder. Children often develop AD during their first year of life. An increased rate of sensitization to both food and aeroallergens has been shown to coexist in patients with AD. Sensitization to well-known allergens such as cow's milk protein can occur on average in 50% of children with AD. In general, quality of life (QoL) is perceived as the quality of an individual's daily life, that is, an assessment of their well-being or lack thereof. QoL is a broad concept that includes such things as standard of living, community, and family life. Patients with skin diseases experience a wide range of symptoms ranging from trivial problems to major handicaps which affect their lives. The misery of living with AD cannot be overstated for it may have a profoundly negative effect on the health-related QoL of children and their families in many cases. Physicians taking care of children with AD should consult parents on how their child's illness has impacted their lifestyle and recommend professional intervention if deemed necessary.

Overview of Plant Chitinases Identified as Food Allergens

Food allergies are induced by proteins belonging to a limited number of families. Unfortunately, relationships between protein structure and capacity to induce the immune response have not been completely clarified yet, which precludes possible improvements in the diagnosis, prevention, and therapy of allergies. Plant chitinases constitute a good example of food allergenic proteins for which structural analysis of allergenicity has only been carried out partially. In plants, there are at least five structural classes of chitinases plus a number of chitinase-related polypeptides. Their allergenicity has been mostly investigated for chitinases of class I, due to both their higher prevalence among plant chitinases and by the high structural similarity between their substrate-binding domain and hevein, a well-known allergen present in the latex of rubber trees. Even if allergenic molecules have been identified for at least three other classes of plant chitinases, the involvement of the different structural motifs in the allergenicity of molecules has been disregarded so far. In this review, we provide a structurally based catalog of plant chitinases investigated for allergenicity, which could be a useful base for further studies aimed at better clarifying the structure-allergenicity relationships for this protein family.

Contact allergy to ingredients of hair cosmetics – a comparison of female hairdressers and clients based on IVDK 2007–2012 data

Cosmetics for bleaching, waving/relaxing and dyeing hair contain well-known allergens, leading to a substantial number of cases of allergic contact dermatitis. To compare the frequency of important contact allergens (i) between two distinct groups of exposed patients, and (ii) with previous surveillance data. On the basis of data collected by the Information Network of Departments of Dermatology (IVDK; www.ivkd.org) between 2007 and 2012 in 824 female hairdressers and 2067 female clients, the current spectrum of contact sensitization to ingredients of hair cosmetics, as contained in different pertinent series, is described. A similar burden of sensitization as in previous analyses was observed, but with some increase in sensitization to oxidative hair dye components in clients. Some allergens mainly affected hairdressers, such as ammonium persulfate (18.7% positive) and glyceryl monothioglycolate (GMTG; still 4.7% positive, with a few cases also in young hairdressers, despite removal from the German market). Hair dyes remain important contact allergens, despite various attempts by the cosmetic industry to introduce hair dyes with lower allergenic potential. The re-emergence of GMTG as an occupational allergen should be considered as a warning signal ('sentinel event') prompting close monitoring.

Vulvar Dermatitis From Allergy to Moist Flushable Wipes

Methylchloroisothiazolinone/methylisothiazolinone is a preservative found in cosmetic and industrial products, and is a common ingredient in moist toilet paper. It is a well-known allergen and is capable of causing allergic contact dermatitis.We present the case of a 58-year-old white woman with a cutaneous vulvar eruption with associated discomfort and pruritus of 6 months in duration. She had been treated with antibiotic and antifungal agents without improvement of symptoms. Careful history taking revealed that the patient was using moist toilet paper. Patch testing confirmed an allergy to methylchloroisothiazolinone, a preservative in the moist toilet paper. After discontinuation of the product and treatment with potent topical steroids, the eruption completely cleared.With the growing use of moist toilet paper among adults, the risk of exposure and potential sensitization is increasing. Health care providers should be aware of the risks of moist toilet paper containing potential allergens because perianal and perineal dermatitis caused by these products may be unrecognized or misdiagnosed. After proper treatment, patients must be educated about alternatives and the importance of label reading.

Fish Allergy: In Review

Globally, the rising consumption of fish and its derivatives, due to its nutritional value and divergence of international cuisines, has led to an increase in reports of adverse reactions to fish. Reactions to fish are not only mediated by the immune system causing allergies, but are often caused by various toxins and parasites including ciguatera and Anisakis. Allergic reactions to fish can be serious and life threatening and children usually do not outgrow this type of food allergy. The route of exposure is not only restricted to ingestion but include manual handling and inhalation of cooking vapors in the domestic and occupational environment. Prevalence rates of self-reported fish allergy range from 0.2 to 2.29% in the general population, but can reach up to 8% among fish processing workers. Fish allergy seems to vary with geographical eating habits, type of fish processing, and fish species exposure. The major fish allergen characterized is parvalbumin in addition to several less well-known allergens. This contemporary review discusses interesting and new findings in the area of fish allergy including demographics, novel allergens identified, immunological mechanisms of sensitization, and innovative approaches in diagnosing and managing this life-long disease.

Electrochemistry/liquid chromatography/mass spectrometry to demonstrate irreversible binding of the skin allergen p‐phenylenediamine to proteins

para-Phenylenediamine (PPD) is a potent and well-known allergen, which is commonly used in hair or fur dyes and can cause severe allergic contact dermatitis. In this work, the skin-sensitizing potential of PPD with respect to the conjugation of proteins was evaluated using an approach without animal testing. Electrochemistry (EC) coupled offline to liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS) was employed to convert the pre-hapten PPD into its reactive hapten analogs. A previous study had already shown that this purely instrumental method is suitable for accelerating and simulating the various oxidation processes, which PPD may undergo, and that the emerging products are prone to react with soft thiol groups of small nucleophiles like glutathione and cysteine. This investigation was extended by successfully demonstrating adduct formation between EC-generated PPD oxidation products and the three proteins β-lactoglobulin A (β-LGA), human serum albumin and human hemoglobin. A tryptic digest of modified β-LGA provided evidence for irreversible protein binding of monomeric PPD, a PPD dimer and the PPD trimer known as Bandrowski's Base. It was shown that the main oxidation product p-phenylene quinone diimine, and the reactive oligomerized species, primarily attack the free thiol function of proteins rather than other nucleophilic amino acid residues. The pre-hapten PPD was efficiently activated upon EC oxidation and the resulting species were further reacted with different proteins leading to diverse hapten-protein complexes. Thereby, problems related to the complex matrix present in conventional in vitro or in vivo methods could effectively be avoided.