Development of a monoclonal antibody-based sandwich ELISA for the detection of ovalbumin in foods

Hen egg is one of the most frequent causes of food allergy in infants and adults. Ovalbumin (OVA) has been identified as a major egg allergen. In order to detect OVA in foods, a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mAbs) was established. The 2 mAbs were selected out of 17 murine hybridomas secreting OVA-specific antibody. Using mAb17 as the capture antibody and mAb15 as the detection antibody, the detection limit of the ELISA method was 0.51 ng/mL, and the linear dynamic range was between 1.95 and 500 ng/mL. The recovery ranged from 85.6 to 115.2%, whereas the intra- and inter-assay coefficients of variation were less than 8.6 and 13.9%, respectively. Sample analysis verified that the produced anti-OVA mAb and the developed ELISA may provide a valuable tool for the sensitive determination of OVA in processed foods and for future studies on the mechanism of how OVA functions in anaphylaxis.     

Quantification in mass units of Bet v 1, the main allergen of Betula verrucosa pollen, by a monoclonal antibody based-ELISA

Background Fagales pollens are considered among the main agents responsible for allergic diseases in many countries of the northern hemisphere and single major allergens have been shown to be responsible for these responses. Objective To develop a solid phase immunoassay for the quantification of Bet v I, the main allergen from Betula verrucosa (birch), and to assess its suitability for quantitating the equivalent major allergen in other Fagales species as well. Methods The assay is based on the use of two different anti-Bet v 1 monoclonal antibodies which were immobilized on the solid phase and. as a primary standard, affinity purified Bet v 1, the protein content of which was determined by amino acid analysis. Results The ELISA proved to measure less than 0.2 ng/mL of Bet v 1 with a practical range of 0.4–40 ng/mL and could be suitable lo quantify the equivalent major allergen in other Fagales species such as Corylus avellana (hazel), Carpinus betulus (horbeam) and Alnus glutinosa (alder). The method was compared witb quantitative electrophoresis and rocket immuno-electrophoresis for the determination of the allergen content in several Betula verrucosa extracts, and a very good quantitative correlation was found. Likewise, the Bet V 1 content exhibited a good correlation (r = 0.87; P < 0.005) with the allergenic potency values obtained by RAST inhibition. Conclusions The results indicate that the Bet v 1-assay could be useful for standardization purposes in Fagales pollen extracts intended for clinical use.     

Establishment of a sandwich ELISA for the determination of beef content in processed foods by using monoclonal antibodies to myoglobin

A sandwich enzyme-linked immunosorbent assay specific to beef myoglobin was developed using monoclonal antibodies to sodium dodecyl sulphate-denatured beef myoglobin and a peptide with an amino acid sequence unique to beef myoglobin. This method was very specific for beef myoglobin, and showed no cross-reactivity with not only pork and chicken myoglobins, but also with other food proteins such as egg, cow's milk, wheat and peanut. The limit of detection and that of quantification for the beef protein was 9.5 and 12.8 ng/ml, respectively. When the content of beef protein in pork- and chicken-based model processed foods containing varying amounts of beef were determined by this method, the recovery of beef protein agreed well with the actual ratio of added beef regardless of the processing conditions. Also, the amount of beef protein in several commercially processed foods could be reasonably determined and confirmed by this method.     

Species-specific measurement of the second group of Dermatophagoides mite allergens, Der p 2 and Der f 2, using a monoclonal antibody-based ELISA

Background The group 2 Dermatophagoides mite allergens. Der p 2 and Der f 2, were known to he highly crossreactive, and previous assays to measure Der p 2 and Der f 2 were not species-specific. Objective The aim of this study was to develop a monoclonal antibody-based ELISA (MoAb-ELISA) to specics-spccifically measure Der p 2 and Der f 2. Methods The MoAb-ELISA lor Der p 2 and Der f 2 was performed using species-specific MoAbs for Der p 2 and Der f 2 and a biotinylated second MoAb which recognized a common epitope on both Der p 2 and Der f 2. Rcsuits The assay was highly specics-spccific, reproducible and sensitive. Thirty-two house dust samples were assayed by the MoAb-ELISA for Der p 2 and Der f 2 and by a previously reported radioimmunoassay for Der 2 with rabbit anti-Der 2 antibodies. The summed values for Der p 2 and Der f 2 by the MoAb-ELISA detnonstrated a good correlation with the Der 2 values using the radioimmunoassay (r = 0.978). Furthermore, the proportion of the Der p 2 level in the total Der 2 level (Der p 2 divided by Der p 2 plus Der f 2) correlated well with that of the D. pteronyssinus mite number to the total Dermalophagoides mite number identificd by species (r = 0.970). Conclusion The MoAb-ELISA for Der p 2 and Der f 2, as well as that for Der p 1 and Der f 1, will be useful for the standardization of mite extracts and for the assessments of mite allergen exposure.     

Quantification of a major bovine allergen by a two-site immunometric assay based on monoclonal antibodies

A two-site immunometric assay based on monoclonal antibodies (mAb) was developed for the measurement of a 20-kDa major allergen of cow. The sensitivity of this assay was (BDA20) 1 ng/ml. It was used to measure airborne allergen concentrations in 10 Finnish cowsheds. The mean concentration of the BDA20 measured at two stationary sites was 280 ng/m³. Concentrations varied more than 10-fold among cowsheds (54–804 ng/m³). The mean intertest coefficient of variation was 8.2%, and the mean intratest variation 4.1 %.     

A novel monoclonal antibodies-based sandwich ELISA for detection of serotype 4 fowl adenovirus

As a major causative agent for hepatitis-hydropericardium syndrome (HPS) in chickens, serotype 4 fowl adenovirus (FAdV-4) has caused huge economic losses in the poultry industry globally. However, there is no commercial diagnostic test for FAdV-4 antigens. To generate a rapid approach for specific detection of FAdV-4, a monoclonal antibodies (mAbs)-based sandwich ELISA was developed. In this ELISA, a purified mAb 4A3 and a HRP-labelled mAb 3C2 specific to the fiber-2 of FAdV-4 were used as the capture antibody and detection antibody respectively. Specificity assay revealed the ELISA only reacted with FAdV-4, not with other avian viruses tested. Sensitivity assay showed the limit of detection of the ELISA was 1000 TCID₅₀/ml and 12.5?ng/ml for the FAdV-4 and the purified GST-Fiber2 protein respectively. Moreover, the ELISA could be efficiently applied in detecting the FAdV-4 in tissue samples from a clinically-diseased chicken flock. All these data demonstrated that the ELISA developed here provides a promising tool for rapid and efficient diagnosis of clinical infection with FAdV-4.     

Group 5 determination in Pooideae grass pollen extracts by monoclonal antibody-based ELISA. Correlation with biologic activity

A solid-phase, monoclonal antibody-based ELISA was set up to quantitale group 5 allergens in pollen extracts of wild and cultivated Pooideae grasses. The method was able to evaluate group 5 concentration in mass units with a sensitivity in the ng/ml range and a practical working range of 1–100 ng/ml. The group 5 ELISA was compared with rocket immunoelectrophoresis for determination of allergen levels in several Phleumpratense extracts, and a very good quantitative correlation was found ( r =0.98; P <0.0001). A highly significant correlation ( r >0.8) was also obtained in comparing allergenic potency determined by RAST inhibition to group 5 content in several wild and cultivated grass species. The results proved the usefulness of the method in the standardization of Pooideae pollen extracts employed in diagnosis and treatment.     

Development of a monoclonal antibody-based ELISA to detect Escherichia coli O157:H7

A pair of monoclonal antibodies (mAb) from 10 murine hybridomas secreting Escherichia coli O157:H7 (E. coli O157:H7)-specific mAbs were selected for the development of the sandwich ELISA to detect E. coli O157:H7. On the basis of pairwise interaction analysis, mAb-1 was selected as a capture antibody while mAb-6 was used as a detection antibody. The buffer system which provided the greatest difference between the specific E. coli O157:H7-positive antigen and the negative control was chosen. This sandwich ELISA showed good linearity when the concentration of E. coli O157:H7 was in the range of 10⁵–10⁸ cfu/mL, and the sensitivity was 1×10⁴ cfu/mL. With 8-h enrichment of bacteria, this ELISA was found to detect 0.4 cfu/g E. coli O157:H7 in artificially contaminated green tea samples.     

Characterization of the human T cell response to rye grass pollen allergens Lol p 1 and Lol p 5

BACKGROUND: Knowledge of dominant T cell epitopes of major allergens recognized by allergic individuals is required to improve efficacy and safety of allergen immunotherapy. Rye grass pollen (RGP) is the most important source of seasonal aeroallergens in temperate climates and Lol p 1 and Lol p 5 are the two major IgE-reactive allergens. This study aimed to characterize the T cell response to these allergens using a large panel of RGP-sensitive individuals. METHODS: Short-term RGP-specific T cell lines (TCL) were generated from 38 RGP-sensitive subjects and stimulated with Lol p 1 and/or Lol p 5 allergens and synthetic 20-mer peptides. Proliferative responses were determined by 3H-thymidine uptake and IL-5 and IFN-gamma in culture supernatants analysed by ELISA. RESULTS: Of 17 subjects tested for reactivity to both allergens 16 (94%) responded to Lol p 1 and/or Lol p 5, establishing these as major T cell-reactive allergens. Sites of T cell reactivity were spread throughout the allergen molecules but regions of high reactivity were found. For Lol p 1 these spanned residues 19-38, 109-128, 154-173, 190-209, and for Lol p 5 37-56, 100-119, 145-164, 154-173, 190-209, 217-236 and 226-245. IL-5 and IFN-gamma were produced by T cells cultured with proliferation-inducing peptides. CONCLUSIONS: T cell responses to RGP major allergens have been extensively characterized, providing fundamental information for developing T cell-targeted immunotherapy for RGP allergy.     

Quantification of group 5 grass pollen allergens in house dust.

BACKGROUND: It is widely known and accepted that grass pollen is a major outdoor cause of hay fever. However, it is of virtual importance for grass pollen allergic patients with symptoms all the year round to know the concentration of grass pollen allergens in their homes. OBJECTIVE: The main objective of this study was to quantify the amount of grass pollen allergen in mass units (microg Phl p 5) in dust settled indoors and to detect the distribution of allergenic activity in different sampling locations of homes. Furthermore, we studied the seasonal fluctuation of allergen content in dust samples. METHODS: We adapted the two site binding assay for detection of group 5 grass pollen allergens in samples from randomly selected homes in Hamburg (n = 371), Erfurt (n = 396), Hettstedt (n = 353), Zerbst (n = 289) and Bitterfeld (n = 226), Germany. Dust samples were collected from floor of living room (LR), bedroom (BR) or children's room (CR) and mattress (MA) during period of June 1995 to August 1998. The amount of the major grass group 5 allergens was detected in microg/g dust. RESULTS: Phl p 5 was detected in 67% of the samples analysed (n = 4760). The range was between undetectable (< 0.03 microg/g dust) and 81 microg/g dust. Phl p 5 levels were significantly higher in the dust from LR (geometric mean 0.117 microg/g dust) or BR/CR floors (geometric mean 0.098 microg/g dust) than in mattresses (geometric mean 0.043 microg/g dust). We observed seasonal fluctuation of indoor Phl p 5 levels with peak in June but also annual differences. Thus Phl p 5 content indoors reflects also the different quantities of pollen counts of annual courses. During pollination period we found two times higher Phl p 5 levels (0.172 microg/g dust, P < 0.001) than outside of grass pollination season (0.095 microg/g dust). The indoor Phl p 5 levels outside of season seem to be independent of pollination before. We suppose that settled pollen grains or allergenic material from outdoor particles carried indoors via footwear and clothes accumulates in house dust. CONCLUSION: Although we not known how the allergens in settled dust are equilibrated with those in the air, the considerable high level of Phl p 5 in indoor dust even during periods when no grass pollen is present in the atmosphere may be an important cause of pollen-allergy symptoms outside of season.     

Quantification of profilins by a monoclonal antibody-based sandwich ELISA.

Profilins are plant allergens responsible for cross-reactivities in pollen and fruit-allergic patients. A two-site enzyme-linked immunosorbent assay has been developed for the quantification of profilins and its suitability for quantifying profilin in different plant extracts has been evaluated. The assay is based on two profilin-specific monoclonal antibodies (mAbs) with different epitope specificities. These antibodies were immobilized on ELISA plates and incubated with samples containing profilin. Bound profilin was detected by a combination of biotinylated profilin-specific antiserum and peroxidase-streptavidin conjugate. The optimized ELISA measured profilin concentrations ranging from 4 to 250 ng/ml and could quantify profilins from plant species of a variety of different botanical families. No reactivity to mites, molds, or crustaceans was detected, suggesting that the immunoassay is plant-specific. The results indicate that this sensitive profilin-assay will be helpful both for quantifying the profilin content of allergenic extracts intended for clinical use and for studying cross-reactivities between pollen extracts.     

Development and validation of a sandwich ELISA for quantification of peanut agglutinin (PNA) in foods

In this study, two anti-peanut agglutinin (PNA) polyclonal antibodies were successfully prepared. Using mouse anti-PNA polyclonal antibody as the capture antibody and rabbit anti-PNA polyclonal antibody as the detection antibody, a novel sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify PNA. The detection limit of the ELISA method was low (0.49 ng/mL), and the linear dynamic range was between 0.78 and 100 ng/mL. The recovery ranged from 93.86% to 139.3%, whereas the intra- and inter-assay coefficients of variation were less than 10.25% and 12.06%, respectively. Sample analysis verified this method as a reliable tool for the detection of PNA in processed foods.     

Generation and characterization of an anti-GP73 monoclonal antibody for immunoblotting and sandwich ELISA

Recently,serum Golgi protein 73(GP73) levels have been found to be elevated in patients with hepatocellular carcinoma(HCC),and GP73 has been proposed as a novel marker for HCC.However,GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent assay(ELISA).Therefore,an anti-GP73 antibody with high specificity was highly demanded.In the present study,by hybridoma screening,we generated an anti-GP73 monoclonal antibody(mAb) designated as 6A2 using recombinant GP73 protein produced by prokaryotic expression.The specificity of 6A2 was evaluated by Western blotting,immunohistochemistry and immunoprecipitation.The results showed that 6A2 recognized GP73 in both native and denatured forms.In addition,we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures,and measured the serum GP73 level of patients using this assay.Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls(P = 0.0036).Furthermore,for the first time,GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls(P = 0.0172).     

单克隆抗体ELISA双抗体夹心法测定人白细胞介素2

本文叙述了测定人白细胞介素2(IL-2)含量的ELISA双抗体夹心法。用辣根过氧化物酶标记IL-2单克隆抗体。聚苯乙烯反应板预先用0.1％戊二醛处理,然后以单克隆抗体包被。本法批内变异系数CV=3.99％,批间变异系数CV=5.96％,标准曲线的测定范围为3.1～100u/ml,检测下限为1.55u/ml。应用本法已测定了2S例正常人和11例恶性肿瘤患者的IL-2含量。     

Major grass pollen allergen Lol p 1 binds to diesel exhaust particles: implications for asthma and air pollution

Background Grass pollen allergens are known to be present in the atmosphere in a range of particle sizes from whole pollen grains (approx. 20 to 55 μim in diameter) to smaller size fractions < 2.5 μ (fine particles, PM2.5). These latter particles are within the respirable range and include allergen-containing starch granules released from within the grains into the atmosphere when grass pollen ruptures in rainfall and are associated with epidemics of thunderstorm asthma during the grass pollen season. The question arises whether grass pollen allergens can interact with other sources of fine particles, particularly those present during episodes of air pollution. Objective We propose the hypothesis that free grass pollen allergen molecules, derived from dead or burst grains and dispersed in microdroplets of water in aerosols, can bind to fine particles in polluted air. Methods We used diesel exhaust carbon particles (DECP) derived from the exhaust of a stationary diesel engine, natural highly purified Lol p 1, immunogold labelling with specific monoclonal antibodies and a high voltage transmission electron -microscopic imaging technique Results DECP are visualized as small carbon spheres, each 30–60 nm in diameter, forming fractal aggregates about 1–2μ in diameter. Here we test our hypothesis and show by in vitro experiments that the major grass pollen allergen, Lol p I. binds to one defined class of fine particles, DECP. Conclusion DECP are in the respirable size range, can bind to the major grass pollen allergen Lol p I under in vitro conditions and represent a possible mechanism by which allergens can become concentrated in polluted air and thus trigger attacks of asthma.     

检测肌糖蛋白C夹心ELISA方法的建立及初步应用

目的:以已经制备的抗肌糖蛋白C(TN-C)的单克隆抗体(mAb)为基础,建立能定量检测肌糖蛋白C浓度的夹心ELISA方法,并初步于临床血清标本检测.方法:将3株mAb(克隆号分别为:1A8、3H7和4D6)制备腹水后纯化,分别与辣根过氧化物酶交联后,两两配对,以重组肌糖蛋白C 蛋白为检测抗原,分析抗体之间最佳组合;利用建立的夹心ELISA方法检测收集的临床骨肉瘤患者和正常人血清标本.结果:包被1A8 mAb,HRP-4D6配对敏感性最高;骨肉瘤患者血清中肌糖蛋白C浓度明显高于正常人.结论:成功建立检测肌糖蛋白C的夹心ELISA方法,并利用其检测到肌糖蛋白C在骨肉瘤患者中的浓度异于正常人.     

Sensitive and highly specific detection of Cronobacter sakazakii based on monoclonal sandwich ELISA

A sensitive and specific monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) was established to detect the Cronobacter sakazakii. A pair of monoclonal antibodies (mAbs) selected from mAbs produced by 11 murine hybridomas was selected for the sandwich ELISA procedure. Targets of two mAbs were 100 kDa and 42 kDa protein extracted from the bacteria, respectively, which were proved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. The limit of detection of this method was established as 1 × 10⁴ cfu/mL, and the linear range from 1 × 10⁵ to 1 × 10⁸ cfu/mL. In the real sample test, 1 cfu/g C. sakazakii was detected in artificially contaminated powdered infant formula with 4 h enrichment.     

自制NSE单抗的鉴定及其双抗夹心ELISA方法的建立

目的:制备并鉴定NSE(Neuron-specific enolase)单克隆抗体,建立可检测NSE蛋白的双抗夹心ELISA方法。方法:用本实验室已表达纯化的NSE融合蛋白免疫BALB/c小鼠,采用杂交瘤技术制备单克隆抗体。采用WB、IP、IF、IHC等方法对获得的NSE单抗进行鉴定及亚型检测。利用辣根过氧化物酶标记纯化后的NSE单抗,建立一个可检测NSE蛋白的双抗夹心ELISA法。结果:通过分析和鉴定,选定2株可稳定分泌抗NSE抗体的杂交瘤细胞株,效价达4.2×107～6.5×107,亚型为IgG2b。免疫印迹结果显示,该抗体不仅能识别细胞内源NSE蛋白,还能识别分泌到细胞培养上清液中的NSE蛋白,此外还可用于免疫荧光及免疫组化检测。文中所建立的双抗夹心ELISA法,最低检测极限为8.85 ng/ml。结论:成功获得了效价高、灵敏度好及特异性强的NSE单抗,建立了一个双抗体夹心ELISA检测系统,具有良好的临床应用前景。     

Spatial clustering of the IgE epitopes on the major timothy grass pollen allergen Phl p 1: importance for allergenic activity

BACKGROUND: The major timothy grass pollen allergen Phl p 1 is one of the most potent and frequently recognized environmental allergens. OBJECTIVE: We sought to study at a molecular and structural level the IgE recognition of Phl p 1 and its relation to allergenic activity. METHODS: Monoclonal human IgE antibody fragments specific for Phl p 1 and group 1 allergens from various grasses were isolated from a combinatorial library made of lymphocytes from patients with grass pollen allergy. Recombinant Phl p 1 fragments and the 3-dimensional structure of Phl p 1 were used to localize the major binding site for the IgE antibodies. A rPhl p 1 fragment containing this binding site was expressed in Escherichia coli, purified, and tested for IgE reactivity and allergenic activity with sera and basophils from patients with grass pollen allergy. RESULTS: Monoclonal antibodies, as well as polyclonal serum IgE, from patients with grass pollen allergy defined a C-terminal fragment of Phl p 1 that represents a sterically oriented portion on the Phl p 1 structure. This Phl p 1 portion bound most of the allergen-specific IgE antibodies and contained the majority of the allergenic activity of Phl p 1. CONCLUSION: IgE recognition of spatially clustered epitopes on allergens might be a general factor determining their allergenic activity. CLINICAL IMPLICATIONS: Geographic distribution of IgE epitopes on an allergen might influence its allergenic activity and hence explain discrepancies between diagnostic test results based on IgE serology and provocation testing. It might also form a basis for the development of low allergenic vaccines.