Results And Discussion

Monoclonal antibodies and sandwich ELISAs for OM

We have established 3 hybridoma clones designated 5C, 7D, and 10D. They produce respective mAbs with characteristic reactivity against OM. MAb 5C is specific to N-OM, mAb 6H is specific to H-OM, and mAb 7D is specific to the carbohydrate moiety of OM, therefore capable to recognize N- and H-OMs equally (Hirose et al., in press). These mAbs did not cross-react with proteins from other sources such as cow's milk, wheat flower, buckwheat flower, soy bean, peanut, yeast extract and meats from beef, pork and chicken on ELISA.

Then, we developed the sandwich ELISA systems capable to distinguish N-OM and/or H-OM (Systems 5C, 6H, and 7D in Fig. 1). MAb 7D was selected as the common first antibody because of its binding capacity to both N- and H-OMs and each biotinylated mAb was used as the second antibody. Property of each sandwich ELISA was dependent on that of the biotinylated second antibody. In the case of biotinylated mAb 5C, System 5C was sensitive to N-OM about 30 times higher than to H-OM. In the case of biotinylated mAb 6H contrarily, System 6H was almost specific to H-OM. Several ng/ml of N- or H-OM was able to be selectively detected by each system. Although a sandwich ELISA is usually unable to set up by a single mAb, it was successful in the case of mAb 7D because of 4 or 5 carbohydrate chains per one molecule of OM. System 7D showed equal reactivity against N-and H-OMs, indicating the possibility to determine total OM content irrespective of the degree of heat denaturation of OM. It is a very important nature required for the allergen detection system because of the allergenicity of H-OM (Hirose et al., in press). The detection limit of System 7D was rather high and about 100 ng/ml for both N- and H-OMs.

Figure 1. ELISAs for the quantitative analysis of N- and H-OMs.

Sandwich ELISAs were constructed with mAb 7D as a common solid phase antibody and 50 |al of biotinylated second mAb(s). In Systems 5C, 6H, and 7D was used 0.5 |4g/ml of each biotinylated mAb. For the oligoclonal ELISA, 0.5 |4g/ml each of biotinylated mAbs 5C and 7D were used in System 5C • 7D, 0.2 |4g/ml of biotinylated mAb 6H and 0.5 ^g/ml of biotinylated mAb 7D in System 6H • 7D, and 0.2 |ag/ml of biotinylated mAb 6H and 0.5 |4g/ml each of biotinylated mAbs 5C and 7D in System 5C • 6H • 7D. N-OM (open circle) and H-OM (closed circle) were used as standards.

Figure 1. ELISAs for the quantitative analysis of N- and H-OMs.

Sandwich ELISAs were constructed with mAb 7D as a common solid phase antibody and 50 |al of biotinylated second mAb(s). In Systems 5C, 6H, and 7D was used 0.5 |4g/ml of each biotinylated mAb. For the oligoclonal ELISA, 0.5 |4g/ml each of biotinylated mAbs 5C and 7D were used in System 5C • 7D, 0.2 |4g/ml of biotinylated mAb 6H and 0.5 ^g/ml of biotinylated mAb 7D in System 6H • 7D, and 0.2 |ag/ml of biotinylated mAb 6H and 0.5 |4g/ml each of biotinylated mAbs 5C and 7D in System 5C • 6H • 7D. N-OM (open circle) and H-OM (closed circle) were used as standards.

Oligoclonalization of ELISA

For the improvement of the sensitivity of system 7D, oligoclonalization of second antibody was examined, because ELISA constructed with polyclonal antibody usually exhibits higher sensitivity than that with mAb. When a mixture of biotinylated mAbs 5C and 7D

was used as a second antibody, sensitivity only to N-OM was improved about 30 times (System 5C-7D in Fig. 1). Contrarily by using that of biotinylated mAbs 6H and 7D, similar enhancement of sensitivity was seen only to H-OM in turn (System 6H-7D in Fig. 1). Then by using that of three biotinylated mAbs, the original sensitivity of System 7D was finally improved about 100 times for both N- and H-OMs (System minimum and essential to get that sensitivity. Increase of biotinylated mAb 6H was inhibitory. We used only mAb 7D as a solid antibody here, because we can determine N- and/or H-OM according to the purpose by selecting proper second antibody from biotinylated mAb 7D, 5C, 6H, or a mixture of them. Optimization of an oligoclonal ELISA should be examined in each case.

Extraction with denaturing reagents

To enhance the overall sensitivity of the assay system, it is important to improve not only determination method but also extraction method. We found that mAb 7D immobilized onto a ELISA plate was stable even in the presence of 0.1% SDS and 0.1M P-mercaptoethanol at 37°C for 60 min (data not shown). It has been known that proteinous samples can be usually well solubilized in the condition of sample preparation for SDS-PAGE (Laemmli, 1970). Then, we decided to extract OM from foods in 1% SDS and 1M P-mercaptoethanol at 100°C for 5 min. After cooling and 20 times dilution with PBS, OM was determined with oligoclonal System 5C-6H-7D (Table 1). There was not big difference between the values extracted with or without denaturing reagents in the case of simple or moderately processed foods. However, 6-30 fold difference were seen for foods processed under complex and/or severe conditions. Extraction with the denaturing reagents was effective even for well soluble and non-heat precipitable protein such as OM, much more for many other proteins that are insoluble or difficult to be soluble in water and heat-precipitable. Moreover, if a food is proved to be not containing allergens or non-allergenic by an in vitro tests because of some masking effects such as modification, aggregation (Kato et al., 2001), and immune complex formation (Hirose et al., 2001), its allergenicity after eating is uncertain. Extraction with denaturing reagents might enable us to determine antigens in foods closer to an absolute quantity by removing masking factors.

Table 1. Determinatin of OM in foods extracted with or without denaturing reagents.

Fresh chicken egg was boiled in water for 20 min or retorted for 20 min at 12ÖG Kamaboko (a traditional Japanese food, semicylindrically shaped fish paste), steamed bun, hard biscuit, and Chinese noodle with labels of egg usage were purchased at a market.After homogenizing whole eggs and foods individually, two gram each was mixed vigorously in 38 ml of PBS with or without 1% SDS and 1M ß-mercaptoethanol (Mer). Samples extracted with denaturing reagents were boiled at 100°Cfor 5 min and cooled. After centrifugation at 13,000g for 10 min, a portion of the supernatant was diluted 20 times with PBS and OM was determined by System 5C-6H-7D in Fig.1. Values were expressed as mg/g.

Food Extraction without SDS & Mer with SDS & Mer

Raw egg

10.3

13.3

Boiled egg

11.2

11.4

Retorted egg

7.5

8.5

Ka maboko

0. 080

0.054

Steamed bun

0.025

0.179

Hard biscuit

0.021

0.298

Chinese noodle

0.004

0.105

There is an international movement for allergen labeling, as seen in the agreement of the Joint FAO/WHO Codex Alimentarius Commission Session in 1999. In consideration of worldwide distribution of foods today, development of an international common method is required for the adequate determination of allergens. Oligoclonal system is suitable for the purpose because it is more stable than polyclonal system in specificity and quantity due to the nature of mAbs.

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