Commercial ELISA kits for soy protein determination: Characterization and compatibility

Abstract

Soy protein has been approved in Japan as a Food for Specified Health Uses to lower serum cholesterol. A foreign-made soy protein ELISA kit has been proposed for quantifying soy protein levels. However, acquisition of this foreign-made product is time-consuming, making the product inconvenient for soy protein quantification in Japan. Therefore, as possible alternatives, we evaluated two commercial domestic ELISA kits, both of which quantify soy protein and specific allergens for food-allergen labeling. In this study, we compared antibody reactivity and soy protein levels in soy milk using these three types of ELISA kits (one foreign-made and two domestic ELISA kits). Per the results, we found that commercial domestic ELISA kit-derived soy protein levels could be converted to the soybean protein levels obtained from the foreign-made soy protein ELISA kit, through multiplication by specific factors.

Introduction

Soybean (Glycine max L.) is a vegetable-protein food with a high protein content and excellent amino acid balance (Singer et al., 2019). As a result, plant-based meat products, with meat-like processing of soybean, have rapidly become popular worldwide (Malav et al., 2015; Singh et al., 2021). Soybean, which contains many biologically active ingredients including isoflavones, saponins, and lecithin, is useful for preventing chronic diseases, and has long been consumed in Asian countries including Japan (Messina, 1995; Birt et al., 2004; Nakai et al., 2020; Kim et al., 2021). There is, therefore, a major global focus on soybean.

Soy proteins also have beneficial physiological effects, such as lowering serum cholesterol in experimental animal and human studies (Sirtori et al., 1977; Nagata et al., 1982; Sugano et al., 1988; Ogawa et al., 1992; Kito et al., 1993; Sugano and Koba, 1993; Potter, 1998; Torres et al., 2006). Based on this fact, soy protein has been approved in Japan as a Food for Specified Health Uses (FOSHU) to lower serum cholesterol levels. Soy protein may also reduce serum triglycerides (Anderson et al., 1995). Specifically, the β-conglycinin (soybean 7S globulin), a soy protein fraction, reduces both triglycerides and visceral fat (Aoyama et al., 2001; Baba et al., 2004; Moriyama et al., 2004; Kohno et al., 2006), as demonstrated with animal and human studies; it has therefore been classified as a novel functional component of soy. The β-conglycinin (7S globulins), is composed of three subunits: α′, α, and β subunit (approx. 75, 73, and 50 kDa, respectively). This major storage protein of soybean is also a major allergen, i.e., Gly m 5 (Krishnan, et al., 2009). The commercial FASPEK ELISA II Soybeans kit (Morinaga Institute of Biological Science, Inc., Yokohama, Japan) efficiently determines β-conglycinin (7S globulin) levels (Moriyama et al., 2020).

The BIOKITS Soya Protein Assay Kit (Neogen, Lansing, MI, USA) has long been used to determine soy protein level when evaluating its cholesterol-lowering effect (Japan Health and Nutrition Food Association, 1999). However, because it is foreign-made, this kit takes time to obtain. Fortunately, there are commercially available and widely used ELISA kits (FASPEK ELISA II Soybeans (Morinaga Institute of Biological Science, Inc., Yokohama, Japan) and FASTKIT ELISA Ver. III Soybeans (Research & Development Center, NH Foods, Tsukuba, Japan) in Japan that can be used to quantify soy protein levels for the detection of contaminants and food-allergen labeling. To evaluate alternatives to the Neogen BIOKITS kit, we compared it with these two Japanese commercial ELISA kits for soy protein determination and verified the similarity of their results.

Materials and Methods

Materials  We purchased the FASPEK ELISA II Soybeans kit (Morinaga Institute of Biological Science, Inc.) and FASTKIT ELISA Ver. III soybeans kit (Research & Development Center, NH Foods Ltd.) for the detection of contaminated soy protein residues containing allergens. The BIOKITS Soya Protein Assay Kit was obtained from Neogen. Electrophoresis-related reagents, such as acrylamide solutions for SDS-PAGE, were obtained from Nacalai Tesque (Kyoto, Japan). Commercial soymilk was donated by a soymilk company (Marusan-Ai Co. Ltd.). The nutritional information of the soymilk was as follows: calories, 139 kcal/200 ml; proteins, 9.2 g/200 ml; fat, 6.2 g/200 ml; and carbohydrate, 11.6 g/200 ml. Soybean 7S globulin was purified from soybeans according to the protocol described by Nagano et al., (Nagano et al., 1992). Western blot-related apparatus and molecular-weight markers for SDS-PAGE were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG antibody and HRP-labeled streptavidin were obtained from Thermo Scientific (Waltham, MA, USA). Immuno-enhancer reagents (CanGetSignal 1 and 2) were obtained from Toyobo (Osaka, Japan). ECL™ western blotting reagent and Hyperfilm-MP™ X-ray films were obtained from GE Healthcare (Piscataway, NJ, USA). ELISA plates were purchased from Asahi Glass (Tokyo, Japan). Polyvinylidene fluoride (PVDF) membrane (Immobilon-P™) was obtained from Millipore (Billerica, MA, USA). TMB (3,3′,5,5′-tetramethylbenzidine) peroxidase substrate was purchased from Kirkegaard & Perry Laboratories (KPL, Gaithersburg, MD, USA). The other chemicals used in this study were of the highest available purity.

Electrophoresis (SDS-PAGE)  To determine the protein composition, SDS-PAGE (Laemmli, 1979) was performed, using a Bio-Rad Mini-Protean-3 system, at 200 V (constant) for 35 min. Gel concentration was 12.5 %. Electrophoresis buffer contained 25 mM Tris-HCl, 191 mM Glycine and 0.1 % SDS. The electrophoresis sample was mixed with three volumes of soymilk and one volume of concentrated sample buffer (250 mM Tris-HCl (pH 6.8), 8 % SDS, 40 % Glycerol, 20 % β-mercaptoethanol and 0.02 % bromophenol blue), and treated in boiling water for 5 min. After electrophoresis, gels were stained with Coomassie Brilliant Blue (CBB) R-350 from GE Healthcare or subjected to western blotting. For CBB-staining of soybean proteins, soy milk proteins (1.7 μg protein/lane) were separated using SDS-PAGE gels. For western blotting, soybean protein levels of 4.4 μg/lane for the Neogen and Morinaga kits, and 1.7 μg/lane for the NH Foods kit, were used. For separation and CBB-staining of purified 7S globulin, the purified sample (0.4 μg/lane) was separated using SDS-PAGE. For western blotting, 0.4 μg/lane of purified soybean 7S globulin was separated for all kits.

Western blotting  Western blotting analysis was conducted by transferring the SDS-PAGE gel onto an Immobilon-P™ PVDF membrane (Millipore) using a semi-dry blotting method (Kyhse-Andersen, 1984) using Trans Blot Turbo™ (Bio-Rad). The membrane was incubated in 10 mM phosphate-buffered saline (PBS) (pH 7.5) containing 0.1 % Tween-20 (PBS-T) and 5 % skim milk for blocking (blocking solution). The membrane was then incubated for 1 h at room temperature in a blocking buffer containing detection antibodies of each ELISA kit. After washing the membranes four times with PBS-T for 10 min, the bound primary antibodies were detected using HRP-conjugated goat anti-rabbit IgG or HRP-labeled streptavidin and an ECL™ western blotting kit (GE Healthcare). The resultant chemiluminescent signals were detected on X-ray film (Hyperfilm™ MP, GE Healthcare). Western blotting experiments were carried out at least three times. Representative images are presented.

Determination of soy protein levels using commercial ELISA kits  Total soy protein levels were determined using the BIOKITS, FASPEK, and FASTKIT commercial ELISA kits, according to their respective manuals. Data analyses were performed using Microplate Manager v. 6.3 (Bio-Rad).

Determination of the reactivity of detection antibodies and the BIOKITS standard protein  To determine the reactivity of each detection antibody of each ELISA kit, and of the standard protein (STD) in the BIOKITS kit, we coated serial dilutions of the STD protein onto a normal ELISA plate and detected the reactivity of each detection antibody from each of the ELISA kits. After serial dilution, the STD (in PBS) was coated onto the normal ELISA plate overnight. The plate was then removed from the STD solution and blocked with a blocking buffer. The detection antibodies from each ELISA kit (diluted in CanGetSignal 1; Toyobo) were then added to the wells, followed by incubation for 1 h at room temperature. After washing the wells with PBS-T three times, we added the HRP-labeled secondary antibodies for the FASPEK kit, or HRP-labeled streptavidin for the FASTKIT kit, both diluted in CanGetSignal 2 (Toyobo). The plate was then incubated for 1 h, washed with PBS-T, and reacted with TMB. Absorbance at 450 nm was measured using a plate reader (Bio-Rad).

Kjeldahl method  Total nitrogen of soymilk samples was obtained using the Kjeldahl method using DK6 Kjeldahl Digestion Units (VELP Scientifica Srl, Usmate, Italy) and the KT 200 Kjeltec™ Distillation unit (FOSS, Denmark). The soy protein equivalent was calculated by multiplying the total nitrogen by a factor of 5.71.

Statistical analysis  Results are expressed as the mean ± SD. Student's t-test was used to determine the significance of differences between the two groups. All experiments were performed at least three times. Differences were considered significantly different at p < 0.05.

Results and Discussion

Detection of soybean proteins using the antibodies from each ELISA kit  Western blotting (Fig. 1A) revealed that the BIOKITS antibody detected protein bands that appeared to be 7S globulin subunits (approx. 75, 73, and 50 kDa), as well as several other proteins (approx. 36 and 20 kDa). The FASPEK antibody detected three subunits that appeared to be 7S globulin (approx. 75, 73, and 50 kDa). The FASTKIT antibody detected mainly proteins of 34 and 18 kDa. These proteins, except for the 7S globulin, are presently unknown.

Fig. 1.

Western blotting analysis of soybean (soy milk) proteins (A) or purified 7S globulin (B) using detection antibodies from three commercial ELISA kits.

1. A) Soybean proteins (in soy milk) were separated by SDS-PAGE and subjected to western blotting using the detection antibodies provided in three commercial ELISA kits. (a) Coomassie Brilliant Blue-staining of soybean proteins separated in SDS-PAGE gels. (b) Western blotting of the soybean proteins detected by each antibody provided in (1) the BIOKITS Soya Protein Assay Kit (Neogen), (2) the FASPEK ELISA II Soybeans kit (Morinaga), and (3) FASTKIT ELISA Ver. III (soy) kit (NH Foods).
2. B) Purified soybean 7S globulin (β-conglycinin) was separated via SDS-PAGE and subjected to western blotting using different detection antibodies provided in the three commercial ELISA kits. (a) Coomassie Brilliant Blue-staining of soybean 7S globulin separated in SDS-PAGE gels. (b) Western blotting analysis of soybean 7S globulin detected by each antibody provided in the Neogen ELISA kit (1), Morinaga ELISA kit (2), and NH Foods ELISA kit (3).

Using partially purified 7S globulin for western blotting (Fig. 1B), the BIOKITS and FASPEK kits, but not the FASTKIT kit, detected three subunits of 7S globulin (approx. 75, 73, and 50 kDa). Therefore, the BIOKITS and FASPEK kits use antibodies that can detect soy proteins, mainly 7S globulin, and have similar reactivity. The FASTKIT kit, in contrast, potentially measures overall soy protein levels, detecting proteins (unknown 34 and 18 kDa proteins) other than 7S globulin. Since BIOKITS and FASPEK kits use antibodies that recognize the same 7S globulin, the reactivity and properties of the ELISA are expected to be similar.

Reactivity of the detection antibodies from each kit to the BIOKITS standard protein  All of the antibodies reacted with the standard protein in a concentration-dependent manner, and their absorbances increased similarly (Fig. 2), indicating that the standard protein in the BIOKITS kit also reacts with the antibodies in the other kits. These results suggest no significant differences in the standard proteins across the three kits.

Fig. 2.

Reactivity of the detection antibodies in each kit to the standard (STD) soybean proteins provided in the Neogen ELISA kit.

The soybean STD proteins (0.5–5000 pg/μl) provided in the Neogen ELISA kit were immobilized on ELISA plates, blocked, and reacted with the detection antibodies in the kits from the other companies [FASPEK ELISA II Soybeans kit; FASTKIT ELISA Ver. III (soy)].

Determination of soybean protein concentration via these kits and the Kjeldahl method  Soymilk soy protein levels (Table 1) were determined using the three kits and the Kjeldahl method, which is widely used to determine crude protein levels. The soy protein levels in Soymilk A were similar using the three methods: 4.74 g/100 g (Kjeldahl), 4.14 g/100 g (BIOKITS), and 4.50 g/100 g (FASPEK). On the other hand, the value measured by FASTKIT was 1.89 g/100 g. The BIOKITS kit, therefore, detected 0.92 times as much soy protein as the FASPEK kit, and 2.19 times as much as the FASTKIT kit. Soymilks B and C showed similar results. For the three soymilk lots, on average, the BIOKITS kit detected 0.92 times as much soy protein as the FASPEK kit, and 2.23 times as much as the FASTKIT kit. The highest values were obtained using the Kjeldahl method, probably because it can measure the content of amino acids and peptides that cannot be detected using the ELISA method.

Table 1.Soy protein concentration in three batches of soymilk, using the three commercial ELISA kits and Kjeldahl method.

	Kjeldahl method	Neogen ELISA kit	Morinaga ELISA kit	NH Foods ELISA kit	Neogen/Morinaga ratio	Neogen/NH Foods ratio
Samples	(g/100 g)	(g/100 g)	(g/100 g)	(g/100 g)	(fold)	(fold)
Soymilk A	4.74	4.14	4.50	1.89	0.92	2.19
Soymilk B	4.81	3.97	4.40	1.81	0.90	2.19
Soymilk C	4.91	4.25	4.58	1.85	0.93	2.30
Average	-----	-----	-----	-----	0.92 ± 0.01	2.23 ± 0.05*

Neogen ELISA kit: “BIOKITS Soya Protein Assay Kit”

Morinaga ELISA kit: “FASPEK ELISA II Soybeans”

NH Foods ELISA kit: “FASTKIT ELISA Ver. III (soy)”

Determination was performed three times. The calculation ratio was expressed as means ± SD of data using three soymilk samples. *: Significant difference (p < 0.05) determined using Student's t-test.

Considering the similarity of these values, and the proteins detected by the three kits, we propose that the FASPEK or FASTKIT soy protein levels can be converted to BIOKITS values by multiplying them by these factors. Because their ratio is closer to 1, the results using BIOKITS appear to be more similar to the FASPEK results than to the FASTKIT results (Table 1).

Although soy milk was used as the soy food in this study, it is unclear whether similar results can be obtained with other processed soy foods. Future studies are needed to determine whether the results of these ELISA kits can reflect the correct soy protein content, especially in the case of fermented soybean foods, since soy protein including soybean allergens (Moriyama et al., 2013), can be degraded in various ways.

There are several ELISA methods (Voller et al., 1978), BIOKITS is a competitive method, and the other two are described in their respective manuals as sandwich methods. It is notable that even though the measurement principles are different, the interchangeability of the assay values is ensured by multiplying by a certain factor.

In this study, we propose that two domestic ELISA kits, FASPEK- or FASTKIT-derived soy protein levels can be converted to BIOKITS levels by multiplying the data by specific factors. This finding proposes useful information for soybean protein measurements.

Acknowledgements  This study was supported in part by a grant from the Agricultural Technology and Innovation Research Institute (ATIRI), Kindai University.

Conflict of interest  There are no conflicts of interest to declare.

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