Embed Size (px)
Citation preview
1
Letters
Competitive immunochromatographic assay for leptosperin as a plausible
authentication marker of manuka honey
Yoji Kato†, ¶, *, Yukako Araki†, Maki Juri†, Akari Ishisaka†, ¶, Yoko Nitta§, Toshio Niwa£,
Noritoshi Kitamoto†, ¶, Yosuke Takimoto¢
† School of Human Science and Environment, and ¶ Research Institute for Food and
Nutritional Sciences, University of Hyogo, Himeji, Hyogo 670-0092, Japan
§ Graduate School of Health and Welfare Science, Okayama Prefectural University, 111
Kuboki, Soja-shi, Okayama 719-1197, Japan
£ Department of Human Health and Nutrition, Shokei Gakuin University, Natori,
Miyagi 981-1295, Japan
¢ Healthcare Systems, Inc., 2-22-8, Chikusa-ku, Nagoya, Aichi 464-0858, Japan
* To whom correspondence should be addressed: Yoji Kato
Tel.: +81-79-292-9413. Fax: +81-79-293-5710.
E-mail: [email protected]
2
ABSTRACT 1
2
Manuka honey is known as one of the premium honeys because of its unique 3
property: a potent antibacterial activity. Leptosperin, methyl syringate 4
4-O-ß-D-gentiobioside, has been specifically identified in manuka honey. 5
Because leptosperin is relatively stable under warmer conditions, measuring 6
leptosperin levels may be applied to authenticate manuka honey. In this 7
study, an immunochromatographic separation and quantification of 8
leptosperin techniques have been developed. The concentration of 9
leptosperin measured by immunochromatography was significantly 10
correlated with the concentration measured by high-performance liquid 11
chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA). 12
Because the immunochromatographic method is rapid and reliable, it could 13
be applied to on-site quality control or inspection of honey samples by a 14
beekeeper, a manufacturer, an inspector, a retailer, or a consumer. 15
16
3
Keywords: Immunochromatography, Leptosperin, Manuka Honey, 17
Authentication 18
19
4
INTRODUCTION 20
21
Manuka honey, which is derived from the nectar of the flower of 22
Leptospermum scoparium in New Zealand, has high antibacterial activity 23
and the measure of this activity aids in the authentication of manuka honey. 24
The major bactericide in the honey is methylglyoxal, a low-molecular weight 25
aldehyde 1, 2. It has been reported that methylglyoxal is generated from 26
dihydroxyacetone, which is a unique component of manuka honey, during 27
storage under moderate warm conditions 3. In general, aldehydes, including 28
methylglyoxal, are known to be highly reactive compounds and are classified 29
as "unstable." These characteristics imply that the antibacterial activity is 30
not constant during storage, transportation, or store display. High 31
antibacterial activity justifies the high market price of this honey. Because 32
methylglyoxal and its precursor are commercially available as a moderately 33
priced reagent, methylglyoxal could be added to honey to artificially enrich 34
the antibacterial activity. A reliable marker for manuka honey is required 35
5
for protecting the consumer and also the brand; however, neither the real 36
antibacterial activity nor the concentration of the bactericide, methylglyoxal, 37
are suitable alone as a stable authentication marker of the honey. 38
We have identified a novel glycoside, leptosperin (leptosin), from 39
manuka honey 4. According to the specific localization of leptosperin in honey 40
from Leptospermum species, Leptospermum scoparium or Leptospermum 41
polygalifolium, the quantification may be relevant to a plausible 42
authentication of manuka honey to protect the quality of manuka honey and 43
its brand image in the market. Indeed, a previous study showed that the 44
leptosperin contents measured by high-performance liquid chromatography 45
(HPLC) are highly correlated with the antibacterial activity and a significant 46
stability of long-term storage under warm conditions 5. A monoclonal 47
antibody specific to leptosperin has been recently developed and applied to 48
“immuno-authentication” by enzyme-linked immunosorbent assay (ELISA) 6. 49
A total of 20 samples plus 10 standards can be analyzed by ELISA in 50
duplicates in one 96-well plate. However, it takes over 4 h to complete the 51
6
ELISA without the antigen pre-absorption process on wells (approximately 52
half a day at 4°C). 53
Immunochromatography has been widely used for detecting viruses, 54
allergens, food poisons, etc. Competitive immunochromatography has been 55
applied to analyze low-molecular weight compounds such as pesticides 7 or 56
mycotoxins 8-10. In this study, an immunochromatographic test kit for 57
leptosperin was developed and honey samples were subjected to this test for 58
the authentication of manuka honey. 59
60
61
7
MATERIALS AND METHODS 62
63
Materials 64
Twenty manuka honey samples were obtained from the Unique Manuka 65
Factor Honey Association (UMFHA). Honey was dissolved in water (0.1 g/ml) 66
and centrifuged (14000g, 10 min). The supernatant was then used for the 67
assay. Leptosperin was isolated and purified from manuka honey as 68
described previously 4 or chemically synthesized according to the published 69
method 11. Leptosperin-conjugated bovine serum albumin (BSA) and 70
anti-leptosperin antibody were prepared as described previously 6. 71
72
Immunochromatography 73
The immunochromatography kit was assembled as follows. The conjugation 74
of the anti-leptosperin antibody to 40 nm-colloidal gold (Roche Diagnostics, 75
Basel, Switzerland) was performed by conventional methods 12. The colloidal 76
gold-conjugated antibody in Tris-based buffer (pH 8.2) was vacuum-dried in 77
8
a glass vial for each test. The test strip is constructed as follows (Figure 1). 78
Using the dispenser system XYZ3050 (BioDot, CA, USA), the 79
leptosperin-BSA (as the trap/test line) and the goat anti-mouse IgG (as the 80
control line) in a phosphate-based buffer (pH 7.4) were applied on the 81
nitrocellulose membrane, HiFlow plus HF120 (Merck Millipore, MA, USA). 82
The membrane with the test and the control line was pasted to the backing 83
sheet, ARcare 9020 (Adhesives Research, UK). The sample pad (glass fiber, 84
G041, Merck Millipore) and the absorbent pad (Cellulose Absorbent Pad 165, 85
Pall, Germany) were pasted to the backing sheet covering the previously 86
pasted nitrocellulose membrane. The assembled sheet was cut to make strips 87
of the size of 5.5 mm × 60 mm and was covered with the plastic cassette 88
(Fujikura Kasei, Tokyo, Japan). 89
The honey sample (0.1 g/ml) was diluted 50 times in water. The 90
sample (or standard) was then further diluted 1:25 with the phosphate-based 91
buffer containing 0.9% NaCl and was added to a vial containing the dried 92
specific antibody conjugated with the colloidal gold. The vial was briefly 93
9
mixed and then 75 µl of the mixture was applied to a sample pad (a sample 94
hole of the kit) and was incubated for 20 min at room temperature. The 95
quantitation was performed by measuring the displayed test line in the 96
window of the cassette with a portable scanner, Immunochromato Reader 97
TA4544 (Fujikura Kasei, Japan). 98
99
Statistics 100
The relationship between two groups was calculated by IBM SPSS Statistics 101
ver. 22 with Spearman’s correlation. 102
103
104
105
10
RESULTS AND DISCUSSION 106
107
The principle of the kit is based on a competitive assay. Leptosperin is mixed 108
with the specific antibody in a sample. The antibody-leptosperin complex 109
and the unreacted antibodies each migrate on the membrane in a plastic 110
cassette. The unreacted antibodies, but not the complex, are captured with 111
immobilized leptosperin-conjugated BSA, visualized at the reddish-violet 112
colored first line. The complex migrates further and will be captured by an 113
antibody to mouse immunoglobulins immobilized on the membrane, leading 114
to the development of the second line which is colored reddish violet (Figure 115
1). The test line is scanned and converted to a number by the reader. The 116
standard curve covers a range from 0.014 µM to at least 10 µM (Figure 2). 117
Next, the 20 certified manuka honey samples were analyzed. The average of 118
leptosperin content in the honey was found to be 1.07 µM, with a minimum 119
of 0.51 µM and a maximum of 1.70 µM. The results were converted in nmol 120
per gram of honey and compared with those of the previous data by HPLC or 121
11
ELISA 5, 6. As shown in Figures 3A and 3B, the immunochromatographic 122
data are significantly correlated with the data obtained by the other methods, 123
indicating the high reliability of the kit (immunochromatography and HPLC, 124
r = 0.952, p < 0.001; immunochromatography and ELISA, r = 0.934, p < 125
0.001). 126
The immunochromatographic test for leptosperin is simple, easy, 127
and rapid. Therefore, this assay is applicable to quantify leptosperin to 128
authenticate manuka honey in a factory laboratory, on-site at an apiary or a 129
retail store, or at home by a consumer. 130
131
ABBREVIATIONS 132
HPLC, high-performance liquid chromatography; ELISA, enzyme-linked 133
immunosorbent assay; UMFHA, Unique Manuka Factor Honey Association; 134
BSA, bovine serum albumin 135
136
137
12
CONFLICT OF INTEREST 138
The authors declare no competing financial interest. 139
140
ACKNOWLEDGMENTS 141
We thank Mr. John Rawcliff (Manager, UMFHA) for supplying 20 certified 142
manuka honey samples. 143
144
Figure legends 145
Fig. 1. Schematic diagram and picture of the competitive 146
immunochromatographic assay test strip for leptosperin. 147
Fig. 2. Standard calibration curve for leptosperin. Seven standards (0.014–10 148
µM) were separately applied to strips, and the developed test line in a strip 149
was scanned by a reader for immunochromatography. 150
Fig. 3. Correlation of immunochromatography with HPLC (A) or ELISA (B) 151
for measurement of leptosperin in manuka honey. The data of HPLC and 152
ELISA were obtained previously 5, 6. 153
154
13
References 155
156
(1) Atrott, J.; Henle, T. Methylglyoxal in manuka honey – Correlation with 157
antibacterial properties. Czech J. Food Sci. 22000099,, 27, S163-S165. 158
(2) Mavric, E.; Wittmann, S.; Barth, G.; Henle, T. Identification and 159
quantification of methylglyoxal as the dominant antibacterial 160
constituent of Manuka (Leptospermum scoparium) honeys from New 161
Zealand. Mol. Nutr. Food Res. 22000088,, 52, 483-489. 162
(3) Adams, C. J.; Manley-Harris, M.; Molan, P. C. The origin of 163
methylglyoxal in New Zealand manuka (Leptospermum scoparium) 164
honey. Carbohydr. Res. 22000099,, 344, 1050-1053. 165
(4) Kato, Y.; Umeda, N.; Maeda, A.; Matsumoto, D.; Kitamoto, N.; Kikuzaki, 166
H. Identification of a novel glycoside, leptosin, as a chemical marker of 167
manuka honey. J. Agric. Food Chem. 22001122,, 60, 3418-3423. 168
(5) Kato, Y.; Fujinaka, R.; Ishisaka, A.; Nitta, Y.; Kitamoto, N.; Takimoto, Y. 169
Plausible authentication of manuka honey and related products by 170
14
measuring leptosperin with methyl syringate. J. Agric. Food Chem. 171
22001144,, 62, 6400-6407. 172
(6) Kato, Y.; Araki, Y.; Juri, M.; Fujinaka, R.; Ishisaka, A.; Kitamoto, N.; 173
Nitta, Y.; Niwa, T.; Takimoto, Y. Immunochemical authentication of 174
manuka honey using a monoclonal antibody specific to a glycoside of 175
methyl syringate. J. Agric. Food Chem. 22001144,, 62, 10672-10678. 176
(7) Kim, Y. A.; Lee, E. H.; Kim, K. O.; Lee, Y. T.; Hammock, B. D.; Lee, H. S. 177
Competitive immunochromatographic assay for the detection of the 178
organophosphorus pesticide chlorpyrifos. Anal. Chim. Acta 22001111,, 693, 179
106-113. 180
(8) Shim, W. B.; Kim, K. Y.; Chung, D. H. Development and validation of a 181
gold nanoparticle immunochromatographic assay (ICG) for the detection 182
of zearalenone. J. Agric. Food Chem. 22000099,, 57, 4035-4041. 183
(9) Sun, Y.; Hu, X.; Zhang, Y.; Yang, J.; Wang, F.; Wang, Y.; Deng, R.; 184
Zhang, G. Development of an immunochromatographic strip test for the 185
rapid detection of zearalenone in corn. J. Agric. Food Chem. 22001144,, 62, 186
15
11116-11121. 187
(10) Liu, G.; Yan, T.; Wang, J.; Huang, Z.; Chen, X.; Jia, G.; Wu, C.; Zhao, 188
H.; Xue, B.; Xiao, L.; Tang, J. Biological system responses to zearalenone 189
mycotoxin exposure by integrated metabolomic studies. J. Agric. Food 190
Chem. 22001133,, 61, 11212-11221. 191
(11) Aitken, H. M. R.; Johannes, M.; Loomes, K. M.; Brimble, M. A. 192
Synthesis of leptosin, a glycoside isolated from mānuka honey. 193
Tetrahedron Lett. 22001133,, 54, 6916-6919. 194
(12) Chun, P. Colloidal gold and other labels for lateral flow immunoassays. 195
In Lateral Flow Immunoassay, Humana Press: New York, 2009. 196
197
