a magnetic particles-based chemiluminescence enzyme immunoassay for rapid detection of ovalbumin
TRANSCRIPT
Accepted Manuscript
A magnetic particles-based chemiluminescence enzyme immunoassay for rapiddetection of ovalbumin
Xiao-Li Feng, Hong-Lin Ren, Yan-Song Li, Pan Hu, Yu Zhou, Zeng-Shan Liu,Dong-Ming Yan, Qi Hui, Dong Liu, Chao Lin, Nan-Nan Liu, Yan-Yan Liu, Shi-Ying Lu
PII: S0003-2697(14)00165-1DOI: http://dx.doi.org/10.1016/j.ab.2014.04.016Reference: YABIO 11713
To appear in: Analytical Biochemistry
Received Date: 6 March 2014Revised Date: 29 March 2014Accepted Date: 15 April 2014
Please cite this article as: X-L. Feng, H-L. Ren, Y-S. Li, P. Hu, Y. Zhou, Z-S. Liu, D-M. Yan, Q. Hui, D. Liu, C.Lin, N-N. Liu, Y-Y. Liu, S-Y. Lu, A magnetic particles-based chemiluminescence enzyme immunoassay for rapiddetection of ovalbumin, Analytical Biochemistry (2014), doi: http://dx.doi.org/10.1016/j.ab.2014.04.016
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1
A magnetic particles-based chemiluminescence enzyme 1
immunoassay for rapid detection of ovalbumin 2
3
Short title: OVA detection by a newly constructed immunoassay 4
Category: Immunology 5
6
Xiao-Li Fenga, 1
, Hong-Lin Rena, 1
, Yan-Song Lia, Pan Hu
a, Yu Zhou
a, Zeng-Shan Liu
a, 7
Dong-Ming Yanb, Qi Hui
c, Dong Liu
a, Chao Lin
a, Nan-Nan Liu
a, Yan-Yan Liu
a, 8
Shi-Ying Lua,
*
9
10
a Key Laboratory of Zoonosis Research, Ministry of Education / Institute of Zoonosis 11
/ College of Veterinary Medicine, Jilin University, Xi An Da Lu 5333, Changchun 12
130062, China 13
b Wuhu Kang Wei Biological Technology Co., Ltd., Wuhu 241000, P. R. China. 14
c Changchun Institute of Biological Products Co., Ltd., Changchun 130062, P. R. 15
China. 16
17
18
19
20
1 The first two authors (Xiao-Li Feng and Hong-Lin Ren) contributed equally to this study.
* Corresponding author. Email: [email protected]; Fax: +86 431 87836722; Tel: +86 431
87836716
2
Abstract 21
22
Egg allergy is an important public health and safety concern, so quantification and 23
administration of food or vaccines containing ovalbumin (OVA) are urgently needed. 24
This study aimed to establish a rapid and sensitive magnetic particles 25
(MPs)-chemiluminescence enzyme immunoassay (MPs-CLEIA) for the determination 26
of OVA. The proposed method was developed on the basis of a double antibodies 27
sandwich immunoreaction and luminol-H2O2 chemiluminescent (CL) system. The 28
MPs served as both the solid phase and separator and the polyclonal antibodies (pAb) 29
anti-OVA coated on MPs were used as capturing antibody, while the HRP-labeled 30
monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the 31
method were evaluated and optimized. The established MPs-CLEIA method had a 32
linear rang from 0.31 to 100 ng/mL with a detection limit of 0.24 ng/mL. The assays 33
showed low reactivities, less than 5% of intra-assay and inter-assay coefficients of 34
variation, and the average recoveries were between 92% and 97%. Furthermore, the 35
developed method was applied in real samples analysis successfully and the 36
correlation coefficient with the commercially available OVA kit was 0.9976. 37
Moreover it was more rapid and sensitive compared to the other methods for testing 38
OVA. 39
Key words: Ovalbumin; Chemiluminescence enzyme immunoassay (CLEIA); 40
Magnetic particles (MPs). 41
42
3
1 Introduction 43
44
Egg allergy is one of the most common food allergy mainly among infants and young 45
children, moreover it is widely reported to be on the rise [1-3]. Egg allergy could 46
cause sever symptoms include urticaria, abdominal pain, diarrhea, nausea, vomiting, 47
itching, even anaphylactic shock [4] and in view of its effect on life quality of the 48
allergic people, appropriate management and strategies are urgently needed. 49
The main allergens in egg include ovalbumin (OVA), lysozyme, ovomucoid, and 50
ovotransferrin, among which OVA accounts for about 54% of the total proteins in egg 51
white [5], therefore OVA could be considered as a detection target for egg allergy. 52
As we known, influenza vaccines are produced in embryonated eggs, leading to a 53
small amount (1 to 7 μg/mL) of egg proteins contained in these vaccines [6,7]，so the 54
OVA content has to be detected after producing these influenza vaccines. 55
CAC (Codex Alimentarius Commission) have declared requirements in “General 56
Standard for the Labeling of Prepackaged Foods ” that the eight categories of food 57
allergens including egg needed to be labeled definitely [8,9]. However, there was no 58
mandatory labeling of food allergens in China. So it was time to take measures to 59
detect and manage the food allergens. 60
At present, there are a few methods reported to detect the allergen OVA including 61
ELISA (enzyme linked immunosorbent assay) [10], radioallergosorbent test [11], 62
counter immunoelectrophoresis [12], histamine releasing test [13], and PCR 63
(polymerase chain reaction) [14]. Although radioallergosorbent test has high 64
4
sensitivity and good accuracy, yet it depends on human sera and human serum IgE. 65
Only contaminating a small proportion of human sera, the antibody specificity is not 66
sure, so this test is difficult to standardize [15]. Counter immunoelectrophoresis test is 67
simple and rapid, however, its resolution ability is low and the accuracy is less than 68
ELISA [16,17]. Histamine releasing test has high sensitivity and specificity, but its 69
sensitivity is affected by freshness of food [10]. PCR also has high sensitivity and 70
stability, however it is not applicable for all allergens detection with relatively high 71
false-positive rate [18,19]. 72
ELISA based on specific antibodies is a rapid, reliable, economic, simple, and 73
sensitive method for identifying and quantifying the target compound in samples [20]. 74
There has been commercially available test kit for OVA, but it is much expensive and 75
transportation was time-consuming. In recent years, chemiluminescence enzyme 76
immunoassay (CLEIA) has been widely used in the research of clinical diagnosis 77
because of its advantages of no radioactive pollution and acceptable sensitivity 78
[21,22]. Yet, a disadvantage of the relatively longer time for immunoassay limits its 79
wide application. The increasing utilization of immunomagnetic beads (MPs) 80
separation techniques may resolve this problem. For the antibody–antigen binding 81
equilibrium could be achieved more rapidly on magnetic beads and the washing 82
process of MPs is much more convenient comparing to the planar surface, such as 83
microplate wells [23,24]. 84
In the present study, a magnetic particles-based chemiluminescence enzyme 85
immunoassay (MPs-CLEIA) using MPs coated polyclonal antibodies (pAb) anti-OVA 86
5
and HRP-labeled monoclonal antibody (mAb) against OVA was established, as shown 87
in Fig. 1, which combined the advantages of both the MPs and CLEIA. Furthermore 88
the methodology parameters were explored and optimized, and the OVA in influenza 89
vaccine and food was determined using the proposed method, finally the results were 90
analyzed and compared to the commercially available OVA kit. 91
92
2 Materials and methods 93
94
2.1 Reagents 95
Egg white albumin (purity of 98%) was obtained from Sigma, magnetic particles 96
(Dynabeads M-280 Tosylactivatted) with diameter 2.8um ± 0.2um were purchased 97
from Dynal Biotech, Chemiluminescent substrate (luminol, H2O2 and ρ-iodophenol 98
solution), complete and incomplete Freund’s adjuvant (CFA and IFA), 99
polythyleneglycol-1000 (PEG), RPMI1640, fetal bovine serum (FBS), HT 100
(hypoxathine/thymidine) and HAT (hypoxathine/aminopterin/thymidine), horseradish 101
peroxidase-conjugated goat anti-mice IgG (HRP-IgG) were all purchased from Sigma. 102
Other chemicals were all analytic grade. 103
104
2.2 Experimental animals 105
Male 6-month-old New Zealand white rabbits (used for polyclonal antibodies 106
production) and female 6-week-old BALB/c mice (used for monoclonal antibodies 107
production) were purchased from experimental animal center of Jilin university. They 108
6
were all raised under comfortable environment with food and water available at any 109
time and a natural light. All animal studies were performed in accordance with the 110
provisions of EU animal management practices (1986.11.24). 111
112
2.3 Preparation of the specific polyclonal anti-OVA antibodies 113
In order to obtain specific antibody against OVA, two New Zealand white rabbits 114
were each immunized with 1mL of OVA (1 mg/mL), the detailed immunization 115
procedures were referenced to literature [25]. At the 10th day after the third 116
immunization, blood from ear vein was obtained and the serum titer was assayed by a 117
double agar immunodiffusion test. Then, the anti-sera were aliquoted into sterile vials 118
and stored at ﹣80 until use. 119
120
2.4 Preparation and characterization of monoclonal antibody against OVA 121
The immunization was carried out according to the method described by Feng et al. 122
[26]. The procedures of cell fusion was performed refer to the literature [27]. Positive 123
hybridoma secreting the mAb against OVA were cloned by limiting dilution and the 124
class of immunoglobulin was determined using an ELISA commercial kit (mouse 125
monoclonal antibody isotyping reagents purchased from Sigma, USA). 126
Then, the selected positive hybridoma were used for ascites production in vitro, the 127
details were carried out as described in literature [28]. 128
129
2. 5 Purification of the antibodies anti-OVA 130
7
Both the polyclonal antibodies and monoclonal antibody against OVA were firstly 131
purified by ammonium sulfate precipitation method to remove most of the hybrid 132
protein and then purified by protein A affinity chromatography columns using ÄKTA 133
purifier 100 system (GE, Sweden). The purified antibodies were then subjected to 134
SDS–PAGE analysis. 135
136
2.6 Immunoassay procedures of MPs-CLEIA (magnetic particles-based 137
chemiluminescence enzyme immunoassay) 138
2.6.1 Preparation of anti OVA pAb-coated MPs and HRP-labeled mAb 139
The preparation of anti OVA pAb-coated MPs was performed mainly according to the 140
manufacturer’s protocols (Dynal BiotechASA). Firstly the polyclonal antibodies were 141
dissolved in solution buffer (0.1 M borate buffer, pH 9.5) and added into the magnetic 142
beads suspension, the mixture was incubated for 16-24 h at room temperature with 143
slow tilt rotation. After incubation, the coated beads were washed for 4 times to 144
remove the unbinding antibodies and redissolved in buffer ( PBS (phosphate buffered 145
saline) with 0.1% (w/v) BSA, pH 7.4) and stored at 4 until use. 146
The HRP-labeled mAb conjugate was made using an activated horseradish peroxidase 147
kit (Pierce) following the manufacture’s instruction. 148
2.6.2 Immunoassay procedures of MPs-CLEIA 149
The proposed immunoassay procedures of MPs-CLEIA in this study were displayed 150
in Fig.1 and the detailed steps were as follows: 151
The MPs coated pAb were served as solid phase and separator. Firstly, the solution of 152
8
MPs-coated pAb were added into blocking buffer (PBS containing 5% defatted milk 153
powder) to block the unbound active sites on MPs and the mixture was stirred gently 154
at room temperature for 30 min. Then the MPs were separated and washed with PBST 155
(PBS containing 0.05% tween 20). After that, the MPs were re-suspended in PBS and 156
added into the ELISA microplate wells. Subsequently, a mixture of 50µL of either 157
OVA standard solution or sample and 50µL of the HRP-labled mAb (final dilution 158
1:2000 in 0.1% of PBST-BSA) was added into each well of the ELISA microplates 159
and incubated at 37 for 30 min. Following that, the MPs were precipitated by 160
placing the ELISA microplates on a magnet, the supernatant was removed and the 161
MPs were washed for at least 3 times with PBST. Then 100 µL of newly prepared 162
chemiluminescent substrate was added into each well of the microplates and 163
incubated at 37 for 20 min. The whole incubation procedure was in the absence of 164
light. Finally the Relative Light Unit (RLU) was detected using chemiluminescence 165
analyzer (TECAN infinity F200 Multi-function microplate, Sweden) and the OVA 166
level in sample was calculated according to the standard curve. 167
168
2.7 Detection of OVA from influenza vaccine and food using the established 169
MPs-CLEIA 170
A total of 10 different batches of influenza vaccines were purchased from Institute of 171
Biological Products in Changchun of China, and the influenza vaccines were tested 172
for its OVA level directly without any pretreatment. 173
Cooked egg whites, cooked duck egg whites, cooked quail egg whites, egg yolk pie, 174
9
Caramel treats, cakes, biscuit contains egg and biscuit contains no egg were purchased 175
from local supermarket in Changchun. Total 8 different kinds of food samples were 176
preprocessed according to the literature [29] before testing for the presence of OVA. 177
The MPs-CLEIA method established in this study and the commercially available 178
OVA kit were both applied to detect the OVA level in the real samples simultaneously, 179
and the results were analyzed using statistical methods. 180
181
3 Results 182
183
3.1 Production and characterization of mAb against OVA 184
Five hybridoma cell lines named 1B4, 4E9, 4E4, 2D7, and 2H8 which could secreted 185
mAb against OVA stably were obtained successfully, among which 1B4, 4E9, and 186
4E4 belonged to IgG2a isotype while 2D7 and 2H8 were IgM. Moreover, 4E9 showed 187
the highest sensitivity and titer, hence 4E9 was selected for the further studies. 188
189
3.2 Purification of the mAb and pAb against OVA 190
As the mAb and pAb were used as capturing and detecting antibody respectively, the 191
purity of the two antibodies would affect the sensitivity and specificity of 192
MPs-CLEIA. The purification effect was shown in Fig. 2, nearly all the other proteins 193
were removed, there were two chains (heavy and light chain) for mAb, however the 194
light chains for pAb were not apparent and there were only some wiped straps, we 195
thought the reason was that light chains of pAb were various and not so stable as 196
10
heavy chains, they may be degraded. The purity of both mAb and pAb could reach to 197
98%, which met the experimental requirements and could be used in the further 198
studies. 199
200
3.3 Development of the MPs-CLEIA based on MPs-coated pAb and HRP-labeled mAb 201
Different experimental parameters were optimized firstly, the best dilution of MPs and 202
pAb was 1 : 2000, the best coating condition was at 4 overnight. With the 203
increase of HRP-labeled antibody concentration, so did the background signal, then 204
the sensitivity of immunoassays may be decreased [30], therefore the optimal working 205
dilution of HRP-labeled mAb was determined at 1 : 4000. Finally the best reaction 206
time of pAb-coated MPs, OVA and HRP-labeled mAb was 20 min at 37 avoiding 207
light and the optimum substrate reaction time was also 20 min at 37 avoiding 208
light . 209
Under the optimal conditions, the calibration curve for OVA standards was 210
constructed, as shown in Fig. 3(A). The standard curve exhibited a high linearity from 211
0.31 ng/mL to 100 ng/mL with the limit of detection (LOD) 0.24 ng/mL and 212
correlation coefficient 0.9964, and the LOD was determined by the average 213
absorbance adding twice the mean of the standard deviation from ten blank wells. 214
215
3.4 Specificity, precision and stability analysis 216
In order to assess the specificity of the proposed method in this study, several other 217
proteins (20μg/mL) including BSA (Bovine Serum Albumin), α casein, β casein, KLH 218
11
(Keyhole Limpet Hemocyanin) were applied to the MPs-CLEIA system, the 219
cross-reactivities with other proteins were negligible, results were shown in Table 1. 220
To confirm the precision of this method, intra-assay and inter-assay were performed 221
using three different concentrations of OVA standards with 6 duplicates for intra-assay 222
while 6 times for inter-assay in 3 days. As we could see from Table 2, both intra- and 223
inter-CV were below 5%. 224
All components that would used to assemble a kit including MPs-coated pAb, 225
HRP-labeled mAb, OVA standards, buffers and chemiluminescence substrate were 226
respectively placed at 4 and 37 for 1, 3, 5, 7 days to test the stability. The 227
results in Table 3 showed that there was nearly no influence on the assay results. 228
229
3.5 Recovery and real sample analysis 230
The recovery studies were carried out using vaccine matrix (free of OVA) spiked with 231
OVA at three different concentrations of 1, 10 and 50 ng/mL. The recovery results 232
were shown in Table 4, and the average recoveries of three different batches were 233
96.34%, 92.33% and 94.90% respectively, with all coefficients of variation (CV) less 234
than 5%. 235
Ten different batches of influenza vaccine and 8 kinds of food samples were 236
determined for their OVA levels using the proposed MPs-CLEIA method and the 237
results were compared with a commercial kit, as shown in Fig. 3(B). A good 238
correlation was obtained between the two methods and the correlation coefficient was 239
0.9926, which indicated that the established MPs-CLEIA method in this study was 240
12
applicable. 241
242
4 Discussion 243
244
In this study, we established a new rapid, sensitive and specific MPs-CLEIA method 245
for analysis of OVA. The proposed assay consisted of anti-OVA pAb-coated MPs, 246
HRP-labeled anti-OVA mAb and a CL detection system. In this immunological 247
“sandwich” reaction, a lot of factors could affect the sensitivity and specificity of the 248
immunoreaction assay including the properties of pAb or mAb, concentration of 249
immunoreagents and the immunoreaction time [21,31]. The pAb and mAb screened in 250
our study were confirmed with high affinity, specificity, and purity of 98%, which 251
were the basic requirements for constructing this sensitive MPs-CLEIA method. In 252
addition, redundant MPs may cause lower sensitivity as MPs could absorb the emitted 253
light [32] and high concentration of HRP-labeled Ab could lead to nonspecific 254
adsorption, the chemiluminescent substrate volume was also an important parameter 255
for it directly related to the chemiluminescence intensity [23]. So we optimized these 256
reaction parameters for detecting OVA. The best reaction time needed to explore too, 257
as the RLU increased with the prolonged incubation time, nevertheless longer 258
immunoreaction time would cause nonspecific absorption and reduced sensitivity. 259
The parameters of this methodology such as sensitivity, specificity, precision, 260
accuracy, stability and so on had all been validated and the results of the real sample 261
analysis indicated the developed method was comparable and acceptable, and could 262
13
be employed in OVA detection satisfactorily. 263
To our knowledge, we were the first to prepare both the pAb and mAb against OVA 264
by ourselves, and then combined MPs and CL detection system to establish a novel 265
double antibodies sandwich immunological method. The MPs-CLEIA had the 266
advantages of both the classical ELISA method and the recently developed CLEIA 267
immunoassay. 268
Comparing with other methods for testing OVA, for example the commercially 269
available kit for OVA detection, there were several advantages of the proposed assay 270
in our study. The first one was rapidity, the entire test time was less than 50 minutes, 271
which including 20 minutes of incubation time, 20 minutes of substrate reaction time 272
and 10 minutes for washing while about 2 hours were needed for the commercially 273
available kit. The second and the most important one was sensitivity, as this 274
immunoassay was a “sandwich” reaction process based on the pAb-coated MPs and 275
HRP-labeled anti-OVA mAb, the immuno-activated magnetic beads working as the 276
solid-phase matrix, which could offer much more active binding sites of the 277
immobilized proteins on their surfaces and facilitate larger linear range in the 278
detection [33]. The MPs-CLEIA in this study had a linearity from 0.31 to 100 ng/mL 279
with LOD 0.24 ng/mL while the linear range of commercially available kit was 0.63 280
to 40 ng/mL with LOD 0.57 ng/mL. Mattarozzi M et al. reported that LC-ESI-MS/MS 281
was applied to determine the OVA in fortified red wine, and the linear range in their 282
devised method was 10-800 ng/mL, the sensitivity was lower than that in our study. 283
[34]. Azarnia S et al. also used LC-ESI-MS/MS and ELISA to detect OVA in egg 284
14
white, whole egg and incurred pasta, however the recoveries were all very low [35]. 285
Furthermore, the MPs-CLEIA was economic and affordable, we estimated the cost for 286
assembling a kit was no more than 1000 yuan while the commercially available kit 287
was 4800 yuan or also. Other methods such as LC-ESI-MS/MS mentioned above 288
required expensive instruments and professional staff, which increased the 289
expenditure greatly. Finally, the MPs-CLEIA was non-radioactive and non-poisonous, 290
which would not pose a threat on the health of experimenters. 291
292
5 Conclusions 293
294
In the present study, we established a chemiluminescence immunoassay method for 295
the rapid and sensitive determination of OVA by combining with magnetic separation 296
and HRP-labeling techniques. As polyclonal antibodies (pAb) and monoclonal 297
antibody (mAb) against OVA were both present in this sandwich-type immunoassay 298
used as separation and detection antibody respectively, the sensitivity and specificity 299
of this method were greatly improved. It was employed for OVA determination and a 300
linear rang from 0.31 to 100 ng/mL with a detection limit of 0.24 ng/mL was obtained 301
in OVA standards. This method was also applied in influenza vaccines and food and 302
the results were compared to the commercially available OVA detection kit, which 303
showed satisfactory consistency. Further studies will be adopted to explore other 304
kinds of immunoassay method that may integrate more new techniques, aiming to 305
improve the stability and sensitivity of CL method for OVA detection. 306
15
307
Acknowledgments 308
309
This work was supported by the National Nature Science Foundation of China (No. 310
31071539); the Fundamental research operating expenditures of Jilin University (No. 311
450060481284); the Key Project of Jilin Province (No. 20120966); Specialized 312
Research Fund for Doctoral Program of Colleges and Universities (No. 313
20120061110078). 314
315
References 316
1. Ciardiello MA, Tamburrini M, Liso M, Crescenzo R, Rafaiani C, Mari A (2013) 317
Food allergen profiling: A big challenge. Food Research International 54 318
(1):1033-1041 319
2. Eggesbø M, Botten G, Halvorsen R, Magnus P (2001) The prevalence of allergy to 320
egg: a population‐ based study in young children. Allergy 56 (5):403-411 321
3. Fishbein AB, Fuleihan RL (2012) The hygiene hypothesis revisited: does exposure 322
to infectious agents protect us from allergy? Current opinion in pediatrics 24 323
(1):98-102 324
4. Poulsen LK, Hansen TK, Nørgaard A, Vestergaard H, Stahl Skov P, 325
Bindslev‐ Jensen C (2001) Allergens from fish and egg. Allergy 56 (s67):39-42 326
16
5. Anet J, Back J, Baker R, Barnett D, Burley R, Howden M (1985) Allergens in the 327
white and yolk of hen’s egg. International Archives of Allergy and Immunology 77 328
(3):364-371 329
6. O'Brien TC, Maloney CJ, Tauraso NM (1971) Quantitation of residual host protein 330
in chicken embryo-derived vaccines by radial immunodiffusion. Applied 331
microbiology 21 (4):780 332
7. James JM, Zeiger RS, Lester MR, Fasano MB, Gern JE, Mansfield LE, Schwartz 333
HJ, Sampson HA, Windom HH, Machtinger SB (1998) Safe administration of 334
influenza vaccine to patients with egg allergy. The Journal of pediatrics 133 335
(5):624-628 336
8. Cowburn G, Stockley L (2005) Consumer understanding and use of nutrition 337
labelling: a systematic review. Public Health Nutrition-Wallingford 8 (1):21-28 338
9. Mills E, Breiteneder H (2005) Food allergy and its relevance to industrial food 339
proteins. Biotechnology advances 23 (6):409-414 340
10. van Hengel AJ (2007) Food allergen detection methods and the challenge to 341
protect food-allergic consumers. Analytical and bioanalytical chemistry 389 342
(1):111-118 343
11. Baumgartner S, Krska R, Welzig E, Mills C, Wichers H, Hoffmann-Sommergruber 344
K (2007) Detecting allergens in foods. Managing allergens in food:228-250 345
12. Wallis C, Melnick JL (1971) Enhanced detection of Australia antigen in serum 346
hepatitis patients by discontinuous counter-immunoelectrophoresis. Applied 347
microbiology 21 (5):867-869 348
17
13. Holzhauser T, Wangorsch A, Vieths S (2000) Polymerase chain reaction (PCR) for 349
detection of potentially allergenic hazelnut residues in complex food matrixes. 350
European food research and technology 211 (5):360-365 351
14. Arlorio M, Cereti E, Coisson J, Travaglia F, Martelli A (2007) Detection of 352
hazelnut (< i> Corylus</i> spp.) in processed foods using real-time PCR. Food 353
Control 18 (2):140-148 354
15. Monti G, Muratore M, Peltran A, Bonfante G, Silvestro L, Oggero R, Mussa G 355
(2002) High incidence of adverse reactions to egg challenge on first known exposure 356
in young atopic dermatitis children: predictive value of skin prick test and 357
radioallergosorbent test to egg proteins. Clinical & Experimental Allergy 32 358
(10):1515-1519 359
16. Hagiescu H (1971) Comparative study of electrophoresis and double diffusion in 360
agarose gel for the detection of Australia antigen. Revue roumaine 361
d'inframicrobiologie 8 (3):203 362
17. Berlin BS, Pirojboot N (1972) A rapid method for demonstration of precipitating 363
antibody against influenza virus by counterimmunoelectrophoresis. Journal of 364
Infectious Diseases 126 (3):345-347 365
18. Poms R, Klein C, Anklam E (2004) Methods for allergen analysis in food: a 366
review. Food additives and contaminants 21 (1):1-31 367
19. Meyer R, Chardonnens F, Hübner P, Lüthy J (1996) Polymerase chain reaction 368
(PCR) in the quality and safety assurance of food: detection of soya in processed meat 369
products. Zeitschrift für Lebensmittel-Untersuchung und Forschung 203 (4):339-344 370
18
20. Feng X-L, Lu S-Y, Liu D, Li L, Wu X-Z, Song J, Hu P, Li Y-S, Tang F, Li Z-H 371
(2013) Direct competitive immunosorbent assay for detection of MEHP in human 372
urine. Chemosphere 373
21. Lin Z, Wang X, Li Z-J, Ren S-Q, Chen G-N, Ying X-T, Lin J-M (2008) 374
Development of a sensitive, rapid, biotin–streptavidin based chemiluminescent 375
enzyme immunoassay for human thyroid stimulating hormone. Talanta 75 (4):965-972 376
22. Zhao L, Lin J-M (2005) Development of a micro-plate magnetic 377
chemiluminescence enzyme immunoassay (MMCLEIA) for rapid-and 378
high-throughput analysis of 17β-estradiol in water samples. Journal of biotechnology 379
118 (2):177-186 380
23. Wang X, Lin J-M, Ying X (2007) Evaluation of carbohydrate antigen 50 in human 381
serum using magnetic particle-based chemiluminescence enzyme immunoassay. 382
Analytica chimica acta 598 (2):261-267 383
24. Wang X, Zhang Q-Y, Li Z-J, Ying X-T, Lin J-M (2008) Development of 384
high-performance magnetic chemiluminescence enzyme immunoassay for 385
α-fetoprotein (AFP) in human serum. Clinica Chimica Acta 393 (2):90-94 386
25. Yao L, Wu Q, Wang D, Kou X, Zhang J (2009) Development of monoclonal 387
antibody-coated immunomagnetic beads for separation and detection of norovirus 388
(genogroup II) in faecal extract samples. Letters in applied microbiology 49 389
(2):173-178 390
19
26. Zhou Y, Zhang Y-Y, Shen Q-F, Lu S-Y, Ren H-L, Li Y-S, Liu Z-S, Pan F-G, Meng 391
X-M, Zhang J-H (2009) Development of a novel antibody probe useful for domoic 392
acid detection. Biosensors and Bioelectronics 24 (10):3159-3163 393
27. Zhou Y, Li Y, Pan F, Liu Z, Wang Z (2009) Identification of tetrodotoxin antigens 394
and a monoclonal antibody. Food Chemistry 112 (3):582-586 395
28. Zhou Y, Li Y-S, Pan F-G, Zhang Y-Y, Lu S-Y, Ren H-L, Li Z-H, Liu Z-S, Zhang 396
J-H (2010) Development of a new monoclonal antibody based direct competitive 397
enzyme-linked immunosorbent assay for detection of brevetoxins in food samples. 398
Food Chemistry 118 (2):467-471 399
29. Qin Q-R (2010) The preparation of anti-ovalbumin monoclonal antibody and the 400
quantitative determination of ovalbumin and gliadin in foods by ELISA. Ocean 401
University of China, 402
30. Pamme N (2006) Magnetism and microfluidics. Lab on a Chip 6 (1):24-38 403
31. Zhang H, Qi S (2011) A rapid and sensitive chemiluminescence immunoassay 404
based on magnetic particles for squamous cell carcinoma antigen in human serum. 405
Clinica Chimica Acta 412 (17):1572-1577 406
32. Li Z, Zhang Q, Zhao L, Li Z, Hu G, Lin J, Wang S (2010) Micro-plate magnetic 407
chemiluminescence immunoassay and its applications in carcinoembryonic antigen 408
analysis. Science China Chemistry 53 (4):812-819 409
33. Zhang Q, Wang X, Li Z, Lin J-M (2009) Evaluation of α-fetoprotein (AFP) in 410
human serum by chemiluminescence enzyme immunoassay with magnetic particles 411
and coated tubes as solid phases. Analytica chimica acta 631 (2):212-217 412
20
34. Mattarozzi M, Milioli M, Bignardi C, Elviri L, Corradini C, Careri M (2014) 413
Investigation of different sample pre-treatment routes for liquid 414
chromatography–tandem mass spectrometry detection of caseins and ovalbumin in 415
fortified red wine. Food Control 38:82-87 416
35. Azarnia S, Boye JI, Mongeon V, Sabik H (2013) Detection of ovalbumin in egg 417
white, whole egg and incurred pasta using LC–ESI-MS/MS and ELISA. Food 418
Research International 52 (2):526-534 419
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Table 1 Cross-reactivity of OVA with other proteins using the proposed MPs-CLEIA method.
Proteins Added concentration
(ng/mL)
Tested concentration
(ng/mL) CR(%)
OVA 10 10 100
BSA (Bovine Serum
Albumin) 50 0.76 1.52
α casein 100 0.89 0.89
β casein 100 0.81 0.81
KLH (Keyhole
Limpet Hemocyanin ) 80 0.57 0.71
Table 2 The intra-assay and inter-assay tests.
Intra-assay and inter-assay tests were performed using 3 different concentrations of OVA
standards.
CV, coefficient of variation. SD, standard deviation.
Times
Added concentration (ng/mL)
1.0 (n=6) 5.0 (n=6) 10.0 (n=6)
Average
(ng/mL) SD
CV
%
Average
(ng/mL) SD CV%
Average
(ng/mL) SD CV%
1 0.95 0.03 3.4 4.89 0.19 3.9 9.95 0.17 1.7
2 0.91 0.04 3.9 4.96 0.12 2.5 9.89 0.29 2.9
3 0.90 0.04 4.1 4.95 0.13 2.6 9.91 0.24 2.4
4 0.89 0.04 4.6 4.90 0.18 3.7 9.86 0.31 3.1
5 0.95 0.03 3.4 4.88 0.20 4.1 9.94 0.19 1.9
6 0.92 0.03 3.7 4.95 0.13 2.6 9.87 0.30 3.0
Total average
(ng/mL) 0.87 4.92 9.90
SD 0.03 0.16 0.25
CV% 3.9 3.2 2.5
Table 3
Stabilit
y of the
reagent
s at
4
and
37
(n=3).
Time
(day)
RLU s1/s0 Correlation coefficient CV %
4 37 4 37 4 37
1 89.8 88.2 0.9978 0.9976 2.41 2.87
3 87.6 87.4 0.9954 0.9951 4.07 3.93
5 89.2 88.0 0.9957 0.9943 3.39 4.85
7 83.5 84.2 0.9949 0.9924 4.85 4.94
Table 4 Recoveries of OVA spiked matrix determined by the established MPs-CLEIA a
Spiked
concentr
ations
(ng/mL)
Recovery b(%)
Coefficient of variation c
(CV% , n=5)
Average recovery
(%)
Batch1 Batch2 Batch3 Batch1 Batch2 Batch3 Batch1 Batch2 Batch3
1 95.40 90.50 95.47 2.99 3.21 4.91
96.34 92.33 94.90 10 95.80 94.72 97.65 3.67 4.13 4.63
50 97.83 91.79 91.57 4.27 3.95 3.05
a The assay was performed as described in the test.
b The values were mens of five times repeat.
c The coefficient variation = SD/mean×100%