Rapid detection of fish major allergen parvalbumin using superparamagnetic nanoparticle-based lateral flow immunoassay.

Food Control. 2012 Aug;26(2):446-52.

Zheng C, Wang XC, Lu Y*, Liu Y.

College of Food Science and Technology, Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai Ocean University, Shanghai, 201306, P. R. China. y-lu@shou.edu.cn

 

Abstract

A quantitative lateral flow immunoassay (LFIA) was developed based on superparamagnetic nanoparticle (SPMNP) probe for fish major allergen parvalbumin (Pa). The SPMNP probe was prepared by coupling monoclonal antibodies against allergen Pa onto the surface of SPMNP. Dispersibility of the obtained SPMNP probewas analyzed. Nitrocellulose membrane and test line coating concentration were optimized to construct the lateral flow system. Results showed that Sartorius CN 140 membrane, 0.8 mg/mL Pa was suitable for strip construction. Furthermore, a calibration curve with good linearity (R2=0.9949) was obtained by plotting magnetic signals against series concentrations of Pa. Signals of the T-line were linear in the range from 0.01 to 100 μg/mL Pa. LODs for qualitative and quantitative detection were 5 μg/mL and 0.046 μg/mL, respectively. The average recoveries in clam and peanut matrices ranged from 84.6% to 97.0%, within an acceptable level (80%~120%). 29 food extract samples were separately tested by LFIA and Western Blot assay. Comparative results indicated that the relative consistency between the two methods was 93.1% (27/29). On the other hand, the magnetic signal analysis results indicated that the detection time of the LFIA method was less than 20 min while Western Blot assay typically takes about 5 h. In conclusion, the LFIA method based on SPMNP probe for allergen Pa detection is rapid, specific and simple. It would significantly improve efficiency for large-scale screening and point-of-care detections of allergen Pa.

 

Supplement:

Food allergies are caused by a wide variety of foods. It is estimated that eight foods account for over 90% of all food allergic reactions. Milk, egg, fish, crustacean shellfish, wheat, soybeans, peanuts and tree nuts are the 8 major allergens listed as the most common causes for food allergy worldwide. The major allergen in a variety of fishes (cod, tuna, mackerel, carp, etc.) is parvalbumin (Pa), which is a water-soluble calcium-binding albumin protein with low molecular weight (typically 9-14 kD) (1).

New methods for Pa detection have been reported in recent years, including PCR, cytochemical detection and various immunoassays such as immunoblotting, enzyme-linked immunosorbent assay, and surface plasmon resonance biosensor. However, all these assays are time-consuming and labour-intensive as they require advanced equipment and professional operating techniques.

A new labelling material – superparamagnetic nanoparticle (SPMNPs) has been developed in recent few years. It provides an alternative to the traditional labelling materials such as colloidal gold, latex, selenium, carbon and liposomes (2) Compared with traditional lateral flow strips which utilize colour intensity to determine the signal strength, SPMNPs-based lateral flow assay can realize quantitative measurement through reading the magnetic signals by a Magnetic Assay Reader (MAR) system. The magnetic signals provided by the SPMNPs were quite stable, making it possible to recheck the testing results (3).

In the article, a competitive format LFIA labeled by SPMNP probe was developed. The main body of the test strip consists of five parts, including support sheet, sample pad, conjugate pad, wick pad and NC membrane, on which the test line and control line were sprayed (Fig. 1). Competitive format was adopted to establish the Pa test strips. Pa and goat anti-mouse secondary antibody at proper concentration were firstly striped onto nitrocellulose (NC) membrane at the rate of 0.8 μL/cm, respectively as T-line and C-line. Next, the NC membrane was dried at 35°C overnight. Striped NC membrane (300 × 25 mm), conjugate pad (300 × 10 mm), treated sample pad (300 × 20 mm), and wick pad (300 × 25 mm) were then pasted onto the base card (300 × 100 mm). Finally, a transparent top cover was pasted on the membrane as a protection sheet. The assembled system was cut into 5 mm-wide strips for detection.

Ying Lu-1

Figure 1. Schematic view of a SPMNPs probe based lateral flow test strip.

The use of SPMNPs and MAR system realized a rapid quantitative detection for Pa, which require little sample manipulation and simple operation. The qualitative and quantitative results of the LFIA can be obtained within 20 min. The developed magnetic LFIA was applicable to different matrices (clam and peanut) and allows for the detection of Pa of different food materials, showing good potential applications in areas such as large-scale screening or point-of-care detections for fish major allergen Pa.

Ying Lu-2Figure 2. Calibration curve for Pa detection by the LFIA. (a) Qualitative results of Pa standard solutions detected by LFIA. 1: negative (PBS); 2: 0.005 μg/ml Pa; 3: 0.01 μg/ml Pa; 4: 0.05 μg/hml Pa; 5: 0.1 μg/ml Pa; 6: 0.5 μg/ml Pa; 7: 1 μg/ml Pa; 8: 5 μg/ml Pa; 9: 10 μg/ml Pa; 10: 50 μg/ml Pa; 11: 100 μg/ml Pa. (b) Calibration curve of Pa standard solutions detected by LFIA. The x-axis is expressed on logarithm.

 

Ying Lu-3

Figure 3. Detection time of the LFIA. △represent negative sample (PBS), ●represent positive sample (1 μg/ml Pa solution).

Table 1. Comparison of 29 food samples tested by the LFIA and Western blot methods.

Ying Lu-4 a For the Western blot result of food sample test: ‘‘–” represents “negative”; “+” represents ‘‘positive”; ‘‘±”represents “weakly positive”.

b P0 value was 80.40; for LFIA result of food sample test, “–” represents “P > P0, negative”; “+” represents “P<50%, positive”; “±”represents “50% ≤P≤ P0, weakly positive”. * The result of western blot was not in accordance with LFIA.

 

References:

  1. Beale, J. E., Jeebhay, M. F., & Lopata, A. L. (2009). Characterisation of purified parvalbumin from five fish species and nucleotide sequencing of this major allergen from Pacific pilchard, Sardinops sagax. Molecular Immunology, 46(15), 2985-2993.
  2. Wang, Y., Xu, H., Wei, M., Gu, H., Xu, Q., & Zhu, W. (2009). Study of superparamagnetic nanoparticles as labels in the quantitative lateral flow immunoassay. Materials Science and Engineering: C, 29(3), 714-718.
  3. Xu, Q., Xu, H., Gu, H., Li, J., Wang, Y., & Wei, M. (2009). Development of lateral flow immunoassay system based on superparamagnetic nanobeads as labels for rapid quantitative detection of cardiac troponin I. Materials Science and Engineering: C, 29(3), 702-707

 

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