J Vet Sci.  2013 Dec;14(4):413-419. 10.4142/jvs.2013.14.4.413.

Development and standardization of a monoclonal antibody-based rapid flow-through immunoassay for the detection of Aphanomyces invadans in the field

• Adil B
• Shankar KM
• Naveen Kumar BT
• Patil R
• Ballyaya A
• Ramesh KS
• Poojary SR
• Byadgi OV
• Siriyappagouder P

• KMID: 1712307
• DOI: http://doi.org/10.4142/jvs.2013.14.4.413

Abstract

A monoclonal antibody-based flow-through immunoassay (FTA) was developed using a nitrocellulose membrane placed on the top of adsorbent pads enclosed in a plastic cassette with a test zone at the center. The FTA could be completed within 10 min. Clear purple dots against a white background indicated the presence of Aphanomyces (A.) invadans. The FTA limit of detection was 7 microg/mL for A. invadans compared to 56 microg/mL for the immunodot. FTA and polymerase chain reaction (PCR) could detect A. invadans in fish tissue homogenates at a 10-11 dilution compared to a 10(-8) dilution by immunodot. In fish suffering from natural cases of epizootic ulcerative syndrome (EUS) collected from Mangalore, India, FTA and PCR could detect A. invadans in 100% of the samples compared to 89.04% detected by immunodot. FTA reagents were stable and produced expected results for 4 months when stored at 4~8degrees C. This rapid test could serve as simple and cost-effective on-site screening tool to detect A. invadans in fish from EUS outbreak areas and in ports during the shipment of live or frozen fish.

Keyword

diagnosis; epizootic ulcerative syndrome; fish; flow-through; monoclonal antibody

MeSH Terms

Animals
Antibodies, Monoclonal/metabolism
Aphanomyces/*isolation & purification
Fish Diseases/*diagnosis/parasitology
Fishes
Immunoassay/*methods/veterinary
India
Infection/diagnosis/parasitology/*veterinary
Polymerase Chain Reaction/methods/veterinary
Reagent Kits, Diagnostic/*standards/veterinary
Antibodies, Monoclonal
Reagent Kits, Diagnostic

Figure

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  Fig. 1 Fish with typical epizootic ulcerative syndrome (EUS) clinical signs collected from Mangalore, India. (A) Terapon spp. (B) Cynoglossus spp. (C) Sillago spp. (D) Mugil spp. (E) Channa spp.

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  Fig. 2 Images of the flow-through immunoassay (FTA). (A) Nitrocellulose membrane in the cassette. (B) Test reagents.

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  Fig. 3 Development of the FTA with MAb C-14 for detecting A. invadans in fish. Homogenates of muscle tissues from fish with EUS were spotted (arrows) onto the nitrocellulose membrane along with pre-dotted A. invadans culture samples as the positive control and muscle homogenates from healthy fish as the polymerase chain reaction (PCR)-negative control.

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  Fig. 4 Comparing sensitivity of the FTA and immunodot. Homogenates of cultured A. invadans were serially diluted to 0.9, 0.45, 0.225, 0.112, 0.056, 0.028, 0.014, 0.007, 0.0035, 0.0017, and 0.008 mg/mL (corresponding to numbers 1 to 11, respectively). For each dilution, 2 µL of supernatant was spotted onto the nitrocellulose membrane along with the positive and negative controls. (A) FTA. (B) Immunodot.

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  Fig. 5 Comparing sensitivity of the FTA, immunodot, and PCR assay using samples from four fish with EUS (a~d). Muscle tissues from the four fish were isolated, homogenized, and divided into two equal aliquots. One aliquot was subjected to serial log dilution and used for (a~d) the FTA and immunodot. DNA was extracted from the other aliquot and subjected to serial log dilution for PCR. Dilutions 1×10-1, 1×10-2, 1×10-3, 1×10-4, 1×10-5, 1×10-6, 1×10-7, 1×10-8, 1×10-9, 1×10-10, 1×10-11, and 1×10-12 are represented as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively. (A) FTA. (B) Immunodot. (C) PCR.

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