“External” antibodies as the simplest tool for sensitive immunochromatographic tests

doi:10.1016/j.talanta.2017.07.027

Talanta

Volume 175, 1 December 2017, Pages 77-81

https://doi.org/10.1016/j.talanta.2017.07.027 Get rights and content

Highlights

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Scheme of immunochromatographic competitive analysis with "external" specific antibodies.
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The concentrations of antibodies and marker may be chosen independently.
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Dilution of the sample with buffer ensures pre-incubation of the antibodies with the sample.
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Detection limits are an order of magnitude lower than the limits of detection of traditional tests with the same reagents.

Abstract

The scheme of immunochromatographic competitive analysis with "external" specific antibodies is offered for the first time. The test strip includes all the necessary components for analysis (membranes, protein antigen conjugates, colored nanoparticles), except specific free unconjugated antibodies that are transferred to the buffer used for sample dilution. Thus, the concentrations of the antibodies and markers can be chosen independently, and dilution of the sample with buffer (a routine procedure for any immunochromatographic test) ensures pre-incubation of the antibodies with the sample. Both factors increase the sensitivity of the test. The proposed scheme has been proven to be effective for the determination of deoxynivalenol and T-2 toxin; detection limits of 500 pg/mL and 50 pg/mL, respectively, were achieved. This is an order of magnitude lower than the limits of detection of traditional tests using the same reagents. The analysis was conducted in water-organic extracts (20% methanol); the duration of the analysis is 10 min (it's the same time as for a traditional test). The proposed approach is universal, and it can be used to detect a variety of compounds.

Graphical abstract

Introduction

Due to its simplicity, low cost and high speed, immunochromatographic analysis (ICA) has become widespread. ICA is used to solve many problems, including diagnosis of diseases, detection of pathogens and toxins, and environmental monitoring [1], [2], [3], [4]. However, existing competitive ICA schemes do not meet the need for fast analysis times. Modern tests should not only be simple and affordable, but also highly sensitive. However, the sensitivities of common competitive ICAs are often insufficient for practical applications [5], [6]. This is due to the polyvalent properties of the antibody-marker conjugates that are used; they have a higher affinity for the protein antigen conjugate immobilized on the membrane surface than for the free antigen of the sample. Often this problem is solved by "brute force", i.e., a complex process for obtaining highly-specific antibodies that are weakly affined to the conjugate, but highly affined to the free antigen [7].

We offer a simpler approach. Traditionally used specific antibodies conjugated to gold nanoparticles (GNPs) were substituted with a combination of native specific antibodies and secondary (anti-species) antibodies conjugated to the GNPs. Multi- and single-stage competitive immunochromatographic test strips were obtained using this indirect labeling. A significant benefit in sensitivity was shown for the systems detecting zearalenone and aflatoxin В1 [7], [8].

In the study presented in this article, a new version of the principle was implemented. The dry test strip already contains all the necessary components (membranes, protein antigens, and GNP conjugates with anti-species antibodies), but specific antibodies are transferred to the sample buffer. Thus, the procedure for determining the antigen does not differ from the traditional approach, and it provides both a pre-incubation and an indirect introduction of the marker.

Trichothecene mycotoxins T-2 toxin (T2T) and deoxynivalenol (DON) were selected as the detection antigens. These contaminants are often found in food and food products, and they represent a significant threat to the health of humans and livestock [9], [10]; this underscores the importance of developing simple, inexpensive, and highly sensitive methods for their detection [11], [12], [13].

Section snippets

Chemicals and instrumentation

Sodium azide, Triton X-100, Tween-20, and chloroauric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA; sial.com). T2T and DON were purchased from Chromresurs (Moscow, Russia; chromresurs.ru). Bovine serum albumin (BSA) was purchased from MP Biomedicals (Santa Ana, CA, USA, mpbio.com). Mouse monoclonal antibodies against T2T were provided by IL-TEST Pushchino (Pushchino, Moscow region, Russia). Mouse monoclonal antibodies against the DON, T2T-BSA, and DON–BSA conjugates were purchased

Description of the immunochromatographic format

The composition of the test strip used for the analysis is shown in Fig. 1. There are several compounds on the plastic base: pad for absorbing the sample, a pad for the conjugate, and a working nitrocellulose membrane. The conjugate of the anti-species antibodies with GNPs is preliminarily applied to the fiberglass pad. In the test zone of the working membrane, the mycotoxin conjugate with the protein is immobilized, and there are mouse non-specific antibodies in the control zone. Specific

Conclusions

In the proposed scheme, two improvements were combined: sample pre-incubation with specific antibodies and indirect labeling. This allowed for a significant decrease in the detection limit. This new system was used for rapid (10 min) assaying of maize aqueous-organic extracts (20% methanol concentration in the final reaction mixture). Instrumental detection limits of 50 pg/mL and 500 pg/mL for T2T and DON, respectively, were reached. The proposed procedure does not differ from the traditional one,

Acknowledgements

The work was financially supported by the Russian Science Foundation (grant 14-14-01131).

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“External” antibodies as the simplest tool for sensitive immunochromatographic tests

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Chemicals and instrumentation

Description of the immunochromatographic format

Conclusions

Acknowledgements

TrAC-Trend Anal. Chem.

Biosens. Bioelectron.

Biosens. Bioelectron.

TrAC-Trends Anal. Chem.

Biosens. Bioelectron.

Biosens. Bioelectron.

Curr. Opin. Biotechnol.

Food Chem.

Food Control

Food Control

Molecules