November 15, 2015 (Vol. 35, No. 20)

Rachel de las Heras Ph.D. Head of Commercial Development Anteo Technologie

Decrease Antibody Usage and Increase Sensitivity

Although long used for an extensive range of analytes including blood protein biomarkers, mycotoxins, pathogens, and most commonly, human chorionic gonadotropin (hCG), the lateral flow immunoassay (also known as LFIA or strip testing) is not without its weaknesses.

Testing by Diagnostic Consulting Network has shown Anteo Mix&Go™ improves upon this process, using half the amount of antibody to achieve five times more sensitivity than the covalently conjugated magnetic particle based assay.

While easy-to-use, relatively fast, and low-cost, conventional lateral flow tests often lack clinical sensitivity and waste significant quantities of antibodies when binding to nanoparticles. Aggregation commonly prevents full exposure of the reactive surface area during the coupling and coating steps, decreasing yield, compromising consistency and assay performance.

A number of approaches have been introduced into lateral flow testing in an attempt to improve sensitivity, handling time, antibody usage, and subsequently, cost. Gold nanoparticles are typically used as the reporter in lateral flow assays, with optical and physical properties making for fast running assays that can be read visually.

An alternative to gold nanoparticles are covalently conjugated magnetic nanoparticles, which have the added benefit of being able to be read visually for a qualitative output and also by magnetic readers for quantitative output.

Anteo Mix&Go has further improved upon the use of covalently conjugated magnetic particles in lateral flow assays, by activating the magnetic particles for a simpler assay development process and more effective assay performance.

Mix&Go is a series of reagents that contain hydrolytic oligomers of polymeric metal ions in an aqueous solution for use as a non-covalent method. This overcomes the limitation of single point chelation, as the polymer of metal ions forms multiple chelation points with both the underlying surface and the biomolecule (Figure 1).

This technology relies on avidity binding (multiple weak bonds) to hold proteins in place strongly, reducing aggregation, antibody usage, and handling time, while improving conjugate flow through testing strips.

Lateral flow testing is made easier with the water-based, ready-to-use Mix&Go activation reagent. Traditional reagent preparation steps are replaced by simply pipetting Mix&Go onto the surface to activate, reducing reagent preparation time by three to four hours.


Figure 1. Mix&Go is a noncovalent method utilizing polymeric metal ions to form multiple chelation points with both the underlying surface and the biomolecule.

Overcoming Lateral Flow Weaknesses

Antibody immobilization is critical to assay performance, and while antibodies are less prone to denaturation than other proteins, even minimal perturbation of the tertiary structure may expose hydrophobic regions, increasing nonspecific protein binding and potentially affecting assay sensitivity.

Mix&Go technology helps overcome these issues by creating an activated surface that gently yet strongly binds proteins using metal chelation rather than passive binding and/or covalent chemistry. Mix&Go was developed, through a screening process, to bind antibodies to particles. A targeted approach, similar to that used in drug design, was used to find a formulation series that preferentially binds to the Fc portion of the antibody. This enhanced antibody orientation and functionality is demonstrated with improved assay performance (Figure 2). 

The metal chelating principle behind Mix&Go is not specific to antibodies and can be applied universally to bind other proteins. To date, Anteo has successfully tested over 100 independent proteins in many varied life science and diagnostic applications including particles (micro and nano) and colloids, biosensor chips, ELISA plates, and microarray slides.

The Mix&Go protocol requires less antibody usage than a covalent method using a coupling reagent such as ethyl-dimethylaminopropyl carbodiimide (EDC). High levels of linearity are achieved, with improved flow of particles through the membrane, and a decrease in background noise from a blocking buffer.

Importantly, Mix&Go reduces aggregation and increases antibody binding capacity and functionality. By decreasing the required antibody loading, sensitivity is increased and costs are reduced due to lowered antibody usage.

Diagnostic Consulting Network (DCN) evaluated the performance of magnetic particles activated using the Mix&Go reagent in a lateral flow assay. This study compared the performance of the Mix&Go activated magnetic particles to covalently conjugated magnetic particles in a lateral flow assay that detects human chorionic gonadotropin (hCG). 


Figure 2. Graph of the reader results from Mix&Go and covalently conjugated magnetic particle hCG assays in urine.

Method

The hCG assay had previously been developed using colloidal gold DCN for internal use and demonstration purposes. The same assay materials (membrane, pads, and antibodies) were used with the Anteo Mix&Go (AMG) Coupling Kit, 200 nm Magnetic Particles and covalently conjugated magnetic particles.

All materials and tests were performed in DCN’s laboratories using magnetic particles conjugated with anti-hCG antibody using the Mix&Go procedure and DCN’s standard covalent conjugation procedure. hCG concentrations from 0 to 250 mIU/mL were prepared in 1% BSA, 0.1% Tween-20 in 1x PBS, and in negative pooled human urine. Tests were performed using 100 μL of sample and test strips with the covalently conjugated and the Anteo Mix&Go conjugated particles dried onto a conjugate pad.

Five replicates for each concentration were tested and results were recorded 15 minutes after the addition of the sample using DCN’s visual grading scale and an ESEQuant (Qiagen) reflectance reader.

The amount of antibody conjugated to the different particles and the amount of particles used per test were optimized to produce the highest sensitivity. The Mix&Go conjugate was tested with an antibody loading of 25 ug of anti-hCG antibody per mg of particles at 0.05% solids. The covalently conjugated particle was tested with an antibody loading of 50 ug of anti-hCG per mg particles at 0.05% solids.


Table 1. Visual and reader results of Mix&Go and covalently conjugated magnetic particle hCG assays with urine.

Results

The limit of detection for the Anteo Mix&Go assay was ~25 mIU/mL in urine for the visual and reader based results. The limit of detection for the covalently conjugated hCG assay was ~100 mIU/mL in urine. As a reference, the limit of the detection for the colloidal gold assay is at ~25 mIU/mL in urine.

NOTE: A visual grade of 2 or higher at the test line is considered positive, while the cut-off for the reader is at 30 mV.


Conclusions

The study confirmed the Anteo Mix&Go based hCG assay is significantly more sensitive than the covalently conjugated magnetic particle based test using the same critical reagents. The hCG assay conducted with the Anteo Mix&Go particles used half the amount of antibody to achieve five times more sensitivity than the covalently conjugated assay.

The variability of the Anteo Mix&Go based hCG assay was lower than the covalently conjugated hCG assay. There was also less nonspecific binding with the Mix&Go particles.

The Mix&Go procedure takes less time with fewer steps than the covalent conjugation procedure. The Anteo Mix&Go particles come pre-activated and are ready for the antibody conjugation. The covalent conjugation procedure requires steps to wash and activate the particles before the addition of the antibody.

Anteo Technologies currently has a kit available with Magnetic nanoparticles pre-activated with Mix&Go for use in lateral flow assays, with a forthcoming pre-activated gold nanoparticle kit due out in 2016.



























Rachel de las Heras, Ph.D. (rachel.delasheras@anteotech.com), is head of commercial development at Anteo Technologies.

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