dotLab mX System Applications

Serological Testing

The detection of infectious diseases and immune disorders by traditional methods such as ELISA or culture screening can often suffer from poor reliability and sensitivity, or are too labor intensive and time consuming to provide timely results at the point of care. Using the dotLab mX System, we have demonstrated that flow-through diffractive optics can significantly overcome many of these barriers and can potentially lead the way to more rapid diagnostic assays suitable for clinical applications. Overall, the unique features of the dotLab mX make it an ideal platform for developing and validating rapid serology assays.

Rapid Serology Example

We have previously demonstrated the effectiveness of the dotLab mX System in the rapid and sensitive detection of serum antibodies against Strongyloides infection from a group of patient serum samples. Strongyloidiasis is a persistent parasitic disease caused by the intestinal nematode Strongyloides stercoralis and is endemic in mainly tropical and subtropical regions of the world. In immunocompromised individuals, the presence of the parasite results in hyperinfection and disseminated disease with an associated mortality of over 80%.  Currently, there are two methods used in the detection of Strongyloides:  microscopic analysis of stool cultures or ELISA-based serology assays. Testing of stool cultures suffers from poor sensitivity as the parasite is not consistently shed in the stool. ELISA assays, on the other hand, require a lot of time and labor to obtain results and are not suitable for processing individual samples as they are obtained.

Using the dotLab mX System, we have been able reduce the time to result to only 30 minutes from many hours or even days needed for traditional ELISA’s and stool culture analyses (see figure 1). In addition, the simplicity of the assay could potentially enable point of care applications and eliminate the need for samples to be delivered to a central laboratory facility. The dotLab assay was also capable of detecting Strongyloides antibodies at very low levels in some individual samples and has shown high specificity relative to other parasitic antibodies.

Figure 1: Representative trace of a Strongyloides assay

Figure 1: Representative trace of a dotLab Strongyloides assay in less than 30 minutes. Biotinylated recombinant antigen was initially immobilized on an avidin-coated dotLab sensor followed by capture of antibodies from serum. Anti-human detection antibody conjugated to gold nanoparticles amplified the signal for Strongyloides antibodies.

For further details of this study, refer to:

IgG Titer and Avidity Example

The ability to distinguish between recent and past infections can have significant clinical utility. For example, Toxoplasma gondii infection in pregnant women can cause major congenital defects or fetal mortality if the primary infection occurs during pregnancy and is left untreated. On the other hand, if the infection is acquired prior to conception, the fetus will escape these deleterious effects. Typically, serological assays are performed to determine the time of infection onset, whereby levels of IgM antibodies (resulting from recent or acute infection) are compared to the levels of IgG antibodies (past or chronic infection). However, for infections such as Toxoplasmosis, these assays are complicated by the persistence of IgM antibodies long after infection onset. In these cases, the overall IgG avidity is measured in order to determine a recent (or acute) versus a chronic infection. A prolonged infection leads to higher overall avidity as a result of affinity maturation where cells producing higher affinity IgG’s are selected for.

Current methods of avidity testing use two separate ELISA assays: one normal and another in the presence of a chaotrope which interferes with the binding of low affinity IgG’s to the antigen. The greater the interference compared to the normal assay, the lower the IgG avidity, thus indicating an acute infection.  The dotLab, however, can perform both of these time-consuming assays in a single, automated run giving both titer and avidity results in less than one hour. Its real time display shows the avidity against a predetermined threshold upon addition of a chaotrope.

Figure 2: A biotinylated recombinant antigen was immobilized on avidin coated sensors. Serum sample was applied to the sensor yielding a direct antibody binding signal. A chaotrope  was added to dissociate the bound antibodies from where the avidity can be determined.

This example illustrates the dotLab’s capabilities for developing rapid avidity assays for infectious diseases with potential applications in clinical development. Additional applications exist in vaccine research where avidity is often used to determine the level of immunity after vaccination. For further details of avidity and titer assays in diagnostics and vaccine research, refer to:

Isotyping Example

Antibody isotype determination is useful for antibody manufacturers, assay developers and for infectious disease research where the isotype distribution can provide additional pathophysiological information. The dotLab mX System can be used to quickly determine the isotype distribution of captured antibodies in a single, automated assay (figure 3).

Figure 3: A biotinylated recombinant antigen was immobilized on avidin coated sensors. Serum sample (6 µL diluted 10-fold) was applied to the sensor yielding a direct antibody binding signal. Sequentially, antibodies against human IgG1, IgG2, IgG3, IgG4 and IgE were applied to the sensor with brief washes between each step. Detectable binding signal during each incubation was indicative of the presence of that particular antibody isotype.

Application Brief: Antibody Isotype Determination

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Direct Pathogen Detection

The ability to directly detect intact pathogens from crude samples has potential utility in food/environmental testing as well as in clinical diagnostics. Most current methods used for pathogen detection require culturing the pathogen to increase their number followed by immunoassay detection, or the amplification of the pathogen genome by polymerase chain reaction (PCR). Both of these methods are cumbersome and require specialized personnel to perform. 

Due to the large size of these pathogens, they are especially well suited for detection using diffractive optics even when present in low titers.  There is also no requirement for sample pre-processing or pre-culturing making the dotLab an ideal platform for rapid pathogen detection. As an example, figure 4 illustrates the use of the dotLab in the direct detection of E. coli O157 bacteria. Figure 5 shows the detection of influenza virus in nasal swab samples. In each case, a secondary detection antibody is added after the sample for both signal amplification and for providing assay specificity against pathogen substrains. In addition, with panelPlus sensors, multiple pathogen screening assays can be performed on a single sample.

Figure 4: Direct detection of E. coli. Biotinylated rabbit anti-goat antibody was immobilized on avidin sensors and used to capture a polyclonal goat antibody against E. coli O157. A sample containing either E. coli O157 (positive) or E. coli O145 (negative) was incubated in the sensor, followed by the addition of a monoclonal antibody against E. coli O157 as a detection antibody.

Figure 5: Detection of influenza virus in nasal swab samples. A biotinylated capture antibody was immobilized on avidin sensors and incubated with a diluted nasal swab sample containing influenza A virus (A/Beijing/262/95 H1N1). Influenza virus binding was directly observed from the nasal swab sample. Viral binding signal was amplified using a secondary antibody and a detection antibody conjugated to 10 nm gold nanoparticles.

Application Briefs:

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Multiplexing

Researchers conducting multivariate diagnostic studies of protein biomarkers benefit from the ability to perform simultaneous quantitative measurements of analytes. However, in many cases, the development of multiplex assays is often complicated by cross-reactivities between assay reagents and analytes. In addition, within a single sample, the analytes of interest are often present in widely varying concentrations which require tedious serial dilutions and multiple runs on the same sample. The dotLab mX System is designed to address both these issues: it simplifies the development and implementation of multiplex analysis and ultimately facilitates the routine analysis of diverse samples for biomarker research.

With the dotLab, real-time monitoring using the panelPlus sensors enables the rapid determination of potential cross-reactivity (cross-talk) between different assays (figure 6). As a result, the dotLab eliminates the need for multiple and time consuming ELISA plates for multiplex assay development.

Figure 6: In this example, a 3-plex assay for CA125, free bhCG and AFP was tested for cross-reactivity. Using panelPlus sensors with antibodies against each of the biomarkers immobilized on different spots, a sample containing high concentrations of CA125 and free bhCG was incubated and the spot for AFP monitored (Panel A). The results showed no signal on the AFP spot indicating that CA125 and free bhCG do not cross-react with AFP antibodies. Similarly, the spots for free bhCG (Panel B) and CA125 (Panel C) following the addition of AFP/CA125 and AFP/free bhCG respectively were monitored for cross-reactivity.

The dotLab’s ability for sequential probing also provides expanded dynamic range for detecting multiple analytes present over widely varying concentrations.  In the figure below (figure 7), a 2-plex assay was performed on c-reactive protein (CRP), which is present in high abundance, and cardiac troponin (cTn), which is low abundant analyte.  Oligonucleotide-conjugated antibodies against CRP and cTn were immobilized on panelPlus sensors. No cross talk was observed when the oligonucleotide-conjugated antibodies were introduced to the sensors (upper left panel). When incubated with a test serum sample, CRP binding was directly detected while no signal was obtained on the cTn spot (upper middle panel). The subsequent addition of an alkaline phosphatase (AP)-linked secondary antibody against cTn and amplification using a precipitating form of AP substrate yielded a detectable signal for cTn with no cross talk with the CRP spot (upper right panel).

Figure 7:Detection of high and low abundance analyte in a single assay. CRP (high abundance) and cTn (low abundance) were detected within their respective linear range of detection in a 2-plex assay using panelPlus sensors.

For a more detailed illustration of the dotLab’s multiplex capabilities, please refer to the following technical notes:

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Protein Complexes

Detection of Protein Complexes

Changes in subunit composition of protein complexes are widely described in various disease states and the ability to easily detect these changes may assist in disease determination and characterization. Traditional approaches require separate assays to be performed for each subunit in the complex. The direct detection of analytes without the use of labels using the dotLab mX System enables the sequential addition of reagents following the capture of protein complexes for subunit characterization in a single assay (figure 8). The System can either replace or be used along side other techniques that are currently employed in studying protein complexes such as multiple ELISAs and mass spectrometry.

Figure 8: Detection of subunits in the cardiac troponin (cTn) complex. A biotinylated antibody against cTnI was used to capture the cTn heterotrimeric complex. Following capture, the sensor was probed sequentially with antibodies against cTnI, cTnC and cTnT with brief washes between incubations. Each of the cTn subunits in the complex was detected in a single assay.

(Lin et al., Development of a qualitative sequential immunoassay for characterizing the intrinsic properties of circulating cardiac troponin I. Clin Chem. 2010, 56:1307-1319)

For applications of the dotLab mX in the study of protein complexes please refer to the following technical notes:

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Immunoassay Development

The development of immunoassays can be a complex and tedious process when performed by traditional endpoint-based methods such as ELISA. However, the dotLab mX Systems offers many features that will simplify the process by providing real time interaction data on an easy to use, fully automated platform. For instance, using its real time monitoring capabilities, the optimal antibody can be determined by comparing the binding curves of different antibodies with an antigen of interest (see figure 9). Similarly, the optimal antibody pairs for sandwich assay can be found in a relatively short period of time. Cross reactivity between immunoassay reagents can also be determined by sequentially probing a capture antibody or other reagents against each other, all in a single run. Overall, the dotLab mX is ideally suited to laboratories involved in developing immunoassays and would assist in the development of new assays or the optimization of existing ones.

Figure 9: A biotinylated antigen was immobilized on avidin-coated sensors. Normalized concentrations of three antibodies were separately applied to the sensors yielding information on their relative binding rates to the immobilized antigen and the levels of binding at equilibrium.

For more details on immunoassay development see the following:

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Quantitation and Signal Amplification

Since the signals generated on the dotLab mX are proportional to the analytes’ concentration, a calibration curve can be generated which can be used to determine unknown concentrations for specific analytes.  Furthermore, the dotLab mX System can quantify the amounts of protein in samples when they are available, unlike alternative methods that are only practical after multiple samples have been accumulated. This capability has direct applications in:

    • On-line protein manufacturing and purification
    • Time sensitive biomarker analysis (eg. in cases where initial results drive the next stage of analysis or experimentation)

The dotLab mX System also allows for the sequential amplification of analytes that are either present in low concentrations or are too small to be detected alone by diffractive optics. Several strategies can be employed to increase the effective signal intensity by simply increasing the analyte’s apparent size on the sensor. Figure 10 summarizes a few commonly used methods for signal amplification that can yield > 7 log units of dynamic range.

Figure 10: A wide range of amplification strategies based on increasing the apparent size of the analyte can be employed on the dotLab System.

For applications of the dotLab mX in the quantitative analysis of proteins, refer to the following technical notes: