from Theron et al. in Nanotechnology in Water Treatment Applications

Immunological methods are based on the specific recognition between antibodies and antigens, and the high affinity that is characteristic of this recognition reaction. Consequently, many different immunoassay methods have become available for both quantitative and qualitative analysis of pathogenic bacteria in water. These include immunocapture of cells or antigens by enzyme-linked immunosorbent assay (ELISA or EIA), or detection of targeted cells by immunofluorescence (IFA). These assays can be performed by a direct or indirect manner. In a direct immunoassay, the monoclonal or polyclonal antibodies, directed against antigens located on the surface of the target pathogen (such as capsid proteins, cell wall or flagellar antigens), are conjugated with a fluorochrome or fluorescent dye. Alternatively, secondary enzymatically- or fluorescently-labelled antibodies directed against the primary antibodies (now serving as antigens) can be used in an indirect immunoassay. The advantage of this procedure is that the secondary antibodies can easily be obtained from a commercial supplier with a range of conjugated fluorochromes and it leads to signal amplification as several labelled secondary antibodies can bind to a single unlabelled primary antibody. The antigen-antibody complex is detected and quantified by the ability of the enzyme to react with a substrate that produces either a coloured product for colorimetry or emits light for luminometry. The immunoassays are often performed on a solid phase to which the pathogen antigens have been applied, such as a membrane filter or the bottom of a microtitre plate well.

Studies have shown that solid-phase enzyme immunoassays generally are too insensitive for direct detection of microbial pathogens in water, as they require a minimum of 103 to 104 target microbes (or their antigens) for detection. In most situations drinking water and its sources rarely contain high enough levels of most target pathogens for direct immunoenzymatic detection. Nevertheless, enumeration of diluted specific cells can be obtained by means of immunomagnetic separation (IMS). Immunomagnetic separation, also termed immunocapture or antibody capture, is a method that uses paramagnetic synthetic beads or other magnetic particles that have been coated with monoclonal or polyclonal antibodies directed against the target microbes to recover the microbes from the sample by antigen-antibody reactions. The retained microbes can be analyzed directly or after they or their nucleic acids have been released or extracted from the antibody and solid phase by various physical or chemical methods. IMS methods have the advantage of selecting, separating and purifying specific target microbes from other microbes and from solutes, based on the specificity of the antigen-antibody reaction. This is a powerful approach for recovering, enriching, purifying and concentrating the target viruses, bacteria and parasites from the sample matrix. However, it is not applicable to some pathogens because of the lack of antisera or the antigenic diversity of a large pathogen group lacking a common antigen and thus requiring many antisera.

As an alternative to the above assays, agglutination methods can be used to detect pathogens by combining dispersed microorganisms with antibodies (on a slide, for example) and allowing for antigen-antibody reactions to produce agglutination (clumping) that can be scored as negative or various degrees of positive. One modification is latex bead agglutination in which antibodies against a specific microbial antigen are attached to latex beads. The beads are reacted with the sample and should the sample contain the specific antigen, agglutination occurs by the reaction of antigens with antibodies on the beads resulting in the beads clumping together. As with enzyme immunoassays, agglutination tests are too insensitive to directly detect and quantify most waterborne pathogens in drinking water and other aquatic samples. The target microbes must first be cultured in order to obtain a sufficient number of them or a sufficient amount of antigen to detect and antigenically characterize them by agglutination methods.

The use of immunological methods for the detection of specific microorganisms is a rapid and simple technique, the accuracy of which mainly depends on the specificity of the antibody. Nevertheless, its application to the detection of specific microorganisms from environmental water samples is limited. While IFA allows specific identification and detection at a single-cell level, it does not provide information on the physiological status or viability of the detected cells. The ELISA is a rapid, simple and quite sensitive test. However, assay limitations are often associated with the specificity of the antibody used, the concentration of both antibody and antigen, and the solid matrix often leads to non-specific binding of the antigen or of the secondary antibody.

Monoclonal antibodies are better suited for biosensors because of their higher specificity. Polyclonal antibodies recognize different epitopes on the same pathogen. False positives can be generated when these antigens are present in other closely-related non-pathogenic microorganisms. The thermal instability of antibodies, in particular monoclonal antibodies, is another drawback when applying them in environmental biosensors. Single domain antibodies (also referred to as nanobodies) have however been developed that are thermostable, even at temperatures as high as 90 degrees C. Their small size, high solubility and refolding capacity are other features that make them ideally situated for biosensing applications.

Tags: Microbial Detection | Pathogen Detection | Biodiagnostics | Biodetection Assays | Biomolecular Detection