The Science Behind BlueGreenTest
The Basics of Immunoassay’s
An Immunoassay is a technique utilized to detect the presence or quantity of a substance, such as hormones, drugs or specific proteins. Due to recent developments, immunoassays are an increasingly popular tool as they offer fast, sensitive and specific results compared to traditional techniques ⁽⁴⁸⁾.
The term Immunoassay refers to any test that, as its core, depends on the binding of an antigen and antibody together. Similar to the locking of two puzzle pieces together, only one specific antibody will bind with an antigen. In general terms, the basis of the test lies through determining whether the substance being tested reacts with the known antibody. Since only one antigen will bind with that specific antibody, it can be verified that the antigen being tested for is present ⁽⁵⁴⁾.
Antigen: Any substance with which a specific antibody will bind to in accordance with the antigen’s molecular structure.
In immunology, an antigen (often a foreign or harmful substance) is a molecule capable of inducing the immune response of the creation of antibodies specific to itself.
Antigens include proteins, peptide (amino acid chains), and polysaccharides (and lipids/nucleic acids only when combined with a protein).
Antibody: A specialised protein (immunoglobulin) which recognises and binds specifically with only one antigen.
In immunology, an antibody will flag the antigen as problematic, or can even neutralize it directly in some cases ⁽⁴²⁾.
In order to indicate whether the antigen has bonded with the antibody, the tests uses reagents to generate a signal from minute amounts of target substances (analyte) in a sample [²⁰]. To simply explain immunoassays, one cannot defer from the simple imagery conjured by David Wild’s brilliant analogy of the magnet on a fishing line [²⁰].
‘Imagine tying a magnet to a piece of fishing line – this magnet is then placed in a stream. Whilst the magnet is sitting in the stream, several miniscule pieces of metal are attracted to the magnet, where they sit, securely affixed. If we relate this to an immunoassay, the magnet is replaced by an antibody, which instead of being fixed to a piece of fishing line, is immobilized on to a plastic surface’.
Due its specificity, only the targeted antigens will bind to the antibodies, even when exposed to a sample containing an infinite number of other particles. In the case of the BlueGreenTest ®, the toxins are in such minute concentrations, that it is not good enough to simply “catch” the toxins, we must have the ability to quantify the amount present in the sample. To do this, the immunoassay process employs the use of a second reagent to generate a signal from the target analyte in the sample. This signal generated enables us to determine the concentration of the target analyte present in the sample ⁽⁵⁵⁾.
Signals are often determined via ‘labelling’ the secondary antibody with a biochemical marker which can be directly measured. There are a variety of mechanisms utilized to label, including covalently attaching either a radioisotope (Radioimmunoassay – RIA), reporter enzyme (Enzyme Linked ImmunoSorbent Assay- ELISA), or a fluorescent compound to the antibody (Immunofluorescent assays – IFA) ⁽⁴⁸⁾
In the case of the BlueGreenTest ®, the signal is displayed as a the vibrancy of the Test ⁽ᵀ⁾ line whereby the concentration of target analyte present is proportional to the strength of the line.
There are two main methods used in Immunoassays: competitive and non-competitive.
In competitive immunoassays, the targeted analyte of interest (antigen) competes with a constant added amount of labelled similar antigen for a limited amount of specific antibody binding sites. The amount of labelled antigens bound with the restricted antibodies is inversely proportional to the amount of analyte of interest present in the specimen, meaning that as the amount of the analyte in the sample increases, the detectable signal decreases ⁽⁵⁶⁾.
Competitive immunoassays can be classified further as:
Simultaneous addition: Where all components are added at once.
Sequential: where the sample is incubated with the antibody before the labelled analogue is added. This assists to improve the sensitivity of the test.
Due to the restricted nature, the assays are highly sensitive and are able to produce a large signal from a small amount of analyte. The sensitivity of each assay is related any inversely proportional to the affinity constant of the antibody used. Additionally, complete purification of the antibody is not essential, and the process is not significantly affected by substances that cross-react with the antibody ⁽⁴⁸⁾
However, competitive immunoassays are comparatively slow and require longer incubation times in order to process (reach equilibrium). Additionally, sensitivity of the test will be impacted by compounds within the sample that affect the equilibrium constant of the reaction ⁽⁴⁷⁾ ⁽⁵²⁾
Non-competitive, or immunometric immunoassays utilize an excess of labelled antibody toward the analyte of interest (the antigen). “Sandwich” immunoassays incorporate two separate antibodies, one labelled, and one un-labelled and fixed to a solid support which is used to extract the analyte of interest from the sample. The second labelled antibody is added and binds to a separate site on the bided analytes- resulting in an antibody “sandwich” with the analyte positioned in the middle of the two antibodies. The amount of analyte is able to be determined through measuring detectable signal from the labelled antibody, whereby the amount of bound labelled antibody is directly proportional to the amount of analyte present in the specimen ⁽⁵⁷⁾.
Non-competitive immunoassays are highly specific and sensitive, being able to theoretically detect just one molecule of a specified antigen. Due to the excess amount of antibody present, non-competitive immunoassays are significantly faster and less prone to influence by substances or conditions affecting the reaction equilibrium compared to competitive immunoassays ⁽⁴⁸⁾
However, non-competitive immunoassays require large amounts of purse, specific antibodies to accurately identify analytes of interest. In addition, sandwich-type immunoassays are limited to the measurement of larger analytes, as they require at least two distinct antibody binding sites ⁽⁵²⁾