Abstract
BACKGROUND:
Immunoassay methods typically involve the use of antibodies, which are either labeled with an enzyme to generate a detectable product or directly tagged with a radioactive or fluorescent substrate.
METHODS:
One approach to enhance the specificity of immuno-detection methods is by employing a combination of different antibodies, such as primary and secondary.
RESULTS:
However, relying solely on one antibody targeting another may not offer the highest level of precision for improving immunoassay specificity; A novel strategy for enhancing the specificity of immunoassay techniques involves directly targeting different epitopes of an antigen.
CONCLUSIONS:
This approach entails utilizing sequential chain reactions facilitated by distinct enzymes bound to various antibodies, each directed at specific epitopes on the antigen. Such an innovative method holds promise for advancing the specificity of immunoassay methods.
Introduction
Immunoassay methods operate based on the interaction of specific antibodies with target antigens to detect a wide range of molecules such as proteins, lipids, and nucleic acids [1] . An antibody with a detectable tag is used for the detection of a specific antigen [2, 3]. Tags typically include enzymes that produce fluorescence signals or they are directly linked to fluorescent chemicals (direct fluorescent antibody (DFA or dFA) which are readily measurable [4].
Regarding the diversity of signals, there are different immunoassay methods including Radioimmunoassay (RIA) [5], Enzyme Immunoassay (EIA) [6], Fluoroimmunoassay (FIA) [7], Chemiluminescence Immunoassay (CLIA) [8], Immunochromatographic Assay (ICA) [9], and Immunohistochemistry (IHC) [10].
Sequential enzymatic reactions used in cholesterol measuring.
In enzyme immunoassay methods, the secondary antibody labeled with an enzyme binds to the primary antibody, producing a detectable signal. The most common enzymes used are horseradish peroxidase (HRP), alkaline phosphatase (AP), and urease. Some other enzymes are also available; however, due to their limited substrate options, they are not very common. These include
The enzymatic chain reactions
The majority of available immunoassay methods use two antibodies: a primary antibody and a secondary antibody. Secondary antibodies play a pivotal role in enhancing sensitivity and amplifying the signal produced during the assay reaction [14]. Applying multiple antibodies targeting different epitopes on an antigen, each tagged with a distinct enzyme that initiates a chain reaction (Fig. 2), is a novel strategy to enhance the method’s specificity by directly binding to several epitopes on the target antigen. In conventional enzyme immunoassays, a secondary antibody is conjugated with an enzyme producing a detectable signal during assessment. The secondary antibody binds to the primary antibody. The most frequent enzymes used as a label are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Although other enzymes, such as
Strategy for enhancing the sensitivity of immunoassay methods by employing multiple antibodies. In this setting, biological samples are subjected to antibodies specific to a particular antigen. These antibodies are bound to different enzymes, and cholesterol serves as the initial substrate. The first enzyme, cholesteryl ester hydrolase (not shown), converts cholesteryl ester into cholesterol and fatty acids. Subsequently, the second enzyme, cholesterol oxidase (not shown), oxidizes cholesterol into cholest-4en-3-one and generates hydrogen peroxide (H2O
Secondary antibodies conjugated with the enzyme responsible for signal production produce a signal at the final reaction. The intensity of this signal indicates the concentration of the target antigen. The last primary antibody in the enzymatic chain reaction provides at least two sites for interaction with two secondary antibodies, leading to strong intensity production of a signal.
Commercially available cholesterol assessment rea-gents contain all the necessary enzymes and components in a single photometric reagent. Typically, these reagents use bacterial cholesteryl ester hydrolase to break down cholesteryl esters into cholesterol and fatty acids (Fig. 1, reaction 1). Subsequently, cholesterol oxidase catalyzes the oxidation of the 3-OH group of cholesterol resulting in the formation of a ketone (Fig. 1, reaction 2). Lastly, the H2O2 generated in the prior step is quantified in a peroxidase-catalyzed reaction, resulting in the production of a detectable color [15, 16] (Fig. 1, reaction 3).
Antibodies labeled with the enzymes used in cholesterol measurement can be employed to detect various epitopes of the target antigen. The first antibody, tagged with cholesteryl ester hydrolase, initiates sequential enzymatic reactions that eventually produce Quinoneimone, a detectable dye. This proposed mechanism may enhance the specificity of immunoassay methods. In these reactions, the product of the first enzyme serves as the substrate for the second enzyme, ultimately generating a detectable signal by the last enzyme (Fig. 2).
Nevertheless, it is worth noting that this method is susceptible to interference from internally produced cholesterol and other substances that can compete with the oxidation reaction. These interfering compounds may include bilirubin, ascorbic acid, and hemoglobin in biological samples [17, 18]. To reduce these potential sources of interference, it is advisable to purify the target proteins in the samples. The purification not only minimizes interference but also improves the accuracy of the method by exposing proteins located in the intercellular spaces or organelles that may remain undetectable by other immunoassay methods [19].
Regarding the reactions involved in cholesterol assessment, each of the three enzymes can theoretically be conjugated with antibodies targeting different epitopes of the target antigen. However, the formation of covalent bonds between enzymes and antigen-specific monoclonal or polyclonal antibodies involves intricate chemobiological engineering. This process must ensure that neither the antigen-binding site of the antibody nor the active site of the enzyme is functionally compromised. Generally, the conjugation process typically involves the following steps [20]:
Activation of the Enzyme: the enzyme like cholesteryl esterase must be chemically activated to introduce reactive groups that can form covalent bonds with the antibody. Common activation methods include the use of crosslinking agents like glutaraldehyde or carbodiimides [21, 22, 23]. Antibody Modification: The antibody may also need to be modified to introduce functional groups that can react with the activated enzyme. This often involves introducing amine, thiol, or carboxyl groups onto the antibody [24]. Conjugation Reaction: The activated enzyme is mixed with the modified antibody under conditions that promote the formation of covalent bonds between the two molecules. This step may require specific buffers, pH conditions, and temperatures to optimize the reaction [25, 26].
In bioassessments, fluorescence produced by chemical dyes typically generates stronger and more sensitive signals compared to enzymatic reactions such as those catalyzed by horseradish peroxidase (HRP). Fluorescent dyes generate high-intensity signals when excited by specific wavelengths, providing strong and clear outputs. This makes fluorescence highly sensitive and capable of detecting low concentrations of target molecules. Enzymatic reactions, such as those involving HRP, produce signals through chemiluminescence. While these can be enhanced with substrates and are useful for many applications, their signal intensity is generally lower and can saturate at higher concentrations, leading to semi-quantitative rather than fully quantitative results.
However, fluorescence-based assays require more sophisticated equipment like fluorometers and specialized imaging devices, which can be a drawback in resource-limited settings. Enzymatic assays are more straightforward, often requiring just a basic luminometer, making them more accessible for routine laboratory use [27, 28, 29, 30]. Despite the advantages of fluorescence-based detection, enzymatic-based assays are popular and produce acceptable sensitivity and specificity, so many bioassay procedures use enzymatic reactions to produce a detectable signal [31].
Overall, the current paper aims to introduce a new method to directly target the epitopes of target antigens and increase the technique’s sensitivity. Another method that uses more than one antibody directly against the target antigen is called “sandwich immunoassay”. In a sandwich immunoassay, the target antigen is captured between two antibodies. The first antibody, immobilized on a solid surface, binds to the antigen, while the second antibody, labeled with a detectable marker, binds to a different part of the antigen. After washing away unbound substances, the detection antibody forms a “sandwich” with the antigen. The detectable marker on the second antibody generates a signal when it binds, indicating the presence of the antigen. The signal intensity correlates with the concentration of the antigen in the sample, allowing for quantification. This method offers high specificity and sensitivity, making it widely used in various fields, including medical diagnostics and research [32]. Regarding the application of more than two antibodies, the method proposed in this paper provides a higher level of specificity.
However, it is important to note that if the secondary enzyme does not be used in the method it may lead to a reduction in signal amplification by secondary; several ssecondary antibodies can bind to a single primary antibody, leading to the attachment or production of numerous reporter molecules. This amplification increases the overall signal, improving the sensitivity of the assay [35, 36]. However, any decrease in the signal intensity can be compensated by considering a secondary antibody conjugated with the last enzyme in the chain reaction which binds to a primary antibody against the epitope of the same antigen (Fig. 3).
Additionally, this method entails high costs associated with using multiple primary antibodies, each conjugated with a specific enzyme. However, if the test’s specificity is critical, it offers a significant improvement by enhancing specificity and reducing the incidence of false positives.
Author contributions
H.A, Y.A and L.A-M contributed to the study’s conception and design. The first draft of the manuscript was written by Y.A and was revised by L.A-M and H.A. Y.A contributed to design of figures. All authors contributed to the article and approved the submitted version.
List of abbreviations
Footnotes
Acknowledgments
We would sincerely appreciate it if any company that has employed the proposed mechanism described in this article for developing new immunoassay methods would kindly consider allocating to us a modest portion of the financial gains generated by its implementation. Such support holds significant value for us and serves as a motivating factor for our continuous efforts in advancing biological methods.
Conflict of interest
None.