Oct. 28, 2024
Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis. Since the principle of immunoassays is based on specific antigen&#;antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic studies by drug monitoring, and the quality control of commercially available products. Berson and Yalow were the first to develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin [ 1 ], a development for which Yalow was awarded the Nobel Prize in Physiology or Medicine in . Even today, after half a century, immunoassays are widely utilized with some modifications from the originally proposed system, e.g., radioisotopes have been replaced with enzymes because of safety concerns regarding the use of radioactivity, which is referred to as enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA). In addition, progress has been made in ELISA with the recent advances in recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. This review article describes ELISA and its applications for the detection of plant secondary metabolites.
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Plant secondary metabolites are plant-produced organic compounds that play an important role in the defense of plants against herbivores, pests, and pathogens, as well as in their adaptation to the environment, although they are not directly involved in the growth and development of organisms [ 3 , 4 ]. Because of their diverse functions, there has been a dramatic increase in their demand in pharmaceuticals, cosmetics, and pesticides, as well as in food additives [ 5 ]. Quality control of these commercial products containing secondary metabolites is crucial as the quality directly affects their potential activity. In addition, Cragg and Newman recently reported that 34% of the currently used drugs originate from natural products [ 6 ]. Meanwhile, simple, selective, and sensitive analytical techniques are also required in pharmacodynamic studies for monitoring effective concentration, side effects, and metabolism, leading to a better quality of life for patients. Thus far, various analytical methods have been developed for such purposes, mainly based on high-performance liquid chromatography (HPLC). However, ELISA exhibits several advantages over such techniques because of its simplicity, selectivity, and sensitivity.
Since the development of radioimmunoassay (RIA) in , there has been a rapid increase in immunoassay techniques using radioactive labels [ 1 ]. However, radioactive labels have been gradually replaced with enzyme labels because of safety concerns associated with radioactivity since the study by Avrameas in , who coupled antigens or antibodies and enzymes using glutaraldehyde [ 2 ]. Currently, ELISA has a higher number of immunoassays compared to RIA.
ELISA is based on the concept of antigen&#;antibody reactions, representing the chemical interaction between antibodies produced by the B cells of leukocytes and antigens. This specific immune response plays an important role in protecting the body from invaders such as pathogens and toxins. Hence, by exploiting this reaction, ELISA permits the highly sensitive and selective quantitative/qualitative analysis of antigens, including proteins, peptides, nucleic acids, hormones, herbicides, and plant secondary metabolites. To detect these molecules, an antigen or antibody is labeled using enzymes, the so-called enzyme immunoassay, in which alkaline phosphatase (ALP) [ 7 ], horseradish peroxidase (HRP) [ 8 ], and β-galactosidase [ 9 &#; 11 ] are commonly used. The antigen in the fluid phase is immobilized on a solid phase, such as a microtiter plate constituting rigid polystyrene, polyvinyl chloride, and polypropylene. Subsequently, the antigen is allowed to react with a specific antibody, which is detected by an enzyme-labeled secondary antibody. The development of color using a chromogenic substrate corresponds to the presence of the antigen. For instance, ALP hydrolyzes p-nitrophenyl phosphate to produce p-nitrophenol, which can be detected at 405 nm (yellow color), and HRP catalyzes the conversion of chromogenic substrates, e.g., 2,2&#;-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 3,3&#;,5,5&#;-tetramethylbenzidine, and o-phenylenediamine into colored products. By using chemiluminescent substrates such as chloro-5-substituted adamantyl-1,2-dioxetane phosphate and luminol for ALP and HRP, respectively, and fluorogenic substrates such as 4-methylumbelliferyl galactoside and nitrophenyl galactoside for β-galactosidase, even more sensitive detection can be achieved. These enzyme&#;substrate reactions are typically completed within 30&#;60 min, and the reaction stops with the addition of an appropriate solution, e.g., sodium hydroxide, hydrochloric acid, sulfuric acid, sodium carbonate, and sodium azide, for individual reactions [ 12 , 13 ]. Finally, colored or fluorescent products are detected using a microtiter plate reader.
Advantages and disadvantages of ELISA are summarized in Table 1 . ELISA exhibits the following advantages: (i) Simple procedure. (ii) High specificity and sensitivity, because of an antigen&#;antibody reaction. (iii) High efficiency, as simultaneous analyses can be performed without complicated sample pre-treatment. (iv) Generally safe and eco-friendly, because radioactive substances and large amounts of organic solvents are not required. (v) Cost-effective assay, as low-cost reagents are used. However, ELISA exhibits the following disadvantages: (i) Labor-intensive and expensive to prepare antibody because it is a sophisticated technique, and expensive culture cell media are required to obtain a specific antibody. (ii) High possibility of false positive or negative results because of insufficient blocking of the surface of microtiter plate immobilized with antigen. (iii) Antibody instability because an antibody is a protein that requires refrigerated transport and storage.
In , Engvall and Perlmann [14] and Van Weemen and Schuurs [15] were the first to develop direct ELISA (Fig. 1), which was the base style for other types of ELISA. Primarily, an antigen or an antibody is immobilized on the surface of microtiter plate. After the surface is blocked with other proteins (e.g., albumin, gelatin, casein, and skimmed-milk [13]) to avoid the non-specific adsorption of other proteins, the corresponding enzyme-labeled antibody or antigen is allowed to react with the immobilized targets, followed by color development with appropriate substrates. With an increasing amount of targets, the signal increases. Direct ELISA is suitable for the qualitative analysis of macromolecules.
Direct ELISA to detect antigen (a) and antibody (b). (i) Attach antigen/antibody to solid phase. (ii) Incubate with enzyme-labeled antibody/antigen. (iii) Wash unbound enzyme-labeled antibody/antigen out. (iv) Develop color with substrate
In , Belanger developed competitive ELISA (Fig. 2) to detect rat α-fetoprotein, which involved the development of indirect ELISA and sandwich ELISA [16]. The key event of competitive ELISA is the competitive reaction between targets (antigen or antibody) in the sample and enzyme-labeled targets (antigen or antibody) against corresponding immobilized antibody or antigen. To detect the antigen in competitive ELISA, an enzyme-labeled antigen is used to compete with the target antigens against the immobilized antibody (Fig. 2b). Hence, the higher the amount of antigen in the sample, the lower the amount of enzyme-labeled antigen that binds to the antibody. That is, with an increasing amount of target antigen, the signal decreases. In this case, competitive ELISA is suitable for measuring macromolecules only because a labeling enzyme is required to measure the antigen. If the antigen is a low molecular weight compound (e.g., hapten), resultant hapten&#;enzyme conjugates are not recognized by the immobilized antibody, leading to failure of the analysis. To detect the antibody, the antigen is immobilized, and the competition between the antibody in the sample and enzyme-labeled antibody is observed (Fig. 2a). In this case, both macromolecules and hapten can be detected when hapten is exposed on the surface of the microtiter plate.
Competitive ELISA to detect antigen (a) and antibody (b). (i) Attach antigen/antibody to solid phase. (ii) Incubate antibody/antigen with enzyme-labeled antibody/antigen. (iii) Wash unbound enzyme-labeled antibody/antigen out. (iv) Develop color with substrate
Furthermore, detectable targets (antigen or antibody) can be changed depending on the competitors. When free antigen is used as competitor instead of unlabeled antibody in Fig. 2a, competitive reaction between free antigen and immobilized antigen against enzyme-labeled antibody can be observed, enabling the detection of free antigen (macromolecules and hapten) in the sample in this competitive system, and vice versa when free antibody is used instead of unlabeled antigen in Fig. 2b.
Direct and competitive ELISA methods are simple because only one antibody is required. However, the labeling step is required for each of the ELISA methods, possibly leading to inactivation of the antibody (Table 2).
Characteristics of various types of ELISA
Direct ELISA Competitive ELISA Indirect ELISA Indirect competitive ELISA Sandwich ELISA Advantage Simple because only one antibody is used Higher sensitivity and versatility than direct methods owing to usage of PAb that recognizes different epitopes of primary antibody High specificity as two antibodies possessing different epitopes are used Disadvantage Labeling antibody is necessary for each ELISA, which may result in inactivation of antibody Nonspecific signal is induced through cross-reactivity of secondary antibody To prepare two different antibodies is labor-intensive and expensive Target Macromolecules Macromolecules (Hapten) Macromolecules Macromolecules (Hapten) Generally macromolecules Signal (as target antigen increase) Increase Decrease Increase Decrease Increase Open in a new tabIndirect ELISA systems have been developed on the basis of direct ELISA to evaluate the presence of antibody in antisera (Fig. 3) [17, 18]. The key step of this system is the two-binding process of the primary antibody and enzyme-labeled secondary antibody. That is, the target antigen is indirectly detected by the secondary antibody, which is labeled with the enzyme, or the so-called indirect ELISA. The antigen is primarily immobilized on the surface of the microtiter plate, which blocks the surface with blocking proteins as mentioned above. The primary antibody (in antisera) binding to the immobilized antigen is then allowed to react with the enzyme-labeled secondary antibody, followed by the development of color. The signal increases with an increasing amount of the immobilized target antigen. Indirect ELISA is suitable for measuring macromolecules. With the use of antisera as the primary antibody, the presence of a disease-associated antibody in the antisera can be evaluated; thus, indirect ELISA is effectively used to diagnose endocrine diseases [19, 20].
Indirect ELISA to analyze antibody. (i) Attach antigen to solid phase. (ii) Incubate with primary antibody. (iii) Wash unbound primary antibody out. (iv) Incubate with enzyme-labeled secondary antibody. (v) Develop color with substrate
Indirect competitive ELISA (icELISA) involves the combination of indirect ELISA and competitive ELISA (Fig. 4). The target antigen is immobilized on a solid phase of the microtiter plate and is blocked. Subsequently, free target antigen and antibody are allowed to incubate and there is a competition between the immobilized antigen and free antigen against antibodies. The primary antibody that binds to the immobilized antigen is detected by the enzyme-labeled secondary antibody. Similar to the case in competitive ELISA, in icELISA, the signal decreases with increasing amount of the free antigen. icELISA can be applied for measuring both the macromolecules and hapten when hapten is exposed on the surface of the microtiter plate.
Indirect competitive ELISA to detect antigen. (i) Attach antigen to solid phase. (ii) Incubate free target antigen with primary antibody. (iii) Wash unbound free target antigen and primary antibody out. (iv) Incubate with enzyme-labeled secondary antibody. (v) Develop color with substrate
The use of enzyme-labeled secondary antibodies in indirect methods (e.g., indirect ELISA and icELISA) exhibit advantages over direct methods (direct and competitive ELISA) with respect to sensitivity and versatility [16]. Polyclonal antibody is a type of enzyme-labeled secondary antibody that recognizes different epitopes of the primary antibody, leading to increased sensitivity as compared to direct methods. In addition, a universal secondary antibody can be used if the original animal species of the primary antibody are unified. Thus, the secondary antibody is commercially available, leading to high versatility. Indirect ELISA clearly exhibits disadvantages with respect to the secondary antibody, i.e., the cross-reaction of the secondary antibody should be considered (Table 2).
In this system, the target antigen is detected via anchoring between two antibodies, which recognize different epitopes, or the so-called sandwich system (Fig. 5) [16]. Sandwich ELISA starts from the immobilization of an antibody, called a capture antibody, on the microtiter plate. After blocking the plate surface to avoid non-specific adsorption of other proteins, the antigen in the sample is allowed to react with the immobilized capture antibody, and the antigen bound to the capture antibody is then sandwiched with an enzyme-labeled antibody for color development. This direct system can be modified to the indirect system by using primary and enzyme-labeled secondary antibodies. The signal increases with increasing amount of antigen. As two antibodies containing different epitopes are required against the target antigen, sandwich ELISA is generally suitable for measuring macromolecules with some exceptions. Ciguatoxins, which are produced in the marine dinoflagellate Gambierdiscus toxicus, are the major causative toxins of ciguatera seafood poisoning. Ciguatoxins are structurally classified as ladder-like polyethers with a molecular weight of Da. Oguri et al. divided these polyethers into two parts and prepared different monoclonal antibodies (MAbs) to individually recognize each part for constructing a sandwich ELISA system to measure ciguatoxins [21]. More recently, Boscolo et al. reported a sandwich ELISA method for marine biotoxins, e.g., palytoxins with a molecular weight of Da [22]. The sandwich was formed by using two antibodies obtained from the same antigen with different antibodies, i.e., MAb and PAb, which were used as the capture and primary antibodies, respectively.
Sandwich ELISA for specific detection of antigen. (i) Attach capture antibody to solid phase. (ii) Incubate with target antigen. (iii) Wash unbound target out. (iv) Incubate with enzyme-labeled antibody. (v) Develop color with substrate
A highly specific assay can be obtained via a sandwich system because of the use of two antibodies. However, it is an expensive and labor-intensive process to prepare two antibodies. In addition, one more step is required in the sandwich system because immobilization is necessary for capture antibody, which increases the assay time (Table 2).
Advances in DNA technology have enabled the development of unique and interesting immunoassays based on the interaction of variable regions of heavy (VH) and light (VL) chains, which are binding regions for antigens [23]. In the presence of an antigen, the interaction between VH and VL regions is enhanced to form a ternary complex. In the aforementioned report, OS-ELISA started from coating of a solid-phase microtiter plate with streptavidin. After blocking, the VL region conjugated with biotin was allowed to react with streptavidin to immobilize the VL region. In the next process, the phage-displayed VH region was incubated with hen egg lysozyme, which was used as an antigen. Finally, the phage-displayed VH regions forming a ternary complex were detected by the HRP-labeled antibody to develop color. The obtained signal increases with increasing amount of antigen. Currently, this OS-ELISA has been modified to be more easy and effective, and several studies on OS-ELISA for measuring both the macromolecule and hapten have been reported [24&#;27].
In , researchers published details on a new antibody-based test to revolutionize laboratory testing across healthcare, research, and scientific fields. This technique, the enzyme-linked immunosorbent assay (ELISA), provided scientists with an adaptable, sensitive, and reliable testing method that accelerated the pace of discovery and diagnosis.
For over forty years, ELISA kit has been part of modern labs, giving fast answers when testing was long-term or unavailable. ELISA kits offer multiple benefits, including but not limited to pathogen detection analysis of biomarker substances used in characterizing diseases.
This article will highlight 7 key benefits that ELISA kits contribute to contemporary laboratory practice, underscoring their utility in everything from medical labs to academic research groups. From speed to sensitivity to flexibility, we'll see why ELISA remains the go-to testing platform across a vast range of critical analytical applications. Let's explore what makes ELISA such an indispensable technology!
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Elisa kits are top tools in lab tests, famous for their outstanding sensitivity and specificity, which unlock the ability to accurately find and measure substances in biological samples. Their unmatched sensitivity finds tiny substances, making them important in many uses, especially in Elisa Kit's benefits.
These kits are vital in detecting diseases early and carefully watching biomarkers. Their sensitivity finds even tiny bits of specific substances, helping start treatment early. In Elisa assay advantages, this part is crucial in finding biomarkers that show diseases early, assisting doctors to act fast for better patient results.
Also, the strong specificity of Elisa kits is a defining quality, ensuring there's little mixing with other molecules. This precision improves the correctness and trustworthiness of test outcomes in Elisa assay benefits, lessening the possibility of wrong positive or negative results. This correctness is critical in medical places, where exact diagnosis helps treatments, improving patient care.
Do the benefits of Elisa kits go beyond medical use? Yes, this is because of its reach in research and making new things. Their sensitivity and precision help push science forward, making it easier to measure things like biomarkers, proteins, or other items of interest. This exactness helps researchers and scientists understand complicated biological processes and create new treatments.
Also, the flexibility of Elisa kits increases their benefits even more. They can be changed and improved for various things and sample kinds, meeting different needs in Elisa kit benefits and Elisa test benefits in many industries and scientific areas. This changeability makes them essential tools in drugs, biotech, environmental testing, food safety, and more.
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The unmatched flexibility of Elisa kits is a foundation in lab testing, providing a wide range of uses in many scientific areas. For Elisa Kits benefits, their ability goes beyond limits, critical in areas like studying the immune system, cancer, diseases caused by infections, and finding new drugs.
In peptide synthesis, Elisa kits are vital to accurately finding and measuring peptides or genetic signs. Their skill to find antibodies, antigens, cytokines, and hormones gives scientists in these fields a complete set of tools to study how molecules work together, helping improve how we understand diseases and treatments.
Also, in Antibody Sequencing, Elisa kits are important in understanding antibodies and checking their specificity. These kits have many uses that match the different needs of researchers and scientists working on finding and understanding antibodies.
Elisa kits greatly help measure and confirm made proteins in Protein Expression Service. Their flexibility lets researchers accurately measure protein amounts, helping improve how things are made and ensuring the proteins are good quality and work well.
Just as important, the flexibility of Elisa kits goes beyond simple finding tests. Their flexibility allows them to be changed and improved for certain things, sample kinds, or test situations, fitting the unique needs of various scientific studies. This adaptability makes them vital instruments in diverse exploration and creating original items.
Further, the simplicity and expandability of Elisa kits increase their benefits in labs. They offer a consistent and repeatable setup, making quick and correct measurements and speeding up scientific progress in different areas, like making drugs, diagnosing diseases, and basic research.
The affordability and scalability of ELISA confer remarkable value across diverse laboratory contexts. ELISA kits provide out-of-the-box functionality, with pre-measured reagents and straightforward protocols minimizing the need for complex instrumentation. This simplicity translates into reduced costs for equipment, training, and troubleshooting.
Moreover, the flexibility of ELISA enables efficient scaling for anything from high-throughput clinical screening to early-phase research experiments. Testing volumes can be dialed up or down using different sample sizes and plate formats while preserving accuracy and reliability. This modular flexibility makes ELISA ideal for settings like core antibody production facilities where batch sizes fluctuate. Researchers can run small test batches before investing in large-scale amplification.
By eliminating the overhead of specialized tools and complex assays, ELISA kits provide an accessible on-ramp even for labs on tight budgets. The ability to get reliable answers without expensive gear or numerous protocol steps makes ELISA testing cost-efficient and within reach for most scientists.
Financial savings were not the only benefit gained by ELSIA's speed and accuracy; there were also considerable post-procedural benefits in medical diagnostics. ELISA's test result for detection allows for early diagnosis of diseases through accurate identification decisions on treatment with the best patient welfare. It will enable cost-effectiveness in delivering cheap and efficient quality healthcare instead of expensive, unnecessary costs arising from misdiagnosis or diagnosis delay.
Thus, from budgeting to scalability, and even in terms of cost avoidance from incorrect outcomes, ELISA demonstrates excellent value for money. The flexibility to balance accuracy, speed, and affordability epitomizes this crucial laboratory method's remarkable cost-effectiveness advantage.
The affordability of Elisa kits goes further than just thinking about finances. Their effectiveness in giving correct results quickly speeds up choices in medical places, helping treatments happen quickly and making patients better.
ELISA tests can provide results in as little as 30-90 minutes. This makes Elisa's kits stand out. A trait that sets them apart in lab tests and the various benefits of Elisa kits. Providing results relatively quickly compared to other lab methods, these kits streamline processes, ensuring fast tests while maintaining accuracy and exactness. This rapidity is vital, notably in critical settings such as medical examinations, where prompt decisions regarding diagnosis and treatment are critical for individuals.
In Elisa examination benefits, the more straightforward procedures and enhanced steps of Elisa examinations aid in obtaining outcomes more swiftly. The straightforward manner in which these examinations operate speeds up processes and reduces the probability of errors, establishing them as a preferred option in locations requiring both rapidity and accuracy.
In Antibody Production, the speed of Elisa tests is very important in understanding and confirming antibodies. Quick testing helps researchers check how specific and functional the antibodies are, speeding up the creation and confirmation of antibodies for different uses.
Also, indulging in Recombinant Antibody Production, the speed of Elisa tests helps a lot in watching and improving processes. These tests allow fast and accurate measurements, helping quickly check how much protein is made and its quality.
The importance of the Elisa test speed also goes to Protein Synthesis Companies. In these institutions, the quick testing abilities of Elisa tests speed up checking genetic material, peptides, or proteins, ensuring their quality and functionality are confirmed quickly.
Further, in Protein Expression & Production Services, the quick results of Elisa tests help understand and confirm already made proteins. This speeds up improving making processes and finding any differences quickly, making sure good proteins are made.
The speed of Elisa tests is a crucial benefit in many science and medical places. Their skill to give quick and correct outcomes in Elisa kits, Making Protein Businesses, Antibody Analysis Businesses, and similar industries helps speed up important choices, making things more efficient, faster research, and better patient care.
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Elisa kits enable accurate quantitative analysis through their ability to generate standard curves that relate signal intensity to analyte concentration. This allows for precise counting and measurement of biomolecules in samples.
The quantitative capability of Elisa kits is essential for research and industries like custom protein and antibody production. For example, the kits can measure production levels of proteins or genes to help optimize conditions and yield the correct amounts.
The standard curves produced by Elisa kits serve as quantitative guideposts, allowing accurate measurement of antibodies, antigens, hormones, and other analytes in biological samples. This supports understanding biological systems, monitoring diseases, and evaluating treatments.
In custom protein and gene synthesis, the counting ability of Elisa kits is crucial for characterizing and ensuring the quality and consistency of the biomolecules produced. The kits provide quality control by assessing whether the right amounts of proteins or genes are being made.
The exact quantification provided by Elisa kits makes them indispensable tools for many applications requiring accurate counting and measurement of biomolecules. Their quantitative analysis capabilities are essential across research and industries like custom protein, antibody, and gene production.
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The natural simplicity built into Elisa kits is essential to their many benefits, making them a top pick for experienced researchers and new people in labs exploring different.
Another vital attribute of Elisa kits is their ease of use, making testing much more manageable. Such kits are always equipped with simple instructions that one does not require special training for. New people joining peptide-making companies, or the peptide business will, therefore, find it easy since they can conduct quick tests with little assistance.
The set was included in Elisa kits and added to their ease of use. These set methods make testing more accessible, giving a similar and organized way to do things in different labs or research places. This sameness makes results the same and helps compare between labs, which is very important in Peptide Making Prices, where being the same and reliable is important.
Also, the easy-to-use quality of Elisa kits lowers the risk of mistakes or mistakes in testing. The instructions and set ways to make it less likely for people to make mistakes, ensuring even new users can get good results. This is helpful in places like peptide making businesseswhere getting exact measurements is very important for good products and happy customers.
The ability to get Elisa kits and the ease of using them isn't just for skilled researchers and lab workers. They're also for schools, small research labs, and businesses looking into Peptide Making Prices or Peptide Making Companies. They're easy to use and let more people use excellent testing methods, letting many people use these vital tools for many other science studies.
The fast-processing ability that Elisa kits have is a significant advantage among the many good things about these flexible tools. Especially in labs and businesses handling lots of samples, dealing with many pieces quickly is very important. Elisa kits are great at this, meeting the needs of Elisa kits' good things in labs and different areas because they can be used with machines and can handle many samples simultaneously.
The ability of Elisa kits to work with automatic systems increases how many samples they can process. These kits fit easily with machines, helping process lots of models quickly. This is very important in labs and areas like Making Proteins Companies or Making Special Antibodies Services, where processing lots of samples rapidly is essential for making things and researching.
Elisa kits contribute greatly to laboratory tests and provide multiple advantages, advancing science in many industries. They can sense materials, adjust, operate fast, be economical, calculate, be available, and handle large quantities of samples. They're crucial instruments for researchers, healthcare professionals, and scientists globally.
Elisa kits will get better as tech gets better, making them even more exact, quicker, and valuable in more ways. This makes sure they stay essential in lab tests.
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