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If the goal is to generate a rapid assay for an enzyme of plant source what are the typical options?
i.e. Could one do something like: Generate an antibody to the enzyme and then use it to create an ELISA? Would animal-injection be the way to generate the specific antibody needed?
If not, what is typically done in such cases. How does one go about creating an assay for a new enzyme of plant source. Are there alternative approaches that avoid the antibody creation?
I want to add something on top of Chris answer.
The production of an antibody it is usually a quite slow (and expensive) process, an alternative that worth to consider is phage display (https://en.wikipedia.org/wiki/Phage_display). Once you find the phage that effectively bind your protein of interest, you can use it instead of an antibody in what is called a Phage-ELISA assay. However, mind that the ELISA (or Phage-ELISA) will give you information about the presence and the concentration of the enzyme, it will not give you any hint about its activity.
I think the most promising routes use antibodies. You could either develop an ELISA or do western blot analysis of plant material - both need a good and specific antibody. To generate these, the protein of interest (or at least parts of it) are injected into animals (typically mice or rabbit) and then antibodies are pourified from the blood of these animals. These antibodies are polyclonal, but this approach is rather fast and can be done in a few weeks.
If you want to use a more sustainable source of antibodies, the antibody producing cells from these animals are isolated, immortalized and characterized as single clones to get monoclonal antibodies.
Since you are using an enzyme, you could also think about activity assays. So either a chromogenic or fluorogenic substrate is metabolized, or you could use coupled reactions where your enzyme uses a substrate which is refilled by another reaction - classical examples here are coupled reactions which use NADH or ATP.
It is also possible to measure the metabolic rate if the substrate or the product of your enzyme shows fluorescence. Then you can either measure the decrease of your substrate or the increase of your product.
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ELISA is an abbreviation for "enzyme-linked immunosorbent assay." In 1974, P. Perlmann and E. Engvall developed the test as a substitute for certain radioimmunoassay tests, and eventually, it replaced the western blot test for HIV confirmation. The ELISA test is versatile and medical professionals can perform it easily as compared to other more complicated tests many variations are available commercially.
What is an ELISA test?
An ELISA test uses components of the immune system (such as IgG or IgM antibodies) and chemicals for the detection of immune responses in the body (for example, to infectious microbes). The ELISA test involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules) that may form an antigen-antibody reaction to provide a positive result or, if they do not react, a negative result. Examples of the uses of an ELISA test include diagnosing infections such as HIV (human immunodeficiency virus) and some allergic diseases like food allergies and experimental investigations to identify compounds (antigens from a cell lysate in a wide array of organisms). ELISA tests are also known as an immunosorbent assay or an enzyme immunoassay when an enzyme is bound to another substance as an indicator (can cause a color change, for example).
The test is based on a microtiter plate that has a solid phase substrate (target protein, antigen) at a known concentration fixed to the plate that when exposed to an antibody that has an indicator attached (dye for color change or enzyme-labeled antibody) that can produce a color change. Depending on a standard curve for absorption of enzyme-labeled antibody versus antigen level as related to the dye color change, tests may provide semi-quotative, quantitative, and/or identification of many diverse substances. This type of test is termed a direct ELISA.
There are other types of ELISA tests. Indirect ELISA uses a secondary antibody to attach to the substrate, and the sandwich ELISA that uses the antibody as the substrate fixed to the microtiter plate. For examples and additional details, see http://ruo.mbl.co.jp/bio/e/support/method/elisa.html.
Types of ELISA Tests
Antibody testing is usually done on a blood sample, often using an enzyme-linked assay called an ELISA or EIA. In this test, a person's serum is allowed to react with virus proteins that have been produced in the laboratory. If the person has been infected with HIV, the antibodies in the serum will bind to the HIV proteins, and the extent of this binding can be measured. Negative EIA results are usually available in a day or so.
What is the use of an ELISA test?
ELISA tests primarily detect proteins (as opposed to small molecules and ions such as glucose and potassium). Medical professionals frequently use ELISA tests as blood tests to detect antigens that may be present in the blood. The substances detected by ELISA tests can include hormones, an allergen, viral antigens (dengue fever, for example), bacterial antigens (TB, for example), and antibodies that the body has made in response to infection (antibodies to hepatitis B, for example) or vaccination. They can also identify an infectious disease agent in patients.
What is an ELISA kit?
An ELISA kit is a commercially available ELISA test that usually contains pre-coated polystyrene plates, detection antibodies, and usually all of the chemicals needed to perform an ELISA test. However, people can purchase special kits with substances designated by the customer.
How does ELISA testing work?
There are variations of the ELISA test (see below), but the most utilized type consists of an antibody attached to a solid surface (polystyrene plate). This antibody has affinity for (will latch on to) the substance of interest, such as a hormone, bacteria, or another antibody. For example, human chorionic gonadotropin hormone (HCG), the commonly measured protein that indicates pregnancy, can be detected by ELISA. A mixture of purified HCG linked to an enzyme and the test sample (blood or urine) are added to the test system. If no HCG is present in the test sample, then only the linked enzyme will bind to the solid surface. The more substance of interest that is present in the test sample, the less linked enzyme will bind to the solid surface. The more of the substance of interest is present it will cause a reaction and show up on the test plate in some way, such as a color change of the solution (or like a pregnancy test "two pink lines" or a "+" mark).
Chemiluminescent ELISA substrates
We offer several chemiluminescent substrates for ELISA development with horseradish peroxidase enzyme (HRP) and alkaline phosphatase (AP):
¹ Total number of assays based on 96-well microplate. See product instructions for additional information and assay considerations that determine the number of assays.
² Detection limits and recommended antibody dilutions (based on 1 mg/mL stock) have been generalized as a means to begin optimization. Individual assays may require conditions outside the ranges suggested here.
³ The peak emission wavelength is given for reference. However, for best sensitivity, measure total light output using a luminometer.
Chemiluminescent ELISA substrates at a glance
CSPD and CDP-Star are chemiluminescent 1,2-dioxetane alkaline phosphatase substrates that emit light with a maximum light intensity at a wavelength of 475 nm. These substrates are “glow” substrates and provide a sustained maximum signal over time only after 15–60 minutes depending on temperature. They are supplied at 5 or 25 mM (respectively) in aqueous buffer. Sensitivity can be improved with addition of Sapphire-II or Emerald-II luminescence enhancers.
DynaLight Substrate with RapidGlow Enhancer is a ready-to-use chemiluminescent substrate formulation that has been optimized to achieve faster results in solution-based assays. This substrate is classified as a flash and glow substrate that provides fast and sustained maximum signal as early as 2–10 minutes. The DynaLight Substrate with RapidGlow Enhancer formulation includes 1,2-dioxetane chemiluminescent substrate and a polymeric enhancer that enables ultra-sensitive immunoassay detection by alkaline phosphatase label.
SuperSignal ELISA Pico Chemiluminescent Substrate provides excellent performance for a large range of target protein amounts and is easily optimized to detect with greater sensitivity than entry-level colorimetric substrates. Rapid signal generation with 5–30 minute signal stability depending on HRP concentration.
SuperSignal ELISA Femto Maximum Sensitivity Substrate is one of the most sensitive substrates available for ELISA applications. When properly optimized, the lower detection limit is 1 to 10 orders of magnitude lower than commonly used colorimetric substrates. However, without proper optimization, it is easy to overwhelm the system with protein and enzyme, resulting in high background and possibly negative results.
Immunoassays: Protein Arrays vs. ELISA and Westerns
Studies of biological systems have expanded beyond the ‘one gene, one protein’ paradigm to the field of proteomics, or studying large numbers of proteins that act in a concert of complex biological systems. Thus in the post-genome era, technology has been driven to deliver multiplex assays that allow us to monitor multiple proteins in parallel, resulting in more biologically relevant information. Multiplex assays are used for basic research and drug discovery to look at thousands of proteins, or a ‘proteome’ in a sample. For clinical diagnostics, monitoring only a few proteins along with controls is sufficient. In both cases, however, specificity, sensitivity, sample availability, reagent costs, and convenience of use are major criteria for selecting a protocol.
Immunoassays are still the preferred platform for most protein studies, particularly clinical diagnostics and drug development, where specificity is critical . Antibody-based assays including immune-capture in lateral flow devices, immunoblot (Western blot) and ELISA (Enzyme-Linked Immunosorbent Assay) have been around for decades, and are widely adapted into clinical diagnostics. Most often the first choice is convenience- using the technology that is at hand, and that which has already been set up in the lab or neighboring community. This article describes the relative features and benefits of antibody-based assays, comparing Western, ELISA and protein microarrays.
Western Blots are typically done to determine the presence and integrity of a specific protein. In this assay, a mixture of proteins is first separated based on molecular weight and/or charge by electrophoresis in a gel matrix, then transferred to a membrane and probed with antibody specific to the protein of interest. The relative amount of protein in different samples may be compared and approximated. Because the proteins in a mixture are first separated according to physical properties, the specificity of the Western can be very high, and any cross-reactivity of the detecting antibody with other proteins in the mixture can be distinguished by the known molecular weight of the protein of interest. Western blots are limited to detection of denatured protein because all proteins in the sample are denatured prior to the electrophoresis step. On the downside, Western blots are relatively technically complex, requiring many steps and relatively large sample volume and thus are not easily automated and are relatively time consuming and expensive.
ELISA assays are often performed in 96-well plates and are adaptable to higher throughput than Western blots, but like Western blots offer only monoplex data, or results of a single protein per assay. Unlike the Western assay, an ELISA can be used to detect native proteins, and protein interactions that require intact three-dimensional structure. ELISA assays can be highly quantitative when run with a standard curve of the known protein, and well-characterized antibodies. However, these assays are not highly specific and can give false positives due to cross-reactivity of the detecting antibody with other proteins in the sample. ELISA assays are very useful for looking at protein interactions that require native conformation, and for studying binding competition. In addition, ELISA assays are technically less difficult than Western blots, and can be adapted to higher throughput with automated plate handling and detection systems.
Protein microarrays are more similar to ELISA than Western blot because generally the proteins in a sample are not fractionated prior to the assay. In constructing microarrays, the proteins are deposited in small (100-300 um) spots on a specially coated microscope slide. The slide is often coated with a polymer like nitrocellulose, or gel that increases the binding capacity of the protein. Microarrays offer advantages of higher throughput, multiplex analysis, low reagent consumption, high sensitivity and lower sample requirement compared to either the Western or ELISA assays (see Tables I and II).
Commercially available protein arrays are now available that offer pre-printed arrays of hundreds to thousands of proteins, typically antibodies used to capture proteins much like an ELISA. The manufacture of microarrays is in fact a time-consuming and costly process that is not generally feasible for the individual researcher. More importantly, the antibody content spotted onto microarrays is the most valuable and costly component. However, the amount of antibody required for a microarray as well as the sample requirement is much less than for ELISA. The protein microarray conserves reagents as well as precious samples. Thus for high-throughput, or highly repeated assays the reduced reagent costs, time-savings and sample conservation for microarray can outweigh the relative expense of setup (i.e. printing or purchasing the arrays, see Table II). In addition, the amount of protein or antibody may be limited in some cases (for example, limited amounts of polyclonal antibody or patient sample for diagnostics).
Grace Bio-Labs has developed leading technology for sensitive and high-quality protein microarrays. Our nitrocellulose film-slides provide the highest protein binding capacity, leading to excellent sensitivity (down to XX pg/ml). We have developed a suite of reagents designed to optimize results with these arrays in terms of sensitivity (signal/noise) and reproducibility. If you are interested in developing immunoassays in a protein microarray format, we invite you to partner with us with the content of your choice.
Table I: Relative properties of Western Blot, ELISA and Microarray Assays.
Specific capture antibody is immobilized on high protein-binding plates by overnight incubation. Plates are blocked with irrelevant protein e.g. albumin.
This step is omitted when using Mabtech's pre-coated plates.
Influenza viral infection in the respiratory system—potential ways of monitoring
3.8 Enzyme linked immunosorbent assay
ELISA is the gold standard method to detect a wide range of target molecules assisted with appropriate partner molecules. ELISA is used not only for the detection but also for the basic screening of many important diseases, such as HIV, influenza, and so on. Gopinath et al. 14 have demonstrated ELISA based detection of influenza virus using anti-H3N2 antibody and discriminated against other influenza viruses. Due to its high sensitivity and selectivity, ELISA helps to identify the target molecules, even in the human crude samples (serum, urine, and saliva). Until now, antibodies have commonly been used as a probe to detect biomolecules in ELISA. In general, the binding of the target (antigen) and the probe (antibody) on the ELISA plate was detected by the enzyme (eg, horseradish peroxidase (HRP), alkaline phosphatase) conjugated with a secondary antibody and detected by chromogenic substrates. Biotin-streptavidin conjugation has also been used to improve the ELISA methods. In this case, biotinylated secondary antibody was detected by streptavidin-conjugated enzyme. Various kinds of pattern are used in the ELISA method to improve the detection methods, such as direct, indirect, sandwich, and competitive ELISA. In most of the cases, antibody was used as the probe due its strong binding, stability, and selectivity. The sandwich ELISA is usually conducted by the monoclonal or polyclonal antibody of the specific target ( Fig. 2.7 ). In some cases, only the Fc region of the antibody has been used as the capture molecule on the ELISA plate. After aptamer generation, some researchers have used aptamer as the probe instead of antibody. This method is called aptamer linked immunosorbent assay (ALISA). 46 Since aptamer has a higher sensitivity than the antibody, it is possible to increase the limit of detection when aptamer is used as the probe. In addition, there is a possibility to do a sandwich assay with two different aptamers for the same target. Since both aptamer and antibody are suitable as detection molecules, there is a higher possibility of increasing the limit of detection with the sandwich patterns using aptamer and antibody.
Figure 2.7 . Enzyme Linked Immunosorbent Assay (ELISA)
There are basically three types of ELISAs (direct, indirect, and sandwich). Here a sandwich ELISA is shown as an example.
Veratrum californicum was responsible for large losses of sheep grazing high mountain ranges in central Idaho in the 1950s. Veratrum induces various birth defects including the cyclopic-type craniofacial defect (monkey-faced lambs) that is specifically induced in lambs after pregnant ewes grazed the plant on the 14th day of gestation. The steroidal alkaloids cyclopamine (1) and jervine (2) were isolated from Veratrum and shown to be primarily responsible for the malformations. Cyclopamine (1) and jervine (2) are potent teratogens that inhibit Sonic hedgehog (Shh) signaling during gastrulation-stage embryonic development, producing cyclopia and holoprosencephaly. Although losses to the sheep industry from Veratrum are now relatively infrequent, occasional incidents of toxicoses and craniofacial malformations are still reported in sheep and other species. However, the benefits to biomedical research using cyclopamine (1) as a tool to study human diseases have greatly expanded. A competitive inhibition enzyme-linked immunosorbent assay (ELISA) to detect and measure cyclopamine (1) and jervine (2) was developed using polyclonal antibodies produced in ewes. The limits of detection of the assay were 90.0 and 22.7 pg for cyclopamine (1) and jervine (2), respectively. This assay was used for the detection and measurement of cyclopamine (1) spiked into sheep blood. The simple extraction−ELISA methods developed in this study demonstrate the potential of using these techniques for the rapid screening of biological samples to detect the presence and concentration of cyclopamine (1) and jervine (2) and will be beneficial to pharmacological studies and livestock diagnostics.
Keywords: ELISA enzyme-linked immunoassay cyclopamine jervine Veratrum californicum alkaloids
Author to whom correspondence should be addressed [telephone (435) 752-2941 fax (435) 753-5681 e-mail [email protected]].
Over a half of all known proteins are glycosylated, but we have only recently started to understand the importance of complex oligosaccharides (glycans) attached to protein backbones . This is perhaps not surprising, since the branched structures of sugars make analysis of glycoconjugates significantly more challenging than the analysis of linear DNA and protein sequences. However, significant part of more than a half of all proteins, and of nearly all membrane and extracellular proteins are glycans . To be able to understand the function of glycoproteins, we also have to understand their glycan moieties. This is particularly important in the field of molecular diagnostics, where glycans are proving to be more and more important .
Glycan structures are exceedingly complex and their analysis requires complex time-consuming methods like HPAEC, normal-phase HLPC or mass spectrometry , , . These methods are not convenient in routine clinical laboratory, and simpler methods are needed to enable glycosylation analysis for clinical applications. Since its introduction in the early seventies , enzyme-linked immunosorbent assay (ELISA) has been used for various applications, including the analysis of glycosylation. In addition to methods that use specific antibodies for the analysis of glycosylation, a modified method that uses lectins instead of antibodies (ELLA—enzyme-linked lectin assay) has also been developed . Contrary to the various ELISA methods that are now routinely in clinical use, ELLA methods are being used by a small number of research laboratories (66244 articles with ELISA vs. 86 articles with ELLA in Title or Abstract fields of PubMed between 1971 when ELISA was first used and 2006). Reasons for this situation are very complex, and include both the nature of glycosylation, and the way lectins interact with glycoproteins. Lectins recognize specific carbohydrate structures and (at least in the case of plant lectins that are being used as a tool to analyze glycosylation) generally do not interact with protein backbones to which these structures are attached. Thus, when lectins are being used to study glycosylation, they measure the presence of a corresponding carbohydrate structure on all proteins in the analyzed sample and not only on an individual protein of interest. To be able to analyze glycosylation of a specific protein, this protein has to be either purified or captured with specific antibodies adsorbed to a microtiter plate. Pre-purification of a protein is not practical for diagnostic purposes, thus capture-ELLA would be a method of choice. However, antibodies are also glycosylated which significantly hampers their application for this purpose lectins bind not only to glycans on captured protein, but also to glycans on antibodies used to capture the protein from the sample.
In this study, we have selected transferrin as a model protein for the development of a simple and rapid assay for protein glycosylation. Serum transferrin is particularly an interesting protein for the analysis of sialylation. The majority of transferrin molecules in the circulation are glycosylated by attachment of only two simple biantenary glycans (Fig. 1)  which simplifies the interpretation of experimental results. Changes in the glycosylation of transferrin have been reported in many diseases , , , and are also being used to identify congenital disorders of glycosylation and liver diseases . However, all previously applied methods are time consuming and not applicable in the routine clinical laboratory. Aiming to enable routine analysis of serum transferrin sialylation, we have developed a simple and reliable ELLA method that captures transferrin using periodate-oxidized antibodies and compare its sialylation to standard samples.
ELISA Related Services
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