Tut 3- ELISA Principles and Different ELISA Types and Protocols Flashcards

Question

When would you use sandwich ELISA?

Answer 1

Analysis of complex samples, since the antigen does not need to be purified prior to measurement.

Answer 2

The competition/inhibition ELISA, also known as a blocking ELISA, is perhaps the most complex of all the ELISA techniques. However, each of the above assay types can be adapted to a competitive format. The competitive/inhibition ELISA is predominantly used to measure the concentration of an antigen or antibody in a sample by detecting interference in an expected signal output. Essentially, sample antigen or antibody competes with a reference for binding to a limited amount of labeled antibody or antigen, respectively. The higher the sample antigen concentration, the weaker the output signal, indicating that the signal output inversely correlates with the amount of antigen in the sample.

Answer 3

* Main advantage - no sample processing is required and crude or impure samples can be used * More robust - less sensitive to sample dilution and sample matrix effects than the sandwich ELISA * More consistent - less variability between duplicate samples and assays * Maximum flexibility - it can be based on direct, indirect or sandwich ELISA

Answer 4

Same limitations as base ELISA - as each ELISA technique can be adapted to a competitive format

Answer 5

Commonly used when only one antibody is available for the antigen of interest. It is also suitable for detecting small antigens that cannot be bound by two different antibodies such as in the sandwich ELISA technique.

Answer 6

What do you need to detect? For example, if a large protein with multiple epitopes, such as a cytokine is being detected, then a sandwich ELISA would be most appropriate. However, if a small molecule such as a hapten is being detected then a competitive ELISA would be more appropriate in that instance. What reagents are available for your antigen or antibody of interest? If only one antibody is available for an antigen of interest then a direct or competitive ELISA can be applied. Do you want to measure an immunological response or analyte? If you need to detect or quantitate an analyte, then a sandwich or competitive ELISA can be utilized. However if you need to measure an immunological response, then a direct or indirect ELISA is most suitable for your needs.

Answer 7

The antibodies used in ELISA assays can be monoclonal, polyclonal, or a combination of both. Each antibody type offers distinct advantages in the development of ELISAs, so it is important to appreciate the differences between them and how these can be used to obtain an advantage during ELISA development.

Answer 8

Antibodies, also known as immunoglobulins, are secreted by B cells (plasma cells) to neutralize antigens such as bacteria and viruses. The classical representation of an antibody is a Y-shaped molecule composed of four polypeptides-two heavy chains and two light chains. Each tip of the "Y" contains a paratope (a structure analogous to a lock) that is specific for one particular epitope (similarly analogous to a key) on an antigen, allowing these two structures to bind together with precision. The ability of binding to an antigen has led to their ubiquitous use in a variety of life science and medical science. These antibodies can be classified into two primary types (monoclonal and polyclonal) by the means in which they are created from lymphocytes. Each of them has important role in the immune system, diagnostic exams, and treatments.

Answer 9

Monoclonal antibodies are homogeneous by definition, with specificity for a single epitope or small region of a protein. As a result, they are less likely to interact with closely-related proteins and are not generally expected to trigger non-specific signals in a given immunoassay. Monoclonal antibodies can be used for all antibody-containing steps in all types of ELISAs. They are commonly used in sets as matched pairs in sandwich ELISAs, but can be used for capture or detection, in conjunction with a polyclonal antibody to enhance signal or to provide a greater chance of capturing antigen from a complex solution.

Answer 10

Polyclonal antibodies are complex antibody pools which represent a collection of specificities to various epitopes found in a single antigen. Some epitopes predominate or there may be wide representation of the epitopes available in any given antigen. Polyclonals can vary significantly from batch-to-batch, and must be tested and validated thoroughly.

Answer 11

As a result of their heterogeneity and the wide representation of epitopes present, polyclonal antibodies can be powerful tools for the thorough detection of an antigen, often yielding higher signal levels. It is also rare that they will fail to bind due to a single blocked antibody binding site, antigen configuration change, or misfolding. However, polyclonals are also more likely to share one or more epitopes with closely-related proteins, resulting in higher non-specific signal. One solution to reduce this problem is to use affinity purified or cross-absorbed polyclonal antibodies. Sometimes the detection method for an ELISA is switched from direct to indirect detection, and thus from a monoclonal to a polyclonal, in order to increase assay sensitivity due to higher levels of polyclonal antibody binding to the target antigen. Polyclonal antibodies bring an additional aspect to ELISAs. They can be used as capture and detection antibodies. Antibodies from the same polyclonal batch can both capture the analyte and subsequently also detect it, in a biotin conjugated format.

Answer 12

Polyclonal antibodies (pAbs) are mixture of heterogeneous which are usually produced by different B cell clones in the body. They can recognize and bind to many different epitopes of a single antigen. Polyclonal antibodies are produced by injecting an immunogen into an animal. After being injected with a specific antigen to elicit a primary immune response, the animal is given a secondary even tertiary immunization to produce higher titers of antibodies against the particular antigen. After immunization, polyclonal antibodies can be obtained straight from the serum (blood which has had clotting proteins and red blood cells removed) or purified to obtain a solution which is free from other serum proteins.

Answer 13

Monoclonal antibodies (mAbs) are generated by identical B cells which are clones from a single parent cell. This means that the monoclonal antibodies have monovalent affinity and only recognize the same epitope of an antigen. Unlike polyclonal antibodies, which are produced in live animals, monoclonal antibodies are produced ex vivo using tissue-culture techniques. The process begins with an injection of the desired antigen into an animal, often a mouse, multiple times. Once the animal develops an immune response, the B-lymphocytes are isolated from the animal’s spleen and fused with a myeloma cell line, creating immortalized B cell-myeloma hybridomas. The hybridomas, which are able to grow continuously in culture while producing antibodies, are then screened for desired mAb.

Answer 14

Monoclonal antibody: To establish the highest level of specificity in an assay Polyclonal antibody: To amplify the signal via multiple binding events.

Answer 15

· Short production time and low cost. · Highly stable and tolerant of pH or buffer changes. · High affinity. Since the antibodies bind to more than one epitope, they can help amplify the signal from target protein even with low expression level. This makes these antibodies ideal for immunoprecipitation and chromatin immunoprecipitation. · Tolerant of minor changes of antigen. Polyclonal antibodies are less sensitive to antigen changes (slight denaturation, polymorphism, heterogeneity of glycosylation) than monoclonal antibodies.

Answer 16

· Prone to batch to batch variability. · Multiple epitopes make it important to check immunogen sequence for any cross-reactivity.

Answer 17

· Highly specific recognition of only one epitope of an antigen · Immortal hybridoma cell lines have the ability to produce unlimited quantities of antibodies · High consistency among experiments · Minimal background noise and cross-reactivity · Excellent for affinity purification

Answer 18

· Developing a monoclonal takes time and requires high technical skills. · They can produce large amounts of specific antibodies but may be too specific to detect in across a range of species. · Vulnerable to the change of epitope. Even a slight change in conformation may lead to dramatically reduced binding capacity.

Answer 19

It depends on the different characteristics of monoclonal and polyclonal antibodies. Polyclonal antibodies are ideal reagents in diagnostic assays and hemagglutination reactions due to their ability to recognize different epitopes of a target molecule. The best use of polyclonal antibodies is to detect unknown antigens. Polyclonal antibodies are used as a secondary antibody in immunoassays (e.g. ELISA, western blotting, microarray assays, immunohistochemistry, flow cytometry). Their role is to bind to different epitopes and amplify the signal, leading to better detection. Monoclonal antibodies, in contrast, provide an unlimited source of antibody that is homogeneous and, once characterized, predictable in its behavior. Monoclonal antibodies are often used as primary antibodies in immunoassays due to their ability of specifically binding to a single epitope of an antigen. Through the use of clinical application, some of the disadvantages of using each type of antibody has been nullified. Companies can purify polyclonal antibodies to limit the degree of cross-reactivity in their assays. The combination of monoclonal antibodies leads to the capture of multiple epitopes and expanding its’ specificity.

Answer 20

1) Antigen coating; 2) Blocking all unbound sites to prevent non-specific absorption; 3) Add analyte and incubation; 4) Add labeled antibody and incubation; 5) Add reagents for colouration/ luminescence, thus give a positive result.

Answer 21

For example, by fixing poly-protein G-expressing cells on microplates to improve the coating amount and displayed orientation of capture antibodies we developed a highly sensitive ELISA strategy. One or eight repeated fragment crystallisable (Fc) binding domains of protein G are stably expressed on the surface of BALB/c 3T3 cells which then act as highly antibody-trapping microparticles. The 8pG cell-based microplates were then applied to an anti-IFN-α sandwich ELISA and an anti-CTLA4 competitive ELISA, respectively, and dramatically enhanced their detection sensitivity.

Answer 22

The capture antibodies coated on traditional polystyrene-based microplates exhibit a disorganized orientation due to the hydrophobic interactions between the antibodies and the polystyrene surface. This random display of the capture antibodies coated on traditional polystyrene-based microplate decreases their antigen-capturing avidity, and further limits the detection sensitivity of the assays. By adding a capture coating antigen, a binding domain of a protein being used is able then act as highly antibody-trapping microparticles. This dramatically enhances detection sensitivity.

Answer 23

The capture antibodies coated on traditional polystyrene-based microplates exhibit a disorganized orientation due to the hydrophobic interactions between the antibodies and the polystyrene surface. This random display of the capture antibodies coated on traditional polystyrene-based microplate decreases their antigen-capturing avidity, and further limits the detection sensitivity of the assays. By adding a capture coating antigen, a binding domain of a protein being used is able then act as highly antibody-trapping microparticles. This dramatically enhances detection sensitivity.

Answer 24

Blocking prevents nonspecific interaction, eliminates background signal and improves the signal-to-noise ratio, without altering or obscuring the epitope for antibody binding. A blocking buffer is a solution of irrelevant protein, mixture of proteins, or other compound that passively adsorbs to all remaining binding surfaces of the plate.

Answer 25

The most common blocking agents are protein blockers and non-ionic detergents.

Answer 26

Advantages of non-ionic detergents: - Inexpensive and convenient despite higher concentration requirements - Highly stable, able to be stored as working solutions at room temperature - Increase the effectiveness of washes by encouraging the dissociation of weakly bound molecules and blocking the resulting exposed binding sites (Detergents are primarily useful as a secondary blocking agent; when included in the wash buffer, detergents can actively block sites on the plate surface that become exposed as weakly associated proteins are washed away). Disadvantages: - Ineffective as sole blocking method (detergents are temporary blockers, since they can be stripped by washing with water or aqueous buffer) - May cause the dissociation of molecules bound by noncovalent interactions - May interfere with HRP detection systems - Incompatible with lipopolysaccharides due to their ability to outcompete these molecules

Answer 27

Protein blockers are a permanent blocking solution, and plates only need to be treated once for effective blocking. Protein blockers can also be added to the diluents used in subsequent steps to further reduce background signal. The most common blocking proteins include: bovine serum albumin (BSA), nonfat dry milk, and whole normal serum.

Answer 28

Advantages: - Inexpensive - Effective at concentrations as low as 1-3% - Well documented efficacy - Compatible with protein A Disadvantages: - High lot-to-lot variability due to variable fatty acid content - May cross-react with some classes of antibodies - Less effective at blocking covalent interactions

Answer 29

Advantages: - Inexpensive - Effective at concentrations as low as 0.1-0.5% - Highly stable in dry form - More effective at blocking covalent interactions Disadvantages: - May cross-react with phospho-specific antibodies - Incompatible with alkaline phosphatase - May cause overall higher background

Answer 30

Advantages: - Effective at blocking all nonspecific interactions, including protein-protein interactions - Acts as protein stabilizer Disadvantages: - Cross-reacts with protein A and anti-IgG antibodies - Expensive - Requires up to 10% concentration

Answer 31

Various detection methods for ELISA can be used, but the most prevalent are colorimetric, fluorescent and chemiluminescent. Most commonly, horseradish peroxidase (HRP) or alkaline phosphatase (AP), is conjugated to the antibody. Then, during the detection step of the protocol, a substrate for these enzymes is added that will form either a chromogenic, fluorescent, or chemiluminescent signal. Because this is an enzymatic process, the reaction can be allowed to progress for a period of time, amplifying the signal, and then stopped. The resulting signal is measured on a plate reader.

Answer 32

Blank binding control: Blank Controls are the most common negative control type, but possibly the most inconsistently used terminology. There is some ambiguity as to what exactly is a blank control. Blank empty wells that do not contain or have not seen any liquid at any point are one common form of blank controls, but probably just as common are assay buffer blanks, i.e. wells filled only with the ELISA’s buffer. Blanks are meant to analyze the contribution of background absorbance of the plastic and/or buffer to the signal. They are often omitted in favor of wavelength correction and/or S0 or NSB controls (see below). Some form of blank or background control should be run alongside standards and samples in every assay run, regardless of the ELISA being well established. No secondary antibody control: Secondary/Detection Antibody Control are controls used mostly in direct and sandwich ELISAs. Their purpose is to test nonspecific binding of secondary or detection antibody in the absence of primary or capture antibody, respectively. While these controls can be generally omitted in routine ELISA runs, they might be able to provide valuable insight when troubleshooting sources of high background or the state of decay of assay components. Sample positive control: Positive Controls aim to test the functionality of the assay as well as its feasibility to be used with the type of sample and sample matrix. The following positive controls are commonly used in ELISA: B0 is a specific denomination of zero standard controls in competitive ELISAs. While technically the same as S0, i.e. the absence of any standard or sample analyte, it will – in contrast to S0 in sandwich ELISAs – lead to maximum color development, as the conjugated analyte does not face binding competition and maximal conjugate binding will be achieved. B0 is therefore in contrast to S0 a positive control useful to determine the maximum color development in competitive ELISAs and to control for full functionality of all assay components. It will generally result in values above the maximum detection range of an assay. B0 values can be used to reference the relative binding in a sample or standard to the maximum possible binding and even used to analyze assay readout. This ratio is commonly referred to as percentage of binding, %B, or B/B0. It is therefore recommended to include B0 controls into every assay run of competitive ELISAs, even if the assay and sample/matrix combination is well established. Sample negative control: Negative Matrix Control is a means to determine the signal originating from analyte-independent matrix effects. A sample’s matrix is the entirety of all components contained in the sample, with the exception of the analyte. Especially complex matrices, such as blood samples, can contain a wide variety of components that might interfere with an assay’s signal in one way or another. Nonspecific binding to matrix components can lead to false positive signals, as well as false-negative signals. False-positive signals can be controlled for by measuring a negative matrix control, i.e. a sample matrix that is guaranteed to not contain any analyte. However, while being a useful control, it is often very difficult to impossible to obtain sample matrices that are guaranteed to be analyte-free. Standard: Standards are not commonly conceived as a positive control, but known quantities of analyte added to assay buffer is probably the purest form of a positive control. Not only are standards a useful tool to ensure an assay’s functionality, they are an indispensable part of any fully quantitative ELISA. A series of different known concentrations of analyte, most frequently obtained by serial dilution, are used to generate the assay’s standard curve. An accurate standard curve is an essential component of each assay run to determine the quantity of analyte contained in the unknown samples. While a standard is generally supplied with the assay for commercially available assay kits, it can pose quite some challenge to source suitable analyte standards for custom-made ELISAs.

Answer 33

Weak or no signal: -Primary antibody concentration too low Solution: Increase primary antibody concentration or incubate for longer -Excessive washing Solution: Wash gently with a manual pipette Saturated signal: -Excessive substrate Solution: Decrease concentration or amount of substrate: Follow manufacturer guidelines (The substrate provided with the ELISA kit might require further dilution) -Incubation time too long Solution: Follow the manufacturer guidelines (If the problem persists, try incubating samples at 4°C overnight) High background: -Plate not washed sufficiently Solution: Be sure all wells are filled with buffer during every wash step. Be sure washing apparatus is working properly. Wash 3-5x between steps. Do not add additional washes. After final wash, blot plate forcefully on paper towel to remove residual buffer. Be sure the correct amount of Tween was added to the wash solution (0.01- 0.1% recommended). -Non-specific binding of antibody Solution: Modify blocking buffer used (e.g. substitute casein for BSA). Do not wash after blocking step; dump, blot & go directly to the next step. Low sensitivity: Insufficient target Solution: Reduce dilution factor or concentrate sample Inactive substrate Solution: Run a control to verify reporter enzyme reacts with substrate