ELISA (包括western blot及其比較與應用)
ELISA
Enzyme-Linked ImmunoSorbent Assay, also called ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates enabling much higher sensitivity.
A 96-well microtiter plate being used for ELISA.
Method
The molecule is detected by antibodies that have been made against it; that is, for which it is the antigen. Monoclonal antibodies are often used. The test requires:
* the antibodies fixed to a solid surface, such as the inner surface of a test tube;
* a preparation of the same antibodies coupled to an enzyme. This is one (e.g., β-galactosidase) that produces a colored product from a colorless substrate.
Performing the Test
1. The tubes are filled with the antigen solution (e.g., urine) to be assayed. Any antigen molecules present bind to the immobilized antibody molecules.
2. The antibody-enzyme conjugate is added to the reaction mixture. The antibody part of the conjugate binds to any antigen molecules that were bound previously, creating an antibody-antigen-antibody "sandwich".
3. After washing away any unbound conjugate, the substrate solution is added.
4. After a set interval, the reaction is stopped (e.g., by adding 1 N NaOH) and the concentration of colored product formed is measured in a spectrophotometer. The intensity of color is proportional to the concentration of bound antigen.
ELISA can also be adapted to measure the concentration of antibodies. In this case,
1. The wells are coated with the appropriate antigen.
2. The solution (e.g., serum) containing antibodies is added.
3. After they have had time to bind to the immobilized antigen,
4. an enzyme-conjugated anti-immunoglobulin is added, consisting of an antibody against the antibodies being tested for. For example, if human anti-HIV antibodies are being assayed, then antibodies (raised in a goat or rabbit against human immunoglobulins) are conjugated to the enzyme.
5. After washing away unreacted reagent, the substrate is added.
6. The intensity of the color produced is proportional to the amount of enzyme-labeled antibodies bound (and thus to the concentration of the antibodies being assayed).
Types
"Indirect" ELISA
The steps of the general, "indirect," ELISA for determining serum antibody concentrations are:
1. Apply a sample of known antigen of known concentration to a surface, often the well of a microtiter plate. The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. These samples of known antigen concentrations will constitute a standard curve used to calculate antigen concentrations of unknown samples. Note that the antigen itself may be an antibody.
2. A concentrated solution of non-interacting protein, such as bovine serum albumin (BSA) or casein, is added to all plate wells. This step is known as blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.
3. The plate wells or other surface are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. Since antigen immobilization in this step is due to non-specific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards.
4. The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.
5. Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate-specific enzyme. This step may be skipped if the detection antibody is conjugated to an enzyme.
6. Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.
7. Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.
8. View/quantify the result using a spectrophotometer, spectrofluorometer, or other optical/electrochemical device.
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. A major disadvantage of the indirect ELISA is that the method of antigen immobilization is non-specific; any proteins in the sample will stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface. The sandwich ELISA provides a solution to this problem.
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.
Sandwich ELISA
A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form.
A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:
1. Prepare a surface to which a known quantity of capture antibody is bound.
2. Block any non specific binding sites on the surface.
3. Apply the antigen-containing sample to the plate.
4. Wash the plate, so that unbound antigen is removed.
5. Apply primary antibodies that bind specifically to the antigen.
6. Apply enzyme-linked secondary antibodies which are specific to the primary antibodies.
7. Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
8. Apply a chemical which is converted by the enzyme into a color or fluorescent or electrochemical signal.
9. Measure the absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.
The image to the right includes the use of a secondary antibody conjugated to an enzyme, though technically this is not necessary if the primary antibody is conjugated to an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. The major advantage of a sandwich ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized.
Competitive ELISA
A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:
1. Unlabeled antibody is incubated in the presence of its antigen.
2. These bound antibody/antigen complexes are then added to an antigen coated well.
3. The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")
4. The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.
(Note that some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with your sample antigen (unlabeled). The more antigen in the sample, the less labeled antigen is retained in the well and the weaker the signal).
ELISA Reverse method & device (ELISA-R m&d)
A newer technique uses a solid phase made up of an immunosorbent polystyrene rod with 4-12 protruding ogives. The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous) are carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents.
Advantages:
The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and different antigens for multi-target assays;
The sample volume can be increased to improve the test sensitivity in clinical (saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples;
3. One ogive is left unsensitized to measure the non-specific reactions of the sample;
4. The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required, facilitating ready-to-use lab-kits and on-site kits.
Applications
Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test or West Nile Virus) and also for detecting the presence of antigen. It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs. ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs.
The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity. In an ELISA test, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens have been attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody" — an antibody that binds to other antibodies — is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number; the most controversial aspect of this test is determining the "cut-off" point between a positive and negative result.
One method of determining a cut-off point is by comparison with a known standard. For example, if an ELISA test will be used in workplace drug screening, a cut-off concentration (e.g., 50 ng/mL of drug) will be established and a sample will be prepared that contains that concentration of analyte. Unknowns that generate a signal that is stronger than the known sample are called "positive"; those that generate weaker signal are called "negative."
Some examples:
* screening donated blood for evidence of viral contamination by
o HIV-1 and HIV-2 (presence of anti-HIV antibodies)
o hepatitis C (presence of antibodies)
o hepatitis B (testing for both antibodies and a viral antigen)
o HTLV-1 and -2 (presence of antibodies)
* measuring hormone levels
o HCG (as a test for pregnancy)
o LH (determining the time of ovulation)
o TSH, T3 and T4 (for thyroid function)
o hormones (e.g., anabolic steroids, HGH) that may have been used illicitly by athletes
* detecting infections
o sexually-transmitted agents like HIV, syphilis, and chlamydia
o hepatitis B and C
o Toxoplasma gondii
* detecting allergens in food and house dust
* measuring "rheumatoid factors" and other autoantibodies in autoimmune diseases like lupus erythematosus
* measuring toxins in contaminated food
* detecting illicit drugs, e.g.,
o cocaine
o opiates
o Δ-9-tetrahydrocannabinol, the active ingredient in marijuana
摘要 ELISA (Enzyme-Linked ImmunoSorbant Assay)
The purpose of an ELISA is to determine if a particular protein is present in a sample and if so, how much. There are two main variations on this method: you can determine how much antibody is in a sample, or you can determine how much protein is bound by an antibody. The distinction is whether you are trying to quantify an antibody or some other protein. In this example, we will use an ELISA to determine how much of a particular antibody is present in an individuals blood.
ELISAs are performed in 96-well plates which permits high throughput results. The bottom of each well is coated with a protein to which will bind the antibody you want to measure. Whole blood is allowed to clot and the cells are centrifuged out to obtain the clear serum with antibodies (called primary antibodies). The serum is incubated in a well, and each well contains a different serum (see figure below). A positive control serum and a negative control serum would be included among the 96 samples being tested.
After some time, the serum is removed and weakly adherent antibodies are washed off with a series of buffer rinses. To detect the bound antibodies, a secondary antibody is added to each well. The secondary antibody would bind to all human antibodies and is typically produced in a rodent. Attached to the secondary antibody is an enzyme such as peroxidase or alkaline phosphatase. These enzymes can metabolize colorless substrates (sometimes called chromagens) into colored products. After an incubation period, the secondary antibody solution is removed and loosely adherent ones are washed off as before. The final step is the addition the enzyme substrate and the production of colored product in wells with secondary antibodies bound.
When the enzyme reaction is complete, the entire plate is placed into a plate reader and the optical density (i.e. the amount of colored product) is determined for each well. The amount of color produced is proportional to the amount of primary antibody bound to the proteins on the bottom of the wells.
# Western blot
The western blot (alternatively, immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein. There are now many reagent companies that specialize in providing antibodies (both monoclonal and polyclonal antibodies) against many thousands of different proteins. Commercial antibodies can be expensive, although the unbound antibody can be reused between experiments. This method is used in the fields of molecular biology, biochemistry, immunogenetics and other molecular biology disciplines.
Other related techniques include using antibodies to detect proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA).
The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blotting.
Steps in a Western blot
Tissue preparation
Gel electrophoresis
Immunoblot (Western blot) analysis of proteins separated by SDS-PAGE gradientgel electrophoresis.
The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel.
By far the most common type of gel electrophoresis employs polyacrylamide gels and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS polyacrylamide gel electrophoresis) maintains polypeptides in a denatured state once they have been treated with strong reducing agents to remove secondary and tertiary structure (e.g. disulfide bonds [S-S] to sulfhydryl groups [SH and SH]) and thus allows separation of proteins by their molecular weight. Sampled proteins become covered in the negatively charged SDS and move to the positively charged electrode through the acrylamide mesh of the gel. Smaller proteins migrate faster through this mesh and the proteins are thus separated according to size (usually measured in kilo Daltons, kDa). The concentration of acrylamide determines the resolution of the gel - the greater the acrylamide concentration the better the resolution of lower molecular weight proteins. The lower the acrylamide concentration the better the resolution of higher molecular weight proteins. Proteins travel only in one dimension along the gel for most blots.
Samples are loaded into wells in the gel. One lane is usually reserved for a marker or ladder, a commercially available mixture of proteins having defined molecular weights, typically stained so as to form visible, coloured bands. When voltage is applied along the gel, proteins migrate into it at different speeds. These different rates of advancement (different electrophoretic mobilities) separate into bands within each lane.
It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.
Transfer
Blocking
Detection
During the detection process the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colourimetric reaction and produces a colour. For a variety of reasons, this traditionally takes place in a two-step process, although there are now one-step detection methods available for certain applications.
Analysis
After the unbound probes are washed away, the western blot is ready for detection of the probes that are labeled and bound to the protein of interest. In practical terms, not all westerns reveal protein only at one band in a membrane. Size approximations are taken by comparing the stained bands to that of the marker or ladder loaded during electrophoresis. The process is repeated for a structural protein, such as actin or tubulin, that should not change between samples. The amount of target protein is indexed to the structural protein to control between groups. This practice ensures correction for the amount of total protein on the membrane in case of errors or incomplete transfers.
Colorimetric detection
The colorimetric detection method depends on incubation of the western blot with a substrate that reacts with the reporter enzyme (such as peroxidase) that is bound to the secondary antibody. This converts the soluble dye into an insoluble form of a different color that precipitates next to the enzyme and thereby stains the membrane. Development of the blot is then stopped by washing away the soluble dye. Protein levels are evaluated through densitometry (how intense the stain is) or spectrophotometry.
Chemiluminescent detection
Chemiluminescent detection methods depend on incubation of the western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody. The light is then detected by photographic film, and more recently by CCD cameras which captures a digital image of the western blot. The image is analysed by densitometry, which evaluates the relative amount of protein staining and quantifies the results in terms of optical density. Newer software allows further data analysis such as molecular weight analysis if appropriate standards are used. So-called "enhanced chemiluminescent" (ECL) detection is considered to be among the most sensitive detection methods for blotting analysis.
Radioactive detection
Radioactive labels do not require enzyme substrates, but rather allow the placement of medical X-ray film directly against the western blot which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interest (see image to the right). The importance of radioactive detections methods is declining[citation needed], because it is very expensive, health and safety risks are high and ECL provides a useful alternative.
Fluorescent detection
The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as CCD camera equipped with appropriate emission filters which captures a digital image of the western blot and allows further data analysis such as molecular weight analysis and a quantitative western blot analysis. Fluorescence is considered to be among the most sensitive detection methods for blotting analysis.
Secondary probing
One major difference between nitrocellulose and PVDF membranes relates to the ability of each to support "stripping" antibodies off and reusing the membrane for subsequent antibody probes. While there are well-established protocols available for stripping nitrocellulose membranes, the sturdier PVDF allows for easier stripping, and for more reuse before background noise limits experiments. Another difference is that, unlike nitrocellulose, PVDF must be soaked in 95% ethanol, isopropanol or methanol before use. PVDF membranes also tend to be thicker and more resistant to damage during use.
2-D Gel Electrophoresis
2-dimensional SDS-PAGE uses the principles and techniques outlined above. 2-D SDS-PAGE, as the name suggests, involves the migration of polypeptides in 2 dimensions. For example, in the first dimension polypeptides are separated according to isoelectric point, while in the second dimension polypeptides are separated according to their molecular weight. The isoelectric point of a given protein is determined by the relative number of positively (e.g. lysine and arginine) and negatively (e.g. glutamate and aspartate) charged amino acids, with negatively charged amino acids contributing to a high isoelectric point and positively charged amino acids contributing to a low isoelectric point. Samples could also be separated first under nonreducing conditions using SDS-PAGE and under reducing conditions in the second dimension, which breaks apart disulfide bonds that hold subunits together. SDS-PAGE might also be coupled with urea-PAGE for a 2-dimensional gel.
In principle, this method allows for the separation of all cellular proteins on a single large gel. A major advantage of this method is that it often distinguishes between different isoforms of a particular protein - e.g. a protein that has been phosphorylated (by addition of a negatively charged group). Proteins that have been separated can be cut out of the gel and then analysed by mass spectrometry, which identifies the protein.
Medical diagnostic applications
* The confirmatory HIV test employs a Western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody.
* A Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease').
* Some forms of Lyme disease testing employ Western blotting.
Western Blot vs. ELISA
Sensitivity & Specificity Differences
Western blot is very rarely acceptable for detection of HCPs in your drug substance or drug product samples. Samples downstream in your purification process typically contain HCPs below the sensitivity of western blot. For western blot, you are limited in the amount of total protein you can load and still get good PAGE resolution. When you load final product or samples from downstream in the purification process the vast majority of protein will be the product itself. For example, the maximal load of protein for a PAGE run on a mini gel is on the order of 10 μg/lane. If HCP contamination is 100 ppm, a level typical of many final drug products, then the amount of total HCP in that 10μg of drug would be 1 ng. With the sensitivity of western blot on the order of 1 ng/band it could in theory detect HCP contamination down to 100 ppm if the 100 ppm were a single HCP and not a mixture of several different HCPs. As it turns out there are usually several HCPs that contaminate final product and for this reason western blot is almost always negative for HCP on downstream and final product samples. ELISA demonstrates less interference from drug product and shows sensitivity more than 100 fold lower than western blot. As such, ELISA will typically allow for the detection of total HCP contamination to less than 1 ppm. There are many other fundamental reasons why the sensitivity of western blot is inferior to ELISA.
For example, western blot often requires that the PAGE step be carried out under reducing conditions (DTT or BME followed by boiling) and in the presence of high concentrations of SDS detergent. These procedural components may actually denature or block some of the native HCP epitopes that would be detectable in an ELISA. Incomplete transfer of the proteins out of the PAGE and onto the membrane and adsorption on the membrane at or near antigenic sites will also limit the amount of binding seen by western blot. As you try to increase the sensitivity of western blot it is very common that the specificity of the method is also compromised. What is typically seen is that a non-immunoreactive protein present in very high concentration (e.g. your drug substance) will invariably adsorb some of the excess anti-HCP antibody nonspecifically leading to the erroneous conclusion that the anti-HCP antibody seems to "cross react" with your product. The way to confirm this non-specific binding to your product is to use a non-immune immunoglobulin of the same species and at the same concentration as the anti-HCP antibody. If the intensity of the drug substance band is the same with both the normal goat IgG and the anti-HCP antibody, you can conclude the band is non-specific. Beyond that experiment it should be understood that the specificity of the ELISA method is typically orders of magnitude better than western blot owing in large part to the fact that any protein must be bound simultaneously by both the capture antibody and the detection antibody. For this reason most artifactual product bands in the western will not yield apparent HCP activity in the ELISA method.
ELISA/Western blot tests for HIV
(Update Date: 1/22/2008 Updated by: Kenneth M. Wener, MD, Department of Infectious Diseases, Lahey Clinic, Burlington, MA. Review provided by VeriMed Healthcare Network. A.D.A.M.)
