The square of the diameter of the zone of precipitation is directly proportional to the concentration of antigen. By measuring the zones of precipitation produced by samples of known concentration see the outer ring of samples in Figure 5 , we can prepare a standard curve for determining the concentration of an unknown solution.
The RID assay is a also useful test for determining the concentration of many serum proteins such as the C3 and C4 complement proteins, among others. Figure 5. In this radial immunodiffusion RID assay, an antiserum is mixed with the agar before it is cooled, and solutions containing antigen are added to each well in increasing concentrations wells 1—4.
An antigen solution of an unknown concentration is added to well 5. The zones of precipitation are measured and plotted against a standard curve to determine the antigen concentration of the unknown sample. A flocculation assay is similar to a precipitin reaction except that it involves insoluble antigens such as lipids.
A flocculant is similar to a precipitin in that there is a visible lattice of antigen and antibody, but because lipids are insoluble in aqueous solution, they cannot precipitate. Instead of precipitation, flocculation foaming is observed in the test tube fluid.
Syphilis is a sexually transmitted infection that can cause severe, chronic disease in adults. In addition, it is readily passed from infected mothers to their newborns during pregnancy and childbirth, often resulting in stillbirth or serious long-term health problems for the infant.
Unfortunately, syphilis can also be difficult to diagnose in expectant mothers, because it is often asymptomatic, especially in women. In addition, the causative agent, the bacterium Treponema pallidum , is both difficult to grow on conventional lab media and too small to see using routine microcopy. For these reasons, presumptive diagnoses of syphilis are generally confirmed indirectly in the laboratory using tests that detect antibodies to treponemal antigens.
The antibodies detected in the Wassermann test were antiphospholipid antibodies that are nonspecific to T. Their presence can assist in the diagnosis of syphilis, but because they are nonspecific, they can also lead to false-positive results in patients with other diseases and autoimmune conditions.
The original Wasserman test has been modified over the years to minimize false-positives and is now known as the Venereal Disease Research Lab test, better known by its acronym, the VDRL test. To perform the VDRL test, patient serum or cerebral spinal fluid is placed on a slide with a mixture of cardiolipin an antigenic phospholipid found in the mitochondrial membrane of various pathogens , lecithin, and cholesterol.
The lecithin and cholesterol stabilize the reaction and diminish false positives. Although the VDRL test is more specific than the original Wassermann assay, false positives may still occur in patients with autoimmune diseases that cause extensive cell damage e.
To cause infection, viruses must bind to receptors on host cells. Antiviral antibodies can neutralize viral infections by coating the virions, blocking the binding Figure 6 in Overview of Specific Adaptive Immunity. This activity neutralizes virions and can result in the formation of large antibody-virus complexes which are readily removed by phagocytosis or by antibody binding to the virus and blocking its binding to host cell receptors. This neutralization activity is the basis of neutralization assays , sensitive assays used for diagnoses of viral infections.
Figure 6. In a neutralization assay, antibodies in patient serum neutralize viruses added to the wells, preventing the formation of plaques.
In the assay pictured, the wells with numerous plaques white patches contain a low concentration of antibodies. The wells with relatively few plaques have a high concentration of antibodies. When viruses infect cells, they often cause damage cytopathic effects that may include lysis of the host cells.
Cytopathic effects can be visualized by growing host cells in a petri dish, covering the cells with a thin layer of agar, and then adding virus see Isolation, Culture, and Identification of Viruses. The virus will diffuse very slowly through the agar.
A virus will enter a host cell, proliferate causing cell damage , be released from the dead host cell, and then move to neighboring cells. As more and more cells die, plaques of dead cells will form Figure 6. During the course of a viral infection, the patient will mount an antibody response to the virus, and we can quantify those antibodies using a plaque reduction assay.
To perform the assay, a serial dilution is carried out on a serum sample. Each dilution is then mixed with a standardized amount of the suspect virus. Any virus-specific antibodies in the serum will neutralize some of the virus. The suspensions are then added to host cells in culture to allow any nonneutralized virus to infect the cells and form plaques after several days. Titer is always expressed as a whole number.
Current infections can be identified by waiting two weeks and testing another serum sample. A four-fold increase in neutralizing titer in this second sample indicates a new infection. This assay compares the relative abundance of the various types of serum proteins.
Abnormal protein electrophoresis patterns can be further studied using immunoelectrophoresis IEP. Antisera against selected serum proteins are added to troughs running parallel to the electrophoresis track, forming precipitin arcs similar to those seen in an Ouchterlony assay Figure 7. This allows the identification of abnormal immunoglobulin proteins in the sample. IEP is particularly useful in the diagnosis of multiple myeloma , a cancer of antibody-secreting cells.
Patients with multiple myeloma cannot produce healthy antibodies; instead they produce abnormal antibodies that are monoclonal proteins M proteins. Thus, patients with multiple myeloma will present with elevated serum protein levels that show a distinct band in the gamma globulin region of a protein electrophoresis gel and a sharp spike in M protein on the densitometer scan rather than the normal broad smear Figure 8.
When antibodies against the various types of antibody heavy and light chains are used to form precipitin arcs, the M protein will cause distinctly skewed arcs against one class of heavy chain and one class of light chain as seen in Figure 7.
Figure 7. After electrophoresis, antisera were added to the troughs and the precipitin arcs formed, illustrating the distribution of specific proteins. The skewed arcs arrows help to diagnose multiple myeloma. The advent of electrophoresis ultimately led to researching and understanding the structure of antibodies.
When Swedish biochemist Arne Tiselius — published the first protein electrophoresis results in , [2] he could identify the protein albumin the smallest and most abundant serum protein by the sharp band it produced in the gel. The other serum proteins could not be resolved in a simple protein electrophoresis, so he named the three broad bands, with many proteins in each band, alpha, beta, and gamma globulins.
Two years later, American immunologist Elvin Kabat — traveled to Sweden to work with Tiselius using this new technique and showed that antibodies migrated as gamma globulins. Prior to this discovery, studies on immunoglobulin structure had been minimal, because of the difficulty of obtaining pure samples to study. Sera from multiple myeloma patients proved to be an excellent source of highly enriched monoclonal immunoglobulin, providing the raw material for studies over the next plus years that resulted in the elucidation of the structure of immunoglobulin.
Figure 8. Electrophoresis patterns of myeloma right and normal sera left. The proteins have been stained; when the density of each band is quantified by densitometry, the data produce the bar graph on the right. Both gels show the expected dense band of albumin at the bottom and an abnormal spike in the gamma-globulin region. After performing protein gel electrophoresis, specific proteins can be identified in the gel using antibodies.
This technique is known as the western blot. Following separation of proteins by PAGE, the protein antigens in the gel are transferred to and immobilized on a nitrocellulose membrane. This membrane can then be exposed to a primary antibody produced to specifically bind to the protein of interest. A second antibody equipped with a molecular beacon will then bind to the first. These secondary antibodies are coupled to another molecule such as an enzyme or a fluorophore a molecule that fluoresces when excited by light.
When using antibodies coupled to enzymes, a chromogenic substrate for the enzyme is added. This substrate is usually colorless but will develop color in the presence of the antibody. The fluorescence or substrate coloring identifies the location of the specific protein in the membrane to which the antibodies are bound Figure 9. Typically, polyclonal antibodies are used for western blot assays. They are more sensitive than mAbs because of their ability to bind to various epitopes of the primary antigen, and the signal from polyclonal antibodies is typically stronger than that from mAbs.
Monoclonal antibodies can also be used; however, they are much more expensive to produce and are less sensitive, since they are only able to recognize one specific epitope.
Several variations of the western blot are useful in research. In a southwestern blot , proteins are separated by SDS-PAGE, blotted onto a nitrocellulose membrane, allowed to renature, and then probed with a fluorescently or radioactively labeled DNA probe; the purpose of the southwestern is to identify specific DNA-protein interactions.
Far-western blots are carried out to determine protein-protein interactions between immobilized proteins separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and allowed to renature and non-antibody protein probes. The bound non-antibody proteins that interact with the immobilized proteins in a far-western blot may be detected by radiolabeling, fluorescence, or the use of an antibody with an enzymatic molecular beacon.
Figure 9. Click for a larger image. Antibodies are used to identify specific bands on the protein gel. The top strip is the negative control; the next strip is the positive control.
The bottom two strips are patient serum samples containing antibodies. One of the key functions of antibodies is the activation fixation of complement. When antibody binds to bacteria, for example, certain complement proteins recognize the bound antibody and activate the complement cascade. In response, other complement proteins bind to the bacteria where some serve as opsonins to increase the efficiency of phagocytosis and others create holes in gram-negative bacterial cell membranes, causing lysis.
This lytic activity can be used to detect the presence of antibodies against specific antigens in the serum. Red blood cells are good indicator cells to use when evaluating complement-mediated cytolysis. Hemolysis of red blood cells releases hemoglobin, which is a brightly colored pigment, and hemolysis of even a small number of red cells will cause the solution to become noticeably pink Figure This characteristic plays a role in the complement fixation test , which allows the detection of antibodies against specific pathogens.
The complement fixation test can be used to check for antibodies against pathogens that are difficult to culture in the lab such as fungi, viruses, or the bacteria Chlamydia. To perform the complement fixation test, antigen from a pathogen is added to patient serum.
If antibodies to the antigen are present, the antibody will bind the antigen and fix all the available complement.
When red blood cells and antibodies against red blood cells are subsequently added to the mix, there will be no complement left to lyse the red cells. Thus, if the solution remains clear, the test is positive. Figure Click to view a larger image. If it does, complement fixation will occur, and there will be no complement available to lyse the antibody-bound sheep red blood cells that are added to the solution in the next step.
If the sample does not contain antibodies to the antigen, hemolysis of the sheep blood cells will be observed. In the Ouchterlony assay, we see a sharp precipitin arc form between antigen and antiserum. The maximum amount of precipitate forms when antigen and antibody are present in similar molar amounts the equivalence zone. In rocket immunoelectrophoresis, antigen migrates in an electric field in a layer of agarose containing an appropriate antibody.
The migration of the antigen toward the anode gives rise to rocket-shaped patterns of precipitation. The area under the rocket is proportional to antigen concentration. The latex agglutination test is a laboratory method to check for certain antibodies or antigens in a variety of body fluids including saliva, urine, cerebrospinal fluid, or blood. DNA is negatively charged, therefore, when an electric current is applied to the gel, DNA will migrate towards the positively charged electrode.
The precipitin test is one of several tests that can be done to determine if you are infected with coccidioides, which causes the disease coccidioidomycosis. Antibodies are specialized proteins that defend the body against bacteria, viruses, and fungi. These and other foreign substances are called antigens. This is known as the zone of equivalence or equivalence point. When the amount of antigen in solution exceeds the amount of antibody, the amount of precipitation will decrease. The height of the rocket, and its area are directly proportional to the amount of antigen in the sample, that is, the height of the precipitin peak depends on the concentration of antigens loaded in the corresponding wells.
Rocket electrophoresis is used mainly for quantitative estimation of antigen in the serum. Rocket Immunoelectrophoresis RIEP also known as electro-immuno diffusion is a simple, quick and reproducible method for determining the concentration of antigen Ag in an unknown sample.
Various concentrations of antigen are loaded side by side in small circular wells along the edge of an agarose gel that contains the specific antibody Ab.
Hence, with increasing antigen concentration, a series of rockets of increasing heights are seen that is proportional to amount of antigen in the well.
Therefore, a direct measurement of the height of rocket will reflect upon the antigen concentration.
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