Laboratory Investigations

Immunofluorescence

The use of immunofluorescence (IF) microscopy for the detection of antibodies in patient serum was one of the first techniques developed in immunopathology. The principle of the test is straightforward. A substrate is mounted on a glass slide and antibodies labeled with a fluorescent tag are used to identify the presence of either antigen in the substrate (direct IF) or of antibody on the substrate (indirect IF) using a fluorescence microscope ( Fig. 10.2 ). Direct IF requires fluorescent-tagged antibodies of known specificity whereas indirect IF requires fluorescent-tagged antibodies to human immunoglobulin (Ig). The substrate used in either technique can be tissue sections, cultured cells, or even microorganisms (e.g., Crithidia luciliae ). IF has several disadvantages of which the most important are that it is time consuming, labor intensive, requires skilled operators, and has a subjective component to interpretation that may cause significant variation in results. For these reasons there has been interest in developing more automated processes.

Enzyme-Linked Immunosorbent Assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) is a commonly used method for detecting the presence of antibody or antigen in a sample. Although there are multiple forms of ELISA, all rely on high specificity antibody–antigen interactions and the use of an automated reader to detect a color change in wells containing a sample and an enzyme-labeled antibody that causes the color change when mixed with an appropriate substrate ( Fig. 10.3 ). Signal strength (color intensity) is related to the amount of labeled antibody present, which in turn is proportional to the amount of antigen or antibody in the sample. The wells used in this assay are contained on a polystyrene plate in large numbers, allowing for the simultaneous testing of numerous samples.

The most basic form of this assay is the direct ELISA, in which a sample containing an antigen to be detected is coated onto the plate wells and a labeled antibody of known specificity is added ( Fig. 10.4A ). This method is useful if the presence of a particular antigen in a sample is in question.

Indirect ELISA is the most common method and involves adding sample to wells coated with a known antigen. Antibodies in the sample that bind to the antigen are detected using enzyme-labeled antihuman Ig antibodies ( Fig. 10.4B ). This method is useful if the presence of a particular antibody in the sample is in question.

A third variant is the sandwich (or “capture”) ELISA, in which the plate wells are coated with a capture antibody targeting a specified antigen. This method may be used to identify the presence of a specific antigen or antibody using direct and indirect detection methods, respectively ( Fig. 10.4C–D ). In a direct sandwich ELISA the presence of a captured sample antigen is detected by the addition of an enzyme-labeled antibody targeting an epitope on the same antigen that does not overlap with that of the capture antibody. In an indirect sandwich ELISA the captured antigen is bound by an intermediate-unlabeled antibody, the presence of which is then detected using an enzyme-labeled antihuman antibody. An advantage of this method is that it avoids the potential disruption of conformational epitopes on sample antigen that may occur in indirect ELISA.

A final variant, the inhibition ELISA, uses competition for labeled antibody between sample and well wall antigen. The resulting reduction in antibody binding to the well wall is an indirect measure of antigen in the sample ( Fig. 10.4E ).

The advantages of ELISA methodology are that it is rapid, objective, able to analyze multiple samples simultaneously, and can be fully automated. It can also provide quantitative results and is highly sensitive. Its main disadvantage is that it tends to be less specific than other assays. ^,

Radioimmunoassay

Radioimmunoassay (RIA) is a highly sensitive way to measure the concentration of antigen in a sample. In this assay a quantity of the antigen of interest is tagged with a radioactive isotope (typically of iodine-125 or iodine-131) and mixed with a known amount of its cognate antibody. Sample is then added and any antigen in the sample matching the radiolabeled antigen will compete for binding to the added antibody, effectively drawing antibody away from the labeled antigen. Bound and unbound antigen are separated and the amount of radioactivity in the unbound fraction measured. The level of radioactivity in this fraction is proportional to the amount of antigen in the sample. A variant of this, the Farr assay, is used for the detection of high-avidity anti–double-stranded DNA (anti-dsDNA) antibodies, which have a higher specificity for the diagnosis of systemic lupus erythematosus (SLE) than low avidity antibodies. With the availability of newer methods of antibody identification, RIA techniques have fallen out of favor because of the need to use and dispose of radioactive material.

Immunoblotting (Western Blotting)

In this technique, protein antigens—typically nuclear and cytoplasmic extracts—are separated according to molecular weight using electrophoresis on a polyacrylamide gel. The separated antigen “bands” are transferred (“blotted”) to a nitrocellulose membrane (or strips), which is then incubated with patient or control serum. Bound antibody is identified by use of labeled antihuman IgG. Antibody present in the patient serum is identified by comparison with the control serum results.

The use of cellular extracts as the source of antigen in immunoblotting has the advantage of not requiring purified antigen for testing, and the separation of proteins by molecular weight allows for the determination of the fine specificities of antibody responses to multisubunit antigens. The disadvantages of this technique are that it is time consuming, relatively expensive, requires the denaturing of proteins prior to gel electrophoresis (and will therefore not detect antibodies against conformational epitopes), and has a poor sensitivity for detecting antibodies against Ro and Scl-70 antigens. ^,

Immunodiffusion and Counterimmunoelectrophoresis

These techniques involve the detection of antibody–antigen binding as a “precipitation band” of immune complexes within a gel, most often composed of agar. In immunodiffusion, a known antigen and a sample of patient serum are placed in separate but closely approximated wells in the gel and allowed to passively diffuse toward one another. Radial immunodiffusion is a related technique in which the agar gel incorporates antibody of known specificity and the sample containing antigen is placed in a well within the gel. Antigen diffuses from the sample into the gel and will precipitate as a ring if bound by the gel antibody. The diameter of this ring is a function of the concentration of the antigen in the sample.

The process in counterimmunoelectrophoresis (CIEP) is similar to immunodiffusion; however, the pH of the gel is adjusted such that antigen will diffuse toward the anode and antibody toward the cathode when an electrical field is placed across the gel. The wells are situated such that antigen and antibody will diffuse through each other when the electric field is active. The role of the electric field is to accelerate movement of antigen and antibody and thereby shorten the time to a result.

The materials required for CIEP are relatively inexpensive and both CIEP and immunodiffusion have high specificity for clinical diagnoses. However, in comparison with other methods (e.g., ELISA) they have a lower sensitivity, their performance and interpretation are more labor intensive and skill dependent, and their turnaround time is longer. ^,

Turbidimetry and Nephelometry

Turbidimetry and nephelometry are techniques designed to measure the turbidity of a fluid sample as a gauge of the amount of particulate matter (e.g., immune complexes) it contains. Turbidimetry measures the amount of light able to pass directly through the sample, whereas nephelometry measures the amount of light scattered by the sample. In both techniques, comparison with standard samples of known turbidity is required. These techniques may be used to identify either antibodies or antigens. Both are automated and relatively simple to perform but are less sensitive than other techniques.

Laser Microbead Arrays

Laser microbead arrays are one of a number of “multiplex” immunodiagnostic techniques that allow the assay of multiple analytes (e.g., autoantibodies) from a single sample. ^, This technique shares some of the principles of ELISA in that it involves the use of labeled “reporter” antibodies to detect binding of patient antibodies to a known antigen. However, rather than being bound to a well wall on a plastic plate, in the most common form of this assay the antigen is bound to a “microbead” color coded to indicate the antigen it carries, and the reporter antibody is labeled with a fluorescent tag rather than an enzyme. Multiple beads bound to different antigens are then mixed with patient serum. Antibodies in the serum will bind to relevant bead-bound antigens, which in turn will be bound by fluorescent-tagged antihuman antibodies. The microbeads are then “read” by lasers of two different wavelengths; one identifying the color of the bead (and hence the antigen it carries) and the other determining the presence or absence of fluorescent-tagged antibody ( Fig. 10.5 ).

The advantages of this method are the number of antibodies that can be measured in parallel, the small volume of sample required, and the speed with which this can be done. The main disadvantage of this method is its reduced sensitivity for some applications. ^,

Antinuclear Antibodies

The term antinuclear antibody (ANA) refers to any of a large group of autoantibodies that recognize cellular antigens found predominantly, although not always exclusively, in the cell nucleus. The first observation hinting at the existence of ANAs was made in 1948 in a series of patients with SLE. It came in the form of what was termed the LE cell , a bone marrow granulocyte that had apparently ingested nuclear material from another cell. Subsequent investigations in vitro revealed that this phenomenon could be induced in bone marrow from healthy subjects by the addition of plasma from patients with SLE and resulted from an element in the globulin fraction of serum with an affinity for cell nuclei. For several decades, demonstration of LE cells retained a role in the diagnosis of SLE, remaining part of the American College of Rheumatology (ACR) classification criteria for SLE until the 1997 revision. Antinuclear antibodies are associated with numerous autoimmune diseases, most importantly SLE, but may also be found in infectious diseases, malignancies and apparently healthy individuals. Table 10.1 lists the reported prevalences of ANA in a variety of diseases.

Immunofluorescence Patterns

The cell nucleus contains thousands of antigens, any one of which could theoretically be the target of an ANA. In SLE alone, over 100 different ANA-target antigens have been reported. The pattern of IF produced by ANA in IIF assays is determined in large part by the location of the target antigen within the cell. A 2015 international consensus statement attempting to standardize the nomenclature for describing ANA IF patterns on HEp2 cells categorizes them into three major groups based on the location of the target antigen or the cell cycle phase in which it appears: nuclear, cytoplasmic, and mitotic. ^, Within each of these groups there are between 5 and 14 descriptors based on the immunofluorescent appearance. In this nomenclature, any antibody that binds to HEp2 cells is considered to be an “ANA,” although some of the responsible antigens are located exclusively in the cytoplasm (e.g., mitochondria) and therefore their cognate antibodies are not actually “antinuclear.” ^{, ,} Many laboratories report these cytoplasmic patterns as an addendum in reports of ANA assays on HEp2 cells. Table 10.2 outlines the proposed nomenclature along with the disease association of each pattern and the responsible target antigen. Fig. 10.6 demonstrates some of the more common IIF patterns seen in clinical practice.

One of the difficulties of the use of ANA IF patterns in the diagnosis of rheumatic diseases is their lack of disease specificity, as evident in Table 10.2 . This derives largely from the fact that, with few exceptions, the patterns are not specific for a particular antigen; multiple antigens may cause the same pattern and the same antigen may give different patterns in different patients. For this reason, in clinical practice once the presence of one or more ANAs has been identified using a screening method, the elucidation of their antigen specificity is generally performed using more targeted assays (see sections titled Antibodies to Extractable Nuclear Antigens and Anti-DNA Antibodies).