The antibodies formed during an immune reaction are important in combating infection, but they hold additional practical value. Characteristics of antibodies such as their quantity or specificity can reveal the history of a patient’s contact with microorganisms or other antigens. This is the underlying basis of serological testing. Serology is the branch of immunology that traditionally deals with in vitro diagnostic testing of serum. Serological testing is based on the familiar concept that antibodies have extreme specificity for antigens, so when a particular antigen is exposed to its specific antibody, it will bind tightly to it. Being able to visualize this interaction provides a powerful tool for detecting, identifying, and quantifying antibodies—or, for that matter, antigens. The scheme works both ways, depending on the situation. An antibody of known specificity can be used to detect or identify an unknown antigen (figure 1), or the opposite can be done, using a known antigen to detect the presence of an antibody (figure2). Modern serological testing has grown into a field that tests more than just serum. Urine, cerebrospinal fluid, whole tissues, and saliva can also be used to determine the immunologic status of patients. These and other immune tests are helpful in confirming a suspected diagnosis or in screening a certain population for disease.
Fig1. Serotyping of unknown bacteria. (a) An unidentified microbial sample is combined with antibodies of a known specificity. In a positive reaction, the antibodies recognize and bind to the microbial sample being tested, causing the cells to agglutinate. (b) In a negative reaction, the antibodies do not recognize the microbial sample, and no agglutination occurs. (c) A positive reaction (left) and a negative reaction (right). (a, b): Barry Chess/McGraw Hill; (c): Barry Chess
Fig2. Serological testing of serum. (a) A patient's serum, with an unknown antibody content, is combined with a single antigen. If the antigen is recognized by antibodies in the serum, a visible reaction occurs (often agglutination, but other reactions are possible). (b) If the patient has no antibodies that recognize the antigen, no reaction occurs. (c) A serological test for the presence of antibodies against five different antigens. The patient's serum contains antibodies against a serotype of Neisseria meningitidis, indicating infection or past vaccination. No antibodies are present that recognize any of the other four antigens tested. (c): Thermo Fisher Scientific Inc.
General Features of Immune Testing
A wide variety of immunological tests exists, but all depend on the in teraction between antigen and antibody. We summarize them under the headings of agglutination, precipitation, complement fixation, fluorescent antibody tests, and immunoassay tests. First we describe the general characteristics of immune testing, and then we look at each type specifically.
The most effective serological tests are both highly specific and highly sensitive (figure 3). Specificity is the property of a test to focus upon only a certain antibody or antigen and not to react with unrelated or distantly related ones. Sensitivity means that the test can detect even very small amounts of antibodies or antigens that are the targets of the test. A test with high sensitivity will diagnose even patients who are weakly positive. Modern systems using monoclonal antibodies have greatly improved specificity, and those using radioactivity, enzymes, and electronics have improved sensitivity.
Fig3. Specificity and sensitivity in immune testing. (a) This test shows specificity in which an antibody (Ab) attaches to one type of antigen, while ignoring all others. (b) Sensitivity is demonstrated by the fact that Ab can detect antigens even when the antigen is present in very low numbers.
Visualizing Antigen–Antibody Interactions
Because the binding of antibody (Ab) to antigen (Ag) cannot be seen without an electron micro scope, these reactions are usually linked to some type of endpoint reaction visible to the naked eye (or light microscope) that tells whether the result is positive or negative. In the case of large antigens such as cells, Ab binds to Ag and creates large clumps or aggregates that are visible macroscopically or microscopically (figure 4a). Smaller Ab-Ag complexes that do not result in readily observable changes will require special indicators in order to be visualized. Endpoints are often revealed by dyes or fluorescent reagents that can tag molecules of interest. Similarly, radioactive isotopes incorporated into antigens or antibodies constitute sensitive tracers that are detectable with photo graphic film.
Fig4. Antigen–antibody testing. (a) Cellular/molecular view of agglutination and precipitation reactions that produce visible antigen–antibody complexes. (b) An agglutination test for determining the antibody titer of serum.
An antigen–antibody reaction can be used to establish a titer, or the concentration of antibodies in serum. Titer is determined by serially diluting a sample in tubes or in a multiple-welled microtiter plate and mixing it with antigen (figure 4b). It is determined by observing the highest dilution of serum that produces a visible reaction with an antigen. The titer is the denominator of this dilution. The more a sample can be diluted and yet still react with antigen, the greater the concentration of antibodies in that sample and the higher its titer. Possible interpretations of test results are discussed in Clinical Connections, “When Positive Is Negative.”
Agglutination and Precipitation Reactions
The essential differences between agglutination and precipitation are in size, solubility, and location of the antigen. In agglutination, the antigens are whole cells such as red blood cells or bacteria with determinant groups on the surface. In precipitation, the antigen is a soluble molecule, such as a protein. In both instances, when Ag and Ab are optimally combined so that neither is in excess, one antigen is interlinked by several antibodies to form an insoluble, three-dimensional aggregate so large that it cannot remain suspended, and it settles out.
Agglutination Tests
Agglutination refers to the cross-linking of antigens (by antibodies) into large clumps, which are visible to the naked eye. Agglutination tests are performed routinely by blood banks to determine ABO and Rh (Rhesus) blood types in preparation for transfusions. In this type of test, antisera containing antibodies against the blood group antigens on red blood cells are mixed with a small sample of blood and read for the presence or absence of clumping. The Widal test is an example of a tube agglutination test for diagnosing salmonelloses and undulant fever. In addition to detecting specific antibody, it gives the serum titer.
Numerous variations of agglutination testing exist. The rapid plasma reagin (RPR) test is one of several tests commonly used to test for antibodies to syphilis. The cold agglutinin test, named for antibodies that react only at lower temperatures (4°C to 20°C), was developed to diagnose Mycoplasma pneumonia. The Weil-Felix reaction is an agglutination test sometimes used in diagnosing rickettsial infections.
In some tests, agglutinating antigens are affixed to the surface of an inert particle. In latex agglutination tests, the inert particles are tiny latex beads. Kits based on agglutination are available for assaying pregnancy hormone in the urine; for identifying Candida yeasts, staphylococci, streptococci, and meningococci; and for diagnosing rheumatoid arthritis.
Precipitation Tests
In precipitation reactions, the soluble antigen is precipitated (made insoluble) by an antibody. While this reaction can be seen in a test tube, the precipitate is easily disrupted. To prevent this, most precipitation reactions are carried out in agar gels. These substrates are sufficiently soft to allow the reactants (Ab and Ag) to freely diffuse, yet firm enough to hold the Ab-Ag precipitate in place. One technique with applications in microbial identification and diagnosis of disease is the double diffusion (Ouchterlony) method (figure 5). It is called double diffusion because it involves diffusion of both antigens and antibodies. The test is performed by punching a pattern of small wells into an agar medium and filling them with test antigens and antibodies. A band forming between two wells indicates that anti bodies from one well have met and reacted with antigens from the other well. Variations on this technique provide a means of identifying unknown antibodies or antigens.
Fig5. Double diffusion precipitation reaction (Ouchterlony test). (a) Small wells are punched into an agar plate. A known antigen is added to the center well, and serum to be tested is added to surrounding wells. Both the antigen and antibodies in the serum diffuse outward, and if the serum contains antibodies that recognize the antigen, they will precipitate, forming a visible band in the agar. (b) In a double diffusion test for the fungus Histoplasma capsulatum, a precipitation line is visible between the central H. capsulatum antigen and sample 2. Samples 1 and 3 show no reaction. (b): Dr. Errol Reiss/CDC
The Western Blot for Detecting Proteins
The Western blot test involves the electro phoretic separation of proteins, followed by an immunoassay to detect these proteins. This test is a counterpart of the Southern blot test for identifying DNA, described in chapter 10. It is a highly specific and sensitive method to identify or verify a particular protein (antibody or antigen) in a sample (figure 6). First, the test material (blood or other tissue in most cases) is electrophoresed in a gel to separate proteins by size. The separated proteins are transferred to a special membrane made of nylon or nitrocellulose, binding the proteins in place and creating a “blot.” The blot is then incubated with a solution containing antibodies that have been labeled with radioactive, fluorescent, or lumines cent molecules. Sites of specific binding will appear as a pattern of bands that can be compared with known positive and negative samples. This is usually the second test used to verify the status of people who are antibody-positive for HIV in the ELISA test, because it tests more types of antibodies and is less subject to misinterpretation and false positives than are other antibody tests. Although time-consuming and expensive when compared to other immunological tests, Western blots are widely used due to their exceptionally high degree of specificity and sensitivity.
Fig6. A Western blot used to detect the presence of antibodies against HIV in the bloodstream. Electrophoresis is used to separate the major antigens of HIV by size, and the antigens are then transferred to a nylon or nitrocellulose membrane. The membrane is incubated with the serum to be tested, and the binding of antigen and antibody produces a colored band. Successive tests on an HIV+ patient over 30 days reveals an increase in band intensity over time, due to continued formation of anti-HIV antibodies. A positive control strip (SRC) displays all the possible positive reactions and acts as a means of comparison. Genelabs Diagnostics Pte Ltd.
Complement Fixation
we introduced the functions of complement as a defense system for destroying foreign cells. An antibody that requires the actions of complement to complete the lysis of its antigenic target cell is termed a lysin or cytolysin. When lysins act in conjunction with the intrinsic complement system on red blood cells, the cells hemolyze (lyse and release their hemoglobin). This lysin-mediated hemolysis is the basis of a type of test called complement fixation, or CF (figure 7). Complement fixation tests are used to diagnose some viral, rickettsial, and fungal infections, but are often replaced by more sensitive and accurate ELISA and genetic tests.
Fig7. Complement fixation test. In this example, two serum samples are being tested for antibodies to a certain infectious agent. In reading this test, one observes the cloudiness of the tube. If it is cloudy, the RBCs are not hemolyzed and the test is positive. If it is clear and pink, the RBCs are hemolyzed and the test is negative.
Complement fixation testing uses four components—antibody, antigen, complement, and sensitized sheep red blood cells—and it is conducted in two stages.
In the first stage, the test antigen is allowed to react with the test antibody (at least one must be of known identity) in the absence of complement. If the antibody and antigen are specific for each other, they form complexes. To this mixture, purified complement proteins from guinea pig blood are added. If the antibody and anti gen have complexed during the previous step, they attach, or fix, the complement to them, thus preventing it from participating in further reactions.
The extent of any complement fixation is determined in the second stage by means of sheep red blood cells (RBCs) with surface lysin molecules. The sheep RBCs serve as an indicator complex that can also fix complement. Contents of the stage 1 tube are mixed with the stage 2 tube and observed with the naked eye for hemolysis.
● If hemolysis does not occur, it means that the complement was used up by the first-stage Ab-Ag complex and that the unknown antigen or antibody was indeed present. This result is considered positive.
● If hemolysis does occur, it means that unfixed complement from tube 1 reacted with the RBC complex instead, thereby causing lysis of the sheep RBCs. This result is negative for the antigen or antibody that was the target of the test.
A relative of the CF test, the antistreptolysin O (ASO) titer test, measures the levels of antibody against the streptolysin toxin, an important hemolysin of group A streptococci. A serum sample is exposed to known suspensions of streptolysin and then allowed to incubate with RBCs. Lack of hemolysis indicates antistreptolysin antibodies in the patient’s serum that have neutralized the streptolysin and prevented hemolysis. This can be a useful procedure to verify scarlet fever, rheumatic fever, and other related streptococcal syndromes.
Point-of-Care and Rapid Diagnostic Tests
An additional advantage seen with serological testing is that many of these tests can be administered and evaluated by someone with minimal training and no access to a traditional laboratory. This al lows testing to take place in the clinic of a pharmacy, in a person’s living room, or in the parking lot of Dodger Stadium. In fact, on the day this sentence was written, 474,292 COVID tests were administered in California. While that may seem like a random factoid, it’s not. It was half a million people receiving health care in the middle of a pandemic, a number only possible because the tests didn’t re quire a hospital, laboratory, doctor, or nurse (whose skills could be better utilized elsewhere).
Point-of-care tests are not new. Home pregnancy tests, along with rapid Streptococcus and Staphylococcus tests, have been around for decades; all three use antibodies to detect specific anti gens and provide an almost immediate result. Such tests—also known as rapid diagnostic tests, or RDTs—now exist for most major infectious agents, including HIV, hepatitis B and C, and of course SARS-CoV-2 (figure 8). And it is not an exaggeration to say that they have revolutionized medicine.
Fig8. Rapid diagnostic tests. (a) A SARS-CoV-2 (COVID 19) rapid antigen test. This test detects viral antigens in a saliva sample. The C line is a control and shows that the test is functioning correctly. The lack of a line in the T (test) portion of the window indicates that the sample does not contain antigens from the virus. (b) A rapid diagnostic panel that identifies antigens from different species of Plasmodium, the agent of malaria, using a small drop of whole blood. (a): staukestock/Shutterstock; (b): Courtesy of Alere, Inc.
To use COVID-19 as an example, rapid antigen tests rely on detection of SARS-CoV-2 viral proteins. Because the test is de pendent on how many viral particles are in the body, it is more accurate if a person is further along in the course of infection— for instance, when they are displaying symptoms—and more likely to produce a false negative result when taken early. Of course, the advantage of the test is its portability and ease of administration; you can get a test virtually anywhere and have the results in just a few minutes. Nucleic acid amplification tests, which include the polymerase chain reaction, are only slightly more complicated to administer, but they require sophisticated laboratory equipment to process, and it typically takes a day or more to receive results. Because these tests amplify viral RNA several million-fold, even a small number of viral particles is likely to be detected, giving the test a near 100% detection rate. Rapid diagnostic tests were integral to interrupting the spread of the SARS-CoV-2 virus, preventing infected people from boarding airplanes or letting them know they should avoid large Thanksgiving get-togethers.
Miscellaneous Serological Tests
A test that relies on changes in cellular activity as seen microscopically is the Treponema pallidum immobilization (TPI) test for syphilis. The impairment or loss of motility of the Treponema spirochete in the presence of test serum and complement indicates that the serum contains anti–Treponema pallidum antibodies. In toxin neutralization tests, a test serum is incubated with the microbe that produces the toxin. If the serum inhibits the growth of the microbe, one can conclude that antitoxins to inactivate the toxin are present.
Serotyping is an antigen–antibody technique for identifying, classifying, and subgrouping certain bacteria into categories called serotypes, using antisera for cell antigens such as the capsule, flagellum, and cell wall. It is widely used in typing Salmonella species and strains and is the basis for identifying the numerous serotypes of streptococci (see figure 1). The Quellung test, which identifies serotypes of the pneumococcus, involves a precipitation reaction in which antibodies react with the capsular polysaccharide. Although the reaction makes the capsule seem to swell, it is actually creating a zone of Ab-Ag complexes on the cell’s surface.
In Vivo Testing
Probably the first immunologic tests were performed not in a test tube but on the body itself. A classic example of one such technique is the tuberculin test, which uses a small amount of purified protein derivative (PPD) from Mycobacterium tuberculosis injected into the skin. The appearance of a red, raised, thickened lesion in 48 to 72 hours can indicate previous exposure to tuberculosis. In practice, in vivo tests employ principles similar to serological tests, except in this case, an antigen or an antibody is introduced into a patient to elicit some sort of visible reaction. Like the tuberculin test, some of these diagnostic skin tests are useful for evaluating infections due to fungi (coccidioidin and histoplasmin tests, for example) or allergens.
Fluorescent Antibody and Immunofluorescent Testing
The property of certain dyes to emit visible light in response to ultraviolet radiation was discussed in chapter 3. This property of fluorescence has found numerous applications in diagnostic immunology. The fundamental tool in immunofluorescent testing is a fluorescent antibody—a monoclonal antibody labeled by a fluorescent dye (fluorochrome).
In direct testing (figure 9) an unknown test specimen or antigen is fixed to a slide and exposed to a fluorescent antibody solution of known composition. If the antibodies are complementary to antigens in the material, they will bind to it. After the slide is rinsed to remove unattached antibodies, it is observed with the fluorescent microscope. Fluorescing cells or specks indicate the presence of Ab-Ag complexes and a positive result. These tests are valuable for identifying and locating antigens on the surfaces of cells or in tissues and in identifying the disease agents of syphilis, gonorrhea, chlamydiosis, whooping cough, Legionnaires’ disease, plague, trichomoniasis, meningitis, and listeriosis.
Fig9. Immunofluorescent testing. Direct Testing: Unidentified antigen (Ag) is directly tagged with fluorescent Ab.
In indirect testing (figure 10) the fluorescent antibodies are antibodies made to react with the Fc region of another antibody (remember that antibodies are proteins and can be recognized as antigens themselves). In this scheme, an antigen of known identity (a bacterial culture, for example) is combined with a test serum of unknown antibody content. The fluorescent antibody solution that can react with the unknown antibody is applied and rinsed off to visualize whether the serum contains antibodies that have affixed to the antigen. A positive test shows fluorescing aggregates or cells, indicating that the fluorescent antibodies have combined with the unlabeled antibodies. In a negative test, no fluorescent complexes will appear. This technique is frequently used to diagnose syphilis and various viral infections.
Fig10. Immunofluorescent testing. Indirect Testing: (a) Ag of known identity is used to assay unknown Ab; a positive reaction occurs when the second Ab (with fluorescent dye) affixes to the first Ab. (b) Indirect immunofluorescent stain of cells infected with two different viruses. Cells with green fluorescing nuclei contain cytomegalovirus; cells with yellow fluorescing nuclei contain adenovirus. (b): CHEMICON® International, Inc.
