Review of Medical Microbiology and Immunology, 13th Edition

64. Antigen-Antibody Reactions in the Laboratory



Types Of Diagnostic Tests


Precipitation (Precipitin)

Radioimmunoassay (RIA)

Enzyme-Linked Immunosorbent Assay (ELISA)

Immunofluorescence (Fluorescent Antibody)

Complement Fixation

Neutralization Tests

Immune Complexes

Hemagglutination Tests

Antiglobulin (Coombs) Test

Western Blot (Immunoblot)

Fluorescence-Activated Cell Sorting (Flow Cytometry)

Antigen–Antibody Reactions Involving Red Blood Cell Antigens

The ABO Blood Groups & Transfusion Reactions

Rh Blood Type & Hemolytic Disease of the Newborn

Self-Assessment Questions

Practice Questions: USMLE & Course Examinations


Reactions of antigens and antibodies are highly specific. An antibody will react only with the antigen that induced it or with a closely related antigen. Because of the great specificity, reactions between antigens and antibodies are suitable for identifying one by using the other. This is the basis of serologic reactions. However, cross-reactions between related antigens can occur, and these can limit the usefulness of the test.

The results of many immunologic tests are expressed as a titer, which is defined as the highest dilution of the specimen (e.g., serum) that gives a positive reaction in the test. Note that a patient’s serum with an antibody titer of, for example, 1/64 contains more antibodies (i.e., is a higher titer) than a serum with a titer of, for example, 1/4.

Table 64–1 describes the medical importance of serologic (antibody-based) tests. Their major uses are in the diagnosis of infectious diseases, in the diagnosis of autoimmune diseases, and in the typing of blood and tissues prior to transplantation.

Table 64–1 Major Uses of Serologic (Antibody-Based) Tests


Microorganisms and other cells possess a variety of antigens and thus induce antisera containing many different antibodies (i.e., the antisera are polyclonal). Monoclonal antibodies excel in the identification of antigens because cross-reacting antibodies are absent (i.e., monoclonal antibodies are highly specific).


Many types of diagnostic tests are performed in the immunology laboratory. Most of these tests can be designed to determine the presence of either antigen or antibody. To do this, one of the components, either antigen or antibody, is known and the other is unknown. For example, with a known antigen such as influenza virus, a test can determine whether antibody to the virus is present in the patient’s serum. Alternatively, with a known antibody, such as antibody to herpes simplex virus, a test can determine whether viral antigens are present in cells taken from the patient’s lesions.


In this test, the antigen is particulate (e.g., bacteria and red blood cells)1 or is an inert particle (latex beads) coated with an antigen. Antibody, because it is divalent or multivalent, cross-links the antigenically multivalent particles and forms a latticework, and clumping (agglutination) can be seen. This reaction can be done in a small cup or tube or with a drop on a slide. One very commonly used agglutination test is the test that determines a person’s ABO blood group (Figure 64–1; see the section on blood groups at the end of this chapter).


FIGURE 64–1 Agglutination test to determine ABO blood type. On the slide at the bottom of the figure, a drop of the patient’s blood was mixed with antiserum against either type A (left) or type B (right) blood cells. Agglutination (clumping) has occurred in the drop on the left containing the type A antiserum but not in the drop containing the type B antiserum, indicating that the patient is type A (i.e., has A antigen on the red cells). The slide at the top shows that the red cells (circles) are cross-linked by the antibodies (“Y” shapes) in the drop on the left but not in the drop on the right. If agglutination had occurred in the right side as well, it would indicate that the patient was producing B antigen as well as A and was type AB.

Precipitation (Precipitin)

In this test, the antigen is in solution. The antibody cross-links antigen molecules in variable proportions, and aggregates (precipitates) form. In the zone of equivalence, optimal proportions of antigen and antibody combine; the maximal amount of precipitates forms, and the supernatant contains neither an excess of antibody nor an excess of antigen (Figure 64–2). In the zone of antibody excess, there is too much antibody for efficient lattice formation, and precipitation is less than maximal.2 In the zone of antigen excess, all antibody has combined, but precipitation is reduced because many antigen–antibody complexes are too small to precipitate (i.e., they are “soluble”).


FIGURE 64–2 Precipitin curve. In the presence of a constant amount of antibody, the amount of immune precipitate formed is plotted as a function of increasing amounts of antigen. In the top part of the figure, the binding of antigen (image) and antibody (Y) in the three zones is depicted. In the zones of antibody excess and antigen excess, a lattice is not formed and precipitation does not occur, whereas in the equivalence zone, a lattice forms and precipitation is maximal. (Modified and reproduced with permission from Stites D, Terr A, Parslow T, eds. Basic & Clinical Immunology. 9th ed. Originally published by Appleton & Lange. Copyright 1997 McGraw-Hill.)

Precipitin tests can be done in solution or in semisolid medium (agar).

Precipitation in Solution

The concept of precipitation in solution is used clinically to measure the amount of immunoglobulins (IgM, IgG, etc.) in the blood plasma. The lab test used is called nephelometry, in which the amount of precipitate formed is measured by optical density of the precipitate. In the test, antibody specific for IgM, IgG, IgA, or IgE is reacted with the patient’s serum and the optical density measured. This value is then compared with a standard curve, which displays the optical density caused by known amounts of the immunoglobulins.

Precipitation in Agar

This is done as either single or double diffusion. It can also be done in the presence of an electric field.

Single Diffusion— In single diffusion, antibody is incorporated into agar and antigen is placed into a well. As the antigen diffuses with time, precipitation rings form depending on the antigen concentration. The greater the amount of antigen in the well, the farther the ring will be from the well. By calibrating the method, such radial immunodiffusion is used to measure IgG, IgM, complement components, and other substances in serum. (IgE cannot be measured because its concentration is too low.)

Double Diffusion—In double diffusion, antigen and antibody are placed in different wells in agar and allowed to diffuse and form concentration gradients. Where optimal proportions (see zone of equivalence, above) occur, lines of precipitate form (Figure 64–3). This method (Ouchterlony) indicates whether antigens are identical, related but not identical, or not related (Figure 64–4).


FIGURE 64–3 Double diffusion in agar. Antigen is placed in the well on the left, and antibody is placed in the well on the right. The antigen and antibody diffuse through the agar and form a precipitate in the zone of equivalence. Close to the antigen-containing well is the zone of antigen excess, and close to the antibody-containing well is the zone of antibody excess. No precipitate forms in the zones of antigen and antibody excess.


FIGURE 64–4 Double-diffusion (Ouchterlony) precipitin reactions. In these Ouchterlony reactions, wells are cut into an agar plate and various antigens and antisera are placed in the wells. The antigens and antibodies diffuse toward each other within the agar, and a line of precipitate forms in the zone of equivalence. Close to the antigen-containing well, a zone of antigen excess exists and no precipitate forms; close to the antibody-containing well, a zone of antibody excess exists and no precipitate forms. A and B are unrelated antigens (i.e., they have no epitopes in common). B and C are related antigens (i.e., they have some epitopes in common but some that are different). For example, chicken lysozyme (well B) and duck lysozyme (well C) share some epitopes because they are both lysozymes but have unique epitopes as well because they are from different species. The line of identity between B and C is caused by the reaction of the anti-B antibody with the shared epitopes on antigens B and C. The spur pointing toward well 4 is caused by the reaction of some of the anti-B antibody with the unique epitopes on antigen B in well 3. These lines of partial identity occur because antibody to B (chicken lysozyme) is polyclonal and has some immunoglobulins that react with the epitopes common to chicken and duck lysozyme and other immunoglobulins that react only with the epitopes unique to chicken lysozyme. (Modified and reproduced with permission from Brooks GF et al. Medical Microbiology. 19th ed. Originally published by Appleton & Lange. Copyright 1991 McGraw-Hill.)

Precipitation in Agar with an Electric Field

Immunoelectrophoresis—A serum sample is placed in a well in agar on a glass slide (Figure 64–5). A current is passed through the agar, and the proteins move in the electric field according to their charge and size. Then a trough is cut into the agar and filled with antibody. As the antigen and antibody diffuse toward each other, they form a series of arcs of precipitate. This permits the serum proteins to be characterized in terms of their presence, absence, or unusual pattern (e.g., human myeloma protein).


FIGURE 64–5 Immunoelectrophoresis. A: Human serum placed in the central well is electrophoresed, and the proteins migrate to different regions (orange ellipses). Antiserum to human serum is then placed in the elongated trough (gray areas). B: Human serum proteins and antibodies diffuse into agar. C: Precipitate arcs (orange lines) form in the agar. (Modified and reproduced with permission from Stites D, Terr A, Parslow T, eds. Basic & Clinical Immunology. 9th ed. Originally published by Appleton & Lange. Copyright 1997 McGraw-Hill.)

Counter-Immunoelectrophoresis— This method relies on movement of antigen toward the cathode and of antibody toward the anode during the passage of electric current through agar. The meeting of the antigen and antibody is greatly accelerated by this method and is made visible in 30 to 60 minutes. This has been applied to the detection of bacterial and fungal polysaccharide antigens in cerebrospinal fluid.

Radioimmunoassay (RIA)

This method is used for the quantitation of antigens or haptens that can be radioactively labeled. It is based on the competition for specific antibody between the labeled (known) and the unlabeled (unknown) concentration of material. The complexes that form between the antigen and antibody can then be separated and the amount of radioactivity measured. The more unlabeled antigen is present, the less radioactivity there is in the complex. The concentration of the unknown (unlabeled) antigen or hapten is determined by comparison with the effect of standards. RIA is a highly sensitive method and is commonly used to assay hormones or drugs in serum. The radioallergosorbent test (RAST) is a specialized RIA that is used to measure the amount of serum IgE antibody that reacts with a known allergen (antigen).

Enzyme-Linked Immunosorbent Assay (ELISA)

This method can be used for the quantitation of either antigens or antibodies in patient specimens. It is based on covalently linking an enzyme to a known antigen or antibody, reacting the enzyme-linked material with the patient’s specimen, and then assaying for enzyme activity by adding the substrate of the enzyme. The method is nearly as sensitive as RIA yet requires no special equipment or radioactive labels (Figure 64–6).


FIGURE 64–6 Enzyme-linked immunosorbent assay (ELISA). The term enzyme-linked refers to the covalent binding (linking) of an enzyme to antibody to human IgG. If the patient has antibodies to the microbial or viral antigen, those antibodies will bind to the microbial or viral antigens. The antibody to human IgG linked to the enzyme will then bind to the patient’s antibodies. Then when the substrate of the enzyme is added, the substrate changes color, indicating that the patient’s serum contained antibodies.

For measurement of antibody, known antigens are fixed to a surface (e.g., the bottom of small wells on a plastic plate), incubated with dilutions of the patient’s serum, washed, and then reincubated with antibody to human IgG labeled with an enzyme (e.g., horseradish peroxidase). Enzyme activity is measured by adding the substrate for the enzyme and estimating the color reaction in a spectrophotometer. The amount of antibody bound is proportional to the enzyme activity. The titer of antibody in the patient’s serum is the highest dilution of serum that gives a positive color reaction.

Immunofluorescence (Fluorescent Antibody)

Fluorescent dyes (e.g., fluorescein and rhodamine) can be covalently attached to antibody molecules and made visible by ultraviolet (UV) light in the fluorescence microscope. Such “labeled” antibody can be used to identify antigens (e.g., on the surface of bacteria such as streptococci and treponemes, in cells in histologic section, or in other specimens) (Figure 64–7). The immunofluorescence reaction is direct when known labeled antibody interacts directly with unknown antigen and indirect when a two-stage process is used. For example, known antigen is attached to a slide, the patient’s serum (unlabeled) is added, and the preparation is washed; if the patient’s serum contains antibody against the antigen, it will remain fixed to it on the slide and can be detected on addition of a fluorescent dye–labeled antibody to human IgG and examination by UV microscopy. The indirect test is often more sensitive than direct immunofluorescence, because more labeled antibody adheres per antigenic site. Furthermore, the labeled antiglobulin becomes a “universal reagent” (i.e., it is independent of the nature of the antigen used because the antibody to IgG is reactive with all human IgG).


FIGURE 64–7 Fluorescent antibody test. A: In the direct fluorescent antibody test, the fluorescent dye is attached directly to the antibody that is interacting with the antigen (dark triangles) on the surface of the cell. B: In the indirect fluorescent antibody test, the fluorescent dye is attached to antibody made against human IgG.

Complement Fixation

The complement system consists of 20 or more plasma proteins that interact with one another and with cell membranes. Each protein component must be activated sequentially under appropriate conditions for the reaction to progress. Antigen–antibody complexes are among the activators, and the complement fixation test can be used to identify one of them if the other is known.

The reaction consists of the following two steps (Figure 64–8): (1) Antigen and antibody (one known and the other unknown; e.g., use a known antigen and determine whether a patient’s serum contains antibodies to that antigen) are mixed, and a measured amount of complement (usually from guinea pig) is added. If the antigen and antibody match, they will combine and use up (“fix”) the complement. (2) An indicator system, consisting of “sensitized” red blood cells (i.e., red blood cells plus anti–red blood cell antibody), is added. If the antibody matched the antigen in the first step, complement was fixed and less (or none) is available to attach to the sensitized red blood cells. The red blood cells remain unhemolyzed (i.e., the test is positive) because the patient’s serum had antibodies to that antigen. If the antibody did not match the antigen in the first step, complement is free to attach to the sensitized red blood cells and they are lysed (i.e., the test is negative).


FIGURE 64–8 Complement fixation. Left: Positive reaction (i.e., the patient’s serum contains antibody). If a known antigen is mixed with the patient’s serum containing antibody against that antigen, then complement (solid circles) will be fixed. Because no complement is left over, the sensitized red cells are not lysed. Right: Negative reaction. If a known antigen is mixed with the patient’s serum that does not contain antibody against that antigen, complement (solid circles) is not fixed. Complement is left over and the sensitized red cells are lysed. Ab, antibody; Ag, antigen.

Complement must be carefully standardized, and the patient’s serum must be heated to 56°C for 30 minutes to inactivate any human complement activity. The antigen must be quantitated. The result is expressed as the highest dilution of serum that gives positive results. Controls to determine whether antigen or antibody alone fixes complement are needed to make the test results valid. If antigen or antibody alone fixes complement, it is said to be anticomplementary.

Neutralization Tests

These use the ability of antibodies to block the effect of toxins or the infectivity of viruses. They can be used in cell culture (e.g., inhibition of cytopathic effect and plaque-reduction assays) or in host animals (e.g., mouse protection tests).

Immune Complexes

Immune complexes in tissue can be stained with fluorescent complement. Immune complexes in serum can be detected by binding to C1q or by attachment to certain (e.g., Raji lymphoblastoid) cells in culture.

Hemagglutination Tests

Many viruses clump red blood cells from one species or another (active hemagglutination). This can be inhibited by antibody specifically directed against the virus (hemagglutination inhibition) and can be used to measure the titer of such antibody. Red blood cells also can absorb many antigens and, when mixed with matching antibodies, will clump (this is known as passive hemagglutination, because the red cells are passive carriers of the antigen).

Antiglobulin (Coombs) Test

Some patients with certain diseases (e.g., hemolytic disease of the newborn [Rh incompatibility] and drug-related hemolytic anemias) become sensitized but do not exhibit symptoms of disease. In these patients, antibodies against the red cells are formed and bind to the red cell surface but do not cause hemolysis. These cell-bound antibodies can be detected by the direct antiglobulin (Coombs) test, in which antiserum against human immunoglobulin is used to agglutinate the patient’s red cells. In some cases, antibody against the red cells is not bound to the red cells but is in the serum and the indirect antiglobulin test for antibodies in the patient’s serum should be performed. In the indirect Coombs test, the patient’s serum is mixed with normal red cells, and antiserum to human immunoglobulins is added. If antibodies are present in the patient’s serum, agglutination occurs.

Western Blot (Immunoblot)

This test is typically used to determine whether a positive result in a screening immunologic test is a true-positive or a false-positive result. For example, patients who are positive in the screening ELISA for human immunodeficiency virus (HIV) infection or for Lyme disease should have a Western blot test performed. Figure 64–9 illustrates a Western blot test for the presence of HIV antibodies in the patient’s serum. In this test, HIV proteins are separated electrophoretically in a gel, resulting in discrete bands of viral protein. These proteins are then transferred from the gel (i.e., blotted) onto filter paper, and the person’s serum is added. If antibodies are present, they bind to the viral proteins (primarily gp41 and p24) and can be detected by adding antibody to human IgG labeled with either radioactivity or an enzyme such as horseradish peroxidase, which produces a visible color change when the enzyme substrate is added.


FIGURE 64–9 Western blot (immunoblot test). In this test, microbial or viral proteins are separated on an acrylamide gel and then transferred (blotted) onto paper. The patient’s serum then interacts with the separated proteins. If antibodies are present in the patient’s serum, they bind to the proteins. The patient’s antibodies are then detected by using labeled antibody to human IgG.

Fluorescence-Activated Cell Sorting (Flow Cytometry)

This test is commonly used to measure the number of the various types of immunologically active blood cells (Figure 64–10). For example, it is used in HIV-infected patients to determine the number of CD4-positive T cells. In this test, the patient’s cells are labeled with monoclonal antibody to the protein specific to the cell of interest (e.g., CD4 protein if the number of helper T cells is to be determined). The monoclonal antibody is tagged with a fluorescent dye, such as fluorescein or rhodamine. Single cells are passed through a laser light beam, and the number of cells that fluoresce is counted by use of a machine called a fluorescence-activated cell sorter (FACS).


FIGURE 64–10 Flow cytometry. At the top of the figure, a cell has interacted with monoclonal antibody labeled with a fluorescent dye. As the cell passes down the tube, ultraviolet light causes the dye to fluoresce and a sensor counts the cell. Farther down the tube, an electrical charge can be put on the cell, which allows it to be deflected into a test tube and subjected to additional analysis.


Many different blood group systems exist in humans. Each system consists of a gene locus specifying antigens on the erythrocyte surface. The two most important blood groupings, ABO and Rh, are described next.

The ABO Blood Groups & Transfusion Reactions

All human erythrocytes contain alloantigens (i.e., antigens that vary among individual members of a species) of the ABO group. A person’s ABO blood group is a very important determinant of the success of both blood transfusions and organ transplants.

The A and B genes encode enzymes that add specific sugars to the end of a polysaccharide chain on the surface of many cells, including red cells (Figure 64–11). People who inherit neither gene are type O. The genes are codominant, so people who inherit both genes are type AB. People who are either homozygous AA or heterozygous AO are type A, and, similarly, people who are either homozygous BB or heterozygous BO are type B.


FIGURE 64–11 ABO blood groups. Structures of the terminal sugars that determine ABO blood groups are shown. Blood group O cells have H antigen on their surface; blood group A cells have N-acetylgalactosamine added to the end of the H antigen; and blood group B cells have galactosamine added to the end of the H antigen. (Reproduced with permission from Stites DP, Stobo JD, Wells JV, eds. Basic & Clinical Immunology. 6th ed. Originally published by Appleton & Lange. Copyright 1987 McGraw-Hill.)

The A and B antigens are carbohydrates that differ by a single sugar. Despite this small difference, A and B antigens do not cross-react. Erythrocytes have three terminal sugars in common on their surface: N-acetylglucosamine, galactose, and fucose. These three sugars form the H antigen (Figure 64–11). People who are blood group O have only the H antigen on the surface of their red cells. People who are blood group A have N-acetylgalactosamine added to the galactose of the H antigen, whereas people who are blood group B have galactose added to the galactose of the H antigen.

There are four combinations of the A and B antigens, called A, B, AB, and O (Table 64–2). A person’s blood group is determined by mixing the person’s blood with antiserum against A antigen on one area on a slide and with antiserum against B antigen on another area (Figure 64–1). If agglutination occurs only with A antiserum, the blood group is A; if it occurs only with B antiserum, the blood group is B; if it occurs with both A and B antisera, the blood group is AB; and if it occurs with neither A nor B antisera, the blood group is O.

Table 64–2 ABO Blood Groups


The plasma contains antibody against the absent antigens (i.e., people with blood group A have antibodies to B in their plasma). These antibodies are formed against cross-reacting bacterial or food antigens, are first detectable at 3 to 6 months of age, and are of the IgM class. Individuals are tolerant to their own blood group antigens, and therefore a person with blood group A does not form antibodies to A antigen. The end result is that antigen and corresponding antibody do not coexist in the same person’s blood. Transfusion reactions occur when incompatible donor red blood cells are transfused (e.g., if group A blood were transfused into a group B person [because anti-A antibody is present]). The red cell–antibody complex activates complement, and a reaction consisting of shock caused by large amounts of C3a and C5a (anaphylatoxins) and hemolysis caused by C5, C6, C7, C8, and C9 (membrane attack complex) occurs (Figure 64–12).


FIGURE 64–12 Transfusion reaction. Top panel: Red blood cells bearing A antigen are transfused into a person who is type B and therefore has antibodies to A antigen. Middle panel: Anti-A antibodies bind to A antigen on the red cells causing agglutination of red cells that can block movement of blood through capillaries causing anoxia to tissue. Bottom panel: Complement is activated by the antigen–antibody complexes and the membrane attack complex lyses the red cells, causing hemolysis and anemia. (Reproduced with permission from Cowan MK, Talaro KP, eds. Microbiology: A Systems Approach. New York: McGraw-Hill; 2009.)

To avoid antigen–antibody reactions that would result in transfusion reactions, all blood for transfusions must be carefully matched (i.e., erythrocytes are typed for their surface antigens by specific sera). As shown in Table 64–2, persons with group O blood have no A or B antigens on their red cells and so are universal donors (i.e., they can give blood to people in all four groups) (Table 64–3). Note that type O blood has A and B antibodies. Therefore when type O blood is given to a person with type A, B, or AB blood, you might expect a reaction to occur. A clinically detectable reaction does not occur because the donor antibody is rapidly diluted below a significant level. Persons with group AB blood have neither A nor B antibody and thus are universal recipients.

Table 64–3 Compatibility of Blood Transfusions Between ABO Blood Groups1


In addition to red blood cells, the A and B antigens appear on the cells of many tissues. Furthermore, these antigens can be secreted in saliva and other body fluids. Secretion is controlled by a secretor gene. Approximately 85% of people carry the dominant form of the gene, which allows secretion to occur.

ABO blood group differences can lead to neonatal jaundice and anemia, but the effects on the fetus are usually less severe than those seen in Rh incompatibility (see next section). For example, mothers with blood group O have antibodies against both A and B antigens. These IgG antibodies can pass the placenta and, if the fetus is blood group A or B, cause lysis of fetal red cells. Mothers with either blood group A or B have a lower risk of having a neonate with jaundice because these mothers produce antibodies to either B or A antigens, respectively, that are primarily IgM, and IgM does not pass the placenta.

Rh Blood Type & Hemolytic Disease of the Newborn

About 85% of humans have erythrocytes that express the Rh(D) antigen [i.e., are Rh(D)+]. When an Rh(D) person is transfused with Rh(D)+ blood or when an Rh(D) woman has an Rh(D)+ fetus (the D gene being inherited from the father), the Rh(D) antigen will stimulate the development of antibodies (Table 64–4). This occurs most often when the Rh(D)+ erythrocytes of the fetus leak into the maternal circulation during delivery of the first Rh(D)+ child (Figure 64–13).

Table 64–4 Rh Status and Hemolytic Disease of the Newborn



FIGURE 64–13 Hemolytic disease of the newborn (erythroblastosis fetalis). Left panel: Fetal red cells (RBCs) bearing the Rh antigen enter the mother’s blood when the placenta separates during the birth of the first Rh-positive child. IgG antibodies to Rh antigen are then produced by the mother. Center panel: During a second pregnancy with an Rh-positive fetus, IgG antibodies pass from the mother into the fetus via the placenta. The antibodies bind to the fetal red cells, complement is activated, and the membrane attack complex lyses the fetal red cells. Right panel: Anemia and jaundice occur in the fetus/newborn. As a result of the anemia, large numbers of erythroblasts are produced by the bone marrow and are seen in the blood of the newborn. (Reproduced with permission from Cowan MK, Talaro KP, eds. Microbiology: A Systems Approach. New York: McGraw-Hill; 2009.)

Subsequent Rh(D)+ pregnancies are likely to be affected by the mother’s anti-D antibody, and hemolytic disease of the newborn (erythroblastosis fetalis) often results. This disease results from the passage of maternal IgG anti-Rh(D) antibodies through the placenta to the fetus, with subsequent lysis of the fetal erythrocytes. The direct antiglobulin (Coombs) test is typically positive (see earlier description of the Coombs test).

The problem can be prevented by administration of high-titer Rh(D) immune globulins (Rho-Gam) to an Rh(D) mother at 28 weeks of gestation and immediately upon the delivery of an Rh(D)+ child. These antibodies promptly attach to Rh(D)+ erythrocytes and prevent their acting as sensitizing antigen. This prophylaxis is widely practiced and effective.


1. Which one of the following laboratory tests would be the best to determine the number of CD4-positive cells in the blood of a patient infected with HIV?

(A) Agglutination

(B) Complement fixation

(C) Enzyme-linked immunosorbent assay (ELISA)

(D) Flow cytometry

(E) Immunoelectrophoresis

2. You have just received a lab report that says your patient is positive for IgM antibody to Borrelia burgdorferi in an enzyme-linked immunosorbent assay (ELISA). This supports your clinical impression that the patient has Lyme disease. Which one of the following best describes how the ELISA was performed? (For brevity, the wash steps have been left out.)

(A) The patient’s serum was reacted with antibody to human mu heavy chain. Then Borrelia antigens labeled with an enzyme were added. Then the enzyme substrate was added, and a color change was observed.

(B) The patient’s serum was reacted with Borrelia antigens. Then antibody to human mu heavy chain labeled with an enzyme was added. Then the enzyme substrate was added, and a color change was observed.

(C) Borrelia antigens were reacted with antibody to human mu heavy chain. Then the patient’s serum labeled with an enzyme was added. Then the enzyme substrate was added, and a color change was observed.

(D) Borrelia antigens were reacted with antibody to human mu heavy chain labeled with an enzyme. Then the patient’s serum was added. Then the enzyme substrate was added, and a color change was observed.

3. Regarding ABO blood groups, which one of the following is the most accurate?

(A) People who are blood group O have the O antigen on the surface of their red cells.

(B) The A and B blood group antigens are located on the surface of red cells but not on the surface of other cells.

(C) The differences between the A and B blood group antigens are dependent on the presence of different D-amino acids on the cell surface.

(D) People who are blood group O do not have antibodies to A and B blood group antigens and thus can be given both type A and type B blood.

(E) The genes that determine ABO blood groups are codominant, so a person who is blood group AB is expressing both genes that encode the enzymes that synthesize the A and the B blood group antigens.

4. Regarding hemolytic disease of the newborn (erythroblastosis fetalis), which one of the following is the most accurate?

(A) Maternal red cells are the source of the antigen that induces the antibody.

(B) It typically occurs when the father is Rh-positive and the mother is Rh-negative.

(C) Maternal IgM anti-Rh antibody enters the fetus and causes damage to the fetal red cells.

(D) Symptomatic disease is more likely to occur in the first child than in the subsequent children.

(E) Administration of Rh antigen to the newborn can prevent symptomatic disease if given early enough.

5. You think your patient has secondary syphilis, and you order a VDRL serological test. The lab reports that the test is negative. If this is a false-negative result due to the “prozone” phenomenon, which one of the following is the most likely explanation?

(A) The patient’s serum has too much antibody, and the reaction is in the zone of antibody excess.

(B) The patient’s serum has too much antigen, and the reaction is in the zone of antigen-excess phase.

(C) The patient’s serum has too little antibody, and the reaction is in the zone of antibody-deficient phase.

(D) The patient’s serum has too little antigen, and the reaction is in the zone of antigen-deficient phase.

(E) The patient’s serum has an amount of antibody that puts it in the zone of equivalence.

6. As part of a murder investigation, the blood group of the victim was determined by analyzing the antibodies in her serum. (Unfortunately, the red cells of the victim were lost by the crime squad, so they had to use her serum.) In this test, red cells known to be either O, A, B, or AB were mixed with her serum and agglutination observed. Based on the results in the following table, what is the blood group of the victim?


(A) Type O

(B) Type A

(C) Type B

(D) Type AB

(E) A laboratory error has occurred, and the test should be repeated.


1. (D)

2. (B)

3. (E)

4. (B)

5. (A)

6. (A)


Questions on the topics discussed in this chapter can be found in the Immunology section of PART XIII: USMLE (National Board) Practice Questions starting on page 713. Also see PART XIV: USMLE (National Board) Practice Examination starting on page 731.

1When red cells are used, the reaction is called hemagglutination.

2The term “prozone” refers to the failure of a precipitate or flocculate to form because too much antibody is present. For example, a false-negative serologic test for syphilis (VDRL) is occasionally reported because the antibody titer is too high. Dilution of the serum yields a positive result.

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