the Complex Mechanisms and Applications of Antigen-Antibody Reactions in Laboratory Settings: A Comprehensive Study

the Complex Mechanisms and Applications of Antigen-Antibody Reactions in Laboratory Settings: A Comprehensive Study

 

INTRODUCTION

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 specific-

ity, 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 (or, in other words,

the smallest concentration) of the specimen (e.g., serum) that

still 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 sero-

logic (antibody-based) tests. Their major uses are in the

diagnosis of infectious diseases, in the diagnosis of autoim-

mune diseases, and in the typing of blood and tissues prior

to transplantation.

Microorganisms and other cells possess a variety of anti-

gens and thus induce antisera containing many different

antibodies (i.e., the antisera are polyclonal). Monoclonal anti-

bodies excel in the identification of antigens because cross-

reacting antibodies are absent (i.e., monoclonal antibodies

are highly specific). Chapter 61 discusses the generation of

specific antibodies, including monoclonal antibodies used for

diagnostic purposes.

TYPES OF DIAGNOSTIC TESTS

Many types of diagnostic tests are performed in the immu-

nology 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 of

a known quantity or concentration 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.

Agglutination

In this test, the antigen is particulate (e.g., bacteria and red

blood cells) 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 lat-

ticework, and clumping (agglutination) can be seen. When the

red blood cells are used as the particulate antigen, the reaction

is called hemagglutination. This reaction can be done in a small

cup or tube or with a drop on a slide. One very commonly used

hemagglutination 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).

Precipitation (Precipitin)

In this test, the antigen is in solution. The antibody cross-links

antigen molecules in variable proportions, and aggregates (pre-

cipitates) form. In the zone of equivalence, optimal propor-

tions 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 maxi-

mal.1

In the zone of antigen excess, all antibody has combined,

1

The 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.

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 the Fc portion

of IgM, IgG, IgA, or IgE is mixed 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 incorpo-

rated 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, earlier) 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).

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 dif-

fuse 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).

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 accel-

erated 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 com-

plexes 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 radioallergo-

sorbent 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 anti-

body, 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).

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 mea-

sured by adding the substrate for the enzyme and estimating the

Immunofluorescence (Fluorescent

Antibody)

Fluorescent dyes (e.g., fluorescein and rhodamine) can be

covalently attached to antibody molecules and made visible by

exposing the sample to light of the correct excitation spectrum

in a 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 reac-

tion is direct when known labeled antibody interacts directly

with unknown antigen and indirect when a two-stage process

is used. For example, in the indirect assay, 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 anti-

body against the antigen, it will remain fixed to it on the slide

and can be detected on addition of a fluorescent dye-labeled

antibody against human IgG and examination by fluorescent

microscopy. The indirect test is often more sensitive than direct

immunofluorescence, because more labeled antibody adheres

per antigenic site, amplifying the signal. Furthermore, the

labeled anti-IgG antibody becomes a “universal reagent” (i.e., it

is independent of the nature of the antigen used, because anti-

IgG is reactive with all human IgG).

Complement Fixation

The complement system consists of 20 or more plasma pro-

teins that interact with one another and with cell membranes

(see Chapter 63). Each protein component must be activated

sequentially under appropriate conditions for the reaction to

progress. Antigen–antibody complexes are among the activators

(e.g. in the classic pathway), and the complement fixation test

can be used to identify one of them if the other is known.

The reaction consists of the following three steps

(Figure 64–8): (1) The patient’s serum is heated to 56°C for

30 minutes to inactivate any complement activity. (2) Either

antigen or antibody (whichever is the “known” quantity in

the reaction) is mixed with the serum, which contains the

“unknown” ingredient. For example, to determine whether a

patient’s serum contains antibodies to a certain antigen, a mea-

sured amount of that antigen is added to the serum. In addition,

a measured amount of complement (usually from guinea pig)

added at this stage. If the antigen and antibody match, they will

combine and use up (“fix”) the complement. (3) An indicator

system, consisting of “sensitized” red blood cells (i.e., red blood

cells plus anti-red blood cell antibody), is added last.

If the antibody matches the antigen in the first step,

complement is fixed and is unavailable to attach and lyse the

sensitized red blood cells. The red blood cells will remain

unhemolyzed (i.e., the test is positive) because the patient’s

serum has antibodies to that antigen and all the complement

is used up in the first step. If the antibody does not match the

antigen in the first step, complement remains free to attach to

the sensitized red blood cells, and they are lysed (i.e., the test is

negative). The result is expressed as the highest dilution (low-

est concentration) of serum that gives positive results.

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

or in host animals. For example, a patient’s serum specimen is

added to a culture of cells. If the cells are killed, meaning there

is virus present, then the culture fluid is collected, split into

sub-aliquots, and mixed with a panel of antibodies specific for

different viruses before inoculating a fresh cell culture. If an

antibody added to the aliquots blocks whatever virus is in that

fluid from infecting the new cells, this identifies the virus in

the culture.

Immune Complexes

Immune complexes in tissue sections can be stained with fluo-

rescent complement, which will bind to the Fc portion of IgM

and IgG (see Chapters 61 and 63). These can be detected using

fluorescent microscopy. 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 cause red blood cells to clump together (active

hemagglutination). This can be inhibited by antibody specifi-

cally directed against the virus (hemagglutination inhibition)

and, like the neutralization tests described earlier, can be used

to measure the titer of inhibitory antibody. Red blood cells also

can absorb many antigens and, when mixed with matching anti-

bodies, 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 against red blood cell antigens but

do not exhibit overt 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 anti-

bodies 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, result-

ing 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 radio-

activity or an enzyme such as horseradish peroxidase, which

produces a visible color change when the enzyme substrate is

added (similar to the process in an ELISA test).

Flow Cytometry & Fluorescence-Activated

Cell Sorting

This test is commonly used to count the number of various types

of immune cells in a sample of blood, bone marrow, or lymphoid

tissue (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 mixed with fluorescently tagged mono-

clonal antibodies specific to different proteins on immune cells

of interest (e.g., CD4 protein, if the number of helper T cells is to

be determined). The monoclonal antibodies have a fluorescent

tag, such as fluorescein or rhodamine, that is excited by a specific

wavelength of light. The flow cytometer instrument passes the

cells one-by-one through a laser beam of the appropriate wave-

length of light. If the antibodies are bound to the cell, the fluores-

cent tag emits a signal that is detected by the instrument, and the

number of cells and their fluorescence intensity are recorded (see

Figure 64–10B).

A more sophisticated instrument called a fluorescence-

activated cell sorter (FACS) does one additional step. A cell

sorter isolates each cell within an individual fluid droplet before

it passes through the laser beam. Cells that are bound by the

fluorescently tagged antibodies are detected and then quickly

sorted away from other cells in the sample by shunting the drop-

lets into separate sample tubes (see Figure 64–10A).

ANTIGEN–ANTIBODY REACTIONS

INVOLVING RED BLOOD CELL

ANTIGENS

Many different blood group systems exist in humans. Each

system consists of a gene locus specifying antigens on the eryth-

rocyte 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, simi-

larly, people who are either homozygous BB or heterozygous

BO are type B.

What do the A, B, and O types refer to? Erythrocytes

have three terminal sugars in common on their surface:

N-acetylglucosamine, galactose, and fucose. These three sugars

form the H antigen (see 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 an extra sugar, N-acetyl-

galactosamine, added to the galactose of the H antigen, whereas

people who are blood group B have an extra galactose added to

the galactose of the H antigen. Thus, the A and B antigens are

carbohydrates that only differ by a single sugar! Despite this

small difference, A and B antigens are different enough that anti-

bodies that bind one antigen do not “cross-react” with the other.

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 deter-

mined 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 (see 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. In the

United States, the approximate percentage of each blood group

is: type O: 45%, type A: 40%, type B: 11%, and type AB: 4%.

Our plasma contains many antibodies against antigens that

are absent, including the blood antigens (i.e., people with blood

group A have antibodies to B in their plasma). How does this

happen? These antibodies are formed against bacterial polysac-

charides, and they happen to cross-react with A or B polysac-

charides. Anti-A and anti-B antibodies are formed through

T-cell–independent B-cell activation and are therefore primarily

of the IgM class (see Chapter 61). They are first detectable at

3 to 6 months of age.

Why do the individuals with A antigens lack anti-A antibod-

ies and those with B antigens lack anti-B antibodies? During the

development of B-cell precursors in the bone marrow, negative

selection causes any precursor clones with antigen receptors that

strongly recognize “self ” antigens to be deleted by apoptosis

(see Chapter 59). The result is that any potential B-cell clones

that make anti-A immunoglobulins in a person with blood

group A are removed from the eventual pool of mature B cells.

Therefore, individuals will always be tolerant to their own

blood group antigens, and the antigens and their corresponding

antibodies 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 was trans-

fused into a group B person, who will have anti-A antibodies).

The anti-A antibodies bind to the donor red cells forming

red cell–antibody complexes. This activates complement (see

Chapter 63), and a cascading reaction of anaphylactic shock

occurs due to large amounts of C3a and C5a (anaphylatoxins)

and hemolysis caused by C5, C6, C7, C8, and C9 (membrane

attack complex) (Figure 64–12).

To avoid antigen–antibody reactions that would result in

transfusion reactions, all blood for transfusions must be care-

fully 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 does have

A and B antibodies. Therefore, when type O blood is given to

a person with type A, B, or AB blood, you might expect that

the antibodies in the type O serum would cause a reaction.

However, a clinically detectable reaction does not actually occur

because the blood used for transfusions is usually packed red

blood cells, and not whole blood. Packed red blood cell trans-

fusions contain extremely small amounts of donor antibody,

and whatever small amount is present is rapidly diluted below

significant level. Persons with group AB blood have neither A

nor B antibody and thus are universal recipients.

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 jaun-

dice and anemia, but the effects on the fetus are usually less

severe than those seen in Rh incompatibility (see next section).

As described earlier, anti-A and anti-B antibodies are usually

of the IgM class and therefore do not cross the placenta (see

Chapter 61). This is because these antibodies are a response to

bacterial polysaccharides encountered in development and only

happen to cross-react with A and B antigens. However, when a

mother and father have different blood groups, there are rare

occasions when the mother’s adaptive immune system can

become sensitized to the father’s blood group. When this occurs,

IgG antibodies are generated against the A and/or B antigens

absent from the mother. These IgG antibodies can pass through

the placenta and, if the fetus has the father’s blood group, can

cause lysis of fetal red cells.

Rh Blood Type & Hemolytic Disease

of the Newborn

About 85% of humans have erythrocytes that express the Rh(D)

antigen on their surface. They are said to be Rh-positive. The

remaining 15% are Rh-negative, that is, they lack the gene for

the Rh(D) protein.

The Rh status of parents is clinically important because a

specific combination can result in hemolytic disease of the

newborn (erythroblastosis fetalis). When an Rh-negative

woman has an Rh-positive fetus (the D gene being inherited

from the father), the Rh(D) antigen on the fetal red blood cells

will sensitize the mother’s adaptive immune response, leading

to development of anti-Rh(D) IgG antibodies (Table 64–4).

This sensitization occurs most often during delivery of the first

Rh(D)-positive child, when Rh(D) erythrocytes of the fetus leak

into the maternal circulation (Figure 64–13).

If the mother does form anti-Rh(D) antibodies in this

way, subsequent Rh(D) pregnancies are at risk of hemolytic

disease of the newborn (erythroblastosis fetalis). This disease

results from the passage of maternal IgG anti-Rh(D) antibod-

ies 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 if the mother’s adaptive

immune system is not allowed to be sensitized to red cells

carrying Rh(D) antigens. This is achieved 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 any 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.