17IMMUNIZATION AND IMMUNE

TESTING

ImmunizationTwo Artificial Methods of Immunity◦Active immunization◦Administration of antigens so patient actively mounts an adaptive

immune response

◦Passive immunization◦Individual acquires immunity through the transfer of antibodies formed

by immune individual or animal

ImmunizationBrief History of Immunization◦Chinese noticed children who recovered from smallpox

did not contract the disease again◦They infected children with material from a

smallpox scab to induce immunity◦This process known as variolation

◦Variolation spread to England and America but was stopped due to risk of death

ImmunizationBrief History of Immunization◦1796 – Edward Jenner discovered process of

vaccination◦1879 – Louis Pasteur developed a vaccine against

Pasteurella multocida◦Antibody transfer developed when it was discovered

vaccines protected through the action of antibodies

ImmunizationBrief History of Immunization◦Many developing nations do

not receive vaccines◦Effective vaccines not

developed for some pathogens

◦Vaccine-associated risks discourage investment in developing new vaccines

Figure 17.1 The effect of immunization in reducing the prevalence of two infectious diseases in the United States.

ImmunizationActive Immunization◦Vaccine types - Attenuated (modified live) vaccines

◦Use pathogens with reduced virulence◦Process of reducing virulence called attenuation◦Can result in mild infections◦Active microbes stimulate a strong immune response◦Can provide contact immunity◦Modified microbes may retain enough residual virulence to cause

disease in susceptible individuals

ImmunizationActive Immunization◦Vaccine types - Inactivated (killed) vaccines

◦Safer than live vaccines◦Whole agent vaccines

◦Deactivated but whole microbes◦Subunit vaccines

◦Antigenic fragments of microbes◦Often require multiple doses to achieve full immunity◦Often contain adjuvants

◦Chemicals added to increase effective antigenicity

ImmunizationActive Immunization◦Vaccine types - Toxoid vaccines

◦Chemically or thermally modified toxins used to stimulate active immunity

◦Useful for some bacterial diseases◦Stimulate antibody-mediated immunity◦Require multiple doses because toxoids possess few antigenic

determinants

Vaccines: Types

Vaccines: Types

ImmunizationActive Immunization◦Vaccine types - Combination vaccines

◦Simultaneous administration of antigens from several pathogens

◦Vaccines using recombinant gene technology

◦Research attempts to make vaccines more effective, cheaper, and safer

◦Recombinant DNA techniques used to improve vaccines

ImmunizationActive Immunization◦Vaccine manufacture

◦Mass-produce many vaccines by growing microbes in culture vessels

◦Viruses are cultured inside chicken eggs◦Individuals with egg allergies must avoid some

vaccines

Figure 17.3 The CDC’s recommended immunization schedule for the general population.

ImmunizationActive Immunization

◦Vaccine safety◦Problems associated with immunization

◦Mild toxicity

◦ Risk of anaphylactic shock

◦ Residual virulence from attenuated viruses

◦ Allegations certain vaccines cause autism, diabetes, and asthma

◦Research has not substantiated these allegations

ImmunizationPassive Immunotherapy◦Administration of antiserum that contains preformed

antibodies◦Provides immediate protection against a recent infection or

ongoing disease◦Antisera have several limitations

◦Can trigger allergic reactions called serum sickness◦Antibodies of antisera are degraded relatively quickly◦Individual not protected from subsequent infections

◦Limitations are overcome through development of hybridomas

The production

of hybridomas

.

Figure 17.5 The characteristics of immunity produced by active immunization (red) and passive immunotherapy (green).

Passiveimmunotherapy

Injection

Boosters Active immunization

Initial inoculation

Serological Tests That Use Antigens and Corresponding Antibodies Serology is the determination of the presence of

specific antigens or antibodies in blood serum Serological tests available to identify a variety of

antigens and antibodies in serum

Serological tests have several uses Monitor the spread of infection within a population Establish diagnosis of disease

Serological Tests That Use Antigens and Corresponding AntibodiesPrecipitation Tests◦One of the simplest of serological tests◦Antigens and antibody mixed in the proper proportion form large complexes

called precipitates◦Antigen-antibody complexes also called immune complexes

No precipitate Precipitate No precipitate

Antibody

Antigen

Amou

nt o

f anti

body

prec

ipita

ted

Increasing amount of antigen

Antibody excess

Optimal proportions

Antigen excess

Figure 17.6 Characteristics of precipitation reactions.

Well containing antibodies against the antigen

Agar

Zone of antigen excess

Zone of optimal precipitation

Zone of antibody excess

Well containing four different antigens

Lines of immune precipitation Well containing a mixture of antibodies, each reacting to a different antigen

Figure 17.7 Immunodiffusion, a type of precipitation reaction.Well containing Line of immune precipitation antigen molecules

Serological Tests That Use Antigens and Corresponding AntibodiesTurbidimetric and Nephelometric Tests◦Turbidimetry and nephelometry measure the cloudiness of a solution◦Turbidimetry measures the light passing through a solution◦Nephelometry measures the light reflected from a solution◦Can be used to quantify the amounts of proteins in serum

Serological Tests That Use Antigens and Corresponding AntibodiesAgglutination Tests◦Agglutination occurs due to the cross-linking of antibodies with

particulate antigens◦Agglutination is the clumping of insoluble particles◦Precipitation involves the aggregation of soluble molecules

◦Reactions can be easy to see and interpret with the unaided eye

◦Hemagglutination◦Agglutination of red blood cells◦Can be used to determine blood type

Negative result: no agglutination of blood cells

Positive result: agglutination of blood cells

A B

Figure 17.8 The use of hemagglutination to determine blood types in humans.Anti-A antibody addedAnti-B antibody added

Blood sample

A

B

Serum added in increasing dilutions

1:1 1:10 1:100 1:1000 1:10,000Control (no specimen added)

Antigen (identical in each well)

Very strong agglutination

No agglutination

Control

Figure 17.9 Titration, the use of agglutination to quantify the amount of antibody in a serumsample.

Serological Tests That Use Antigens and Corresponding Antibodies◦Viral Neutralization

◦Cytopathic effect◦Viruses introduced into appropriate cell cultures will kill the cells

◦Ability of virus to kill culture cells is neutralized when virus is first mixed withantibodies against it

◦Viral neutralization test◦Mixture of virus and serum added to cell culture◦Absence of cytopathic effect indicates presence of antibodies against the

virus in the serum◦Identifies whether individual exposed to a particular virus

Serological Tests That Use Antigens and Corresponding Antibodies

Neutralization Tests◦Viral hemagglutination inhibition test◦Useful for viruses that aren't cytopathic◦Based on viral hemagglutination◦Some viral surface proteins can clump red blood cells

◦A serum sample that contains antibodies against a specific virus will inhibit viral hemagglutination

◦Commonly used to detect antibodies against influenza, measles, and mumps

Serological Tests That Use Antigens and Corresponding AntibodiesThe Complement Fixation Test◦Based on generation of membrane attack complexes

during complement activation◦Used to detect the presence of specific antibodies in

an individual's serum◦Can detect antibody amounts too small to detect by

agglutination◦Replaced by other serological methods

Serological Tests That Use Antigens and Corresponding AntibodiesLabeled Antibody Test◦Uses antibody molecules linked to some "label" that

enables them to be easily detected◦Used to detect either antigens or antibodies

Serological Tests That Use Antigens and Corresponding AntibodiesLabeled Antibody Test◦Fluorescent antibody tests◦Use fluorescent dyes as labels◦Fluorescently labeled antibodies used in two types of tests◦Direct fluorescent antibody tests◦Indirect fluorescent antibody tests

Figure 17.10 A direct fluorescent antibody test.

Figure 17.11 The indirect fluorescent antibody test.

Serological Tests That Use Antigens and Corresponding AntibodiesLabeled Antibody Test◦ELISAs

◦Stands for enzyme-linked immunosorbent assay◦Uses an enzyme as the label

◦Reaction of enzyme with its substrate produces a colored product indicating a positive test

◦Commonly used to detect the presence of serum antibodies

Enzyme

Anti-antibody

Substrate Colored product

4 Enzyme-linked anti-antibody is added and binds to bound antibody.

5 Enzyme’s substrate is added, and reaction produces a visible color change.

3 Patient serum is added; complementary antibody binds to antigen.

2 A protein such as gelatin is added to block the uncoated surface.

1 Antigen is attached to well in plate.

Figure 17.12 The enzyme-linked immunosorbent assay (ELISA).

Serological Tests That Use Antigens and Corresponding Antibodies

Labeled Antibody Test◦ELISAs

◦Advantages of the ELISA◦Can detect either antibody or antigen◦Sensitive◦Can quantify amounts of antigen or antibody◦Easy to perform and can test many samples quickly◦Relatively inexpensive and easy to automate◦Plates coated with antigen can be stored for later testing

Serological Tests That Use Antigens and Corresponding AntibodiesLabeled Antibody Test◦ELISAs◦Antibody sandwich ELISA◦Modification of the ELISA technique◦Commonly used to detect antigen◦Antigen being tested for is "sandwiched" between

two antibody molecules

Figure 17.13 An antibody sandwich ELISA.

Gelatin

SubstrateAntigen inpatient’s serum

Antibody bound to microwell

Enyme-linked antibody

Colored product

Serological Tests That Use Antigens and Corresponding Antibodies

Labeled Antibody Test◦Immunoblots

◦Also called a western blot◦Technique to detect antibodies against multiple antigens◦Used to confirm the presence of proteins

Filter paper

Polyacrylamide gelAbsorbant paper

Patient 1

2

3

4

5

6

Positive controlNegative control

Polypeptides

Figure 17.14 Immunoblotting.Wells containing antigens

Polyacrylamide gel

1 Electrophoresis (not shown)

Polyacrylamidegel2 Blotting

Nitrocellulose membrane

Nitrocellulose (blot) is cut into strips

3 ELISA is performed on strips

Serological Tests That Use Antigens and Corresponding AntibodiesPoint-of-Care Testing◦Simple immunoassays that give results in minutes◦Useful in determining a quick diagnosis◦Common tests

◦ Immunofiltration assay◦ Immunochromatography assay

Serological Tests That Use Antigens and Corresponding AntibodiesPoint-of-Care Testing◦Immunofiltration

◦Rapid ELISA that uses antibodies bound to membrane

filters rather than plates

◦Reduced time to complete the assay

◦Due to the large surface area of the membrane filter

Serological Tests That Use Antigens and Corresponding AntibodiesRecent Developments in Immune Testing

◦Immunochromatography◦Very rapid and easy-to-read ELISAs◦Antigen solution flows through a porous strip

◦Encounters labeled antibody◦Visible line produced when antigen-antibody immune complexes

encounter antibody against them◦Used in pregnancy testing and for rapid identification of some infections

Line of fixedanti-antibody

Anti-antibodies stop movement of antibody- antigen complexes. Color becomes visible because of density of complexes.

Movement of fluid containing complexes of antibodies bound to antigen

Prepared antigenextract from patient’snasal sample

Figure 17.15 Immunochromatographic dipstick.

Zone of antibodies linked to colloidal metal, color too diffuse to see