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Microbiology

20.3 Agglutination Assays

Microbiology 20.3 Agglutination Assays
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  1. Preface
  2. 1 An Invisible World
    1. Introduction
    2. 1.1 What Our Ancestors Knew
    3. 1.2 A Systematic Approach
    4. 1.3 Types of Microorganisms
    5. Summary
    6. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  3. 2 How We See the Invisible World
    1. Introduction
    2. 2.1 The Properties of Light
    3. 2.2 Peering Into the Invisible World
    4. 2.3 Instruments of Microscopy
    5. 2.4 Staining Microscopic Specimens
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  4. 3 The Cell
    1. Introduction
    2. 3.1 Spontaneous Generation
    3. 3.2 Foundations of Modern Cell Theory
    4. 3.3 Unique Characteristics of Prokaryotic Cells
    5. 3.4 Unique Characteristics of Eukaryotic Cells
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. True/False
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  5. 4 Prokaryotic Diversity
    1. Introduction
    2. 4.1 Prokaryote Habitats, Relationships, and Microbiomes
    3. 4.2 Proteobacteria
    4. 4.3 Nonproteobacteria Gram-Negative Bacteria and Phototrophic Bacteria
    5. 4.4 Gram-Positive Bacteria
    6. 4.5 Deeply Branching Bacteria
    7. 4.6 Archaea
    8. Summary
    9. Review Questions
      1. Multiple Choice
      2. True/False
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  6. 5 The Eukaryotes of Microbiology
    1. Introduction
    2. 5.1 Unicellular Eukaryotic Parasites
    3. 5.2 Parasitic Helminths
    4. 5.3 Fungi
    5. 5.4 Algae
    6. 5.5 Lichens
    7. Summary
    8. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  7. 6 Acellular Pathogens
    1. Introduction
    2. 6.1 Viruses
    3. 6.2 The Viral Life Cycle
    4. 6.3 Isolation, Culture, and Identification of Viruses
    5. 6.4 Viroids, Virusoids, and Prions
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. True/False
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  8. 7 Microbial Biochemistry
    1. Introduction
    2. 7.1 Organic Molecules
    3. 7.2 Carbohydrates
    4. 7.3 Lipids
    5. 7.4 Proteins
    6. 7.5 Using Biochemistry to Identify Microorganisms
    7. Summary
    8. Review Questions
      1. Multiple Choice
      2. True/False
      3. Matching
      4. Fill in the Blank
      5. Short Answer
      6. Critical Thinking
  9. 8 Microbial Metabolism
    1. Introduction
    2. 8.1 Energy, Matter, and Enzymes
    3. 8.2 Catabolism of Carbohydrates
    4. 8.3 Cellular Respiration
    5. 8.4 Fermentation
    6. 8.5 Catabolism of Lipids and Proteins
    7. 8.6 Photosynthesis
    8. 8.7 Biogeochemical Cycles
    9. Summary
    10. Review Questions
      1. Multiple Choice
      2. True/False
      3. Matching
      4. Fill in the Blank
      5. Short Answer
      6. Critical Thinking
  10. 9 Microbial Growth
    1. Introduction
    2. 9.1 How Microbes Grow
    3. 9.2 Oxygen Requirements for Microbial Growth
    4. 9.3 The Effects of pH on Microbial Growth
    5. 9.4 Temperature and Microbial Growth
    6. 9.5 Other Environmental Conditions that Affect Growth
    7. 9.6 Media Used for Bacterial Growth
    8. Summary
    9. Review Questions
      1. Multiple Choice
      2. Matching
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  11. 10 Biochemistry of the Genome
    1. Introduction
    2. 10.1 Using Microbiology to Discover the Secrets of Life
    3. 10.2 Structure and Function of DNA
    4. 10.3 Structure and Function of RNA
    5. 10.4 Structure and Function of Cellular Genomes
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. True/False
      3. Matching
      4. Fill in the Blank
      5. Short Answer
      6. Critical Thinking
  12. 11 Mechanisms of Microbial Genetics
    1. Introduction
    2. 11.1 The Functions of Genetic Material
    3. 11.2 DNA Replication
    4. 11.3 RNA Transcription
    5. 11.4 Protein Synthesis (Translation)
    6. 11.5 Mutations
    7. 11.6 How Asexual Prokaryotes Achieve Genetic Diversity
    8. 11.7 Gene Regulation: Operon Theory
    9. Summary
    10. Review Questions
      1. Multiple Choice
      2. True/False
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  13. 12 Modern Applications of Microbial Genetics
    1. Introduction
    2. 12.1 Microbes and the Tools of Genetic Engineering
    3. 12.2 Visualizing and Characterizing DNA, RNA, and Protein
    4. 12.3 Whole Genome Methods and Pharmaceutical Applications of Genetic Engineering
    5. 12.4 Gene Therapy
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. True/False
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  14. 13 Control of Microbial Growth
    1. Introduction
    2. 13.1 Controlling Microbial Growth
    3. 13.2 Using Physical Methods to Control Microorganisms
    4. 13.3 Using Chemicals to Control Microorganisms
    5. 13.4 Testing the Effectiveness of Antiseptics and Disinfectants
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. True/False
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  15. 14 Antimicrobial Drugs
    1. Introduction
    2. 14.1 History of Chemotherapy and Antimicrobial Discovery
    3. 14.2 Fundamentals of Antimicrobial Chemotherapy
    4. 14.3 Mechanisms of Antibacterial Drugs
    5. 14.4 Mechanisms of Other Antimicrobial Drugs
    6. 14.5 Drug Resistance
    7. 14.6 Testing the Effectiveness of Antimicrobials
    8. 14.7 Current Strategies for Antimicrobial Discovery
    9. Summary
    10. Review Questions
      1. Multiple Choice
      2. True/False
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  16. 15 Microbial Mechanisms of Pathogenicity
    1. Introduction
    2. 15.1 Characteristics of Infectious Disease
    3. 15.2 How Pathogens Cause Disease
    4. 15.3 Virulence Factors of Bacterial and Viral Pathogens
    5. 15.4 Virulence Factors of Eukaryotic Pathogens
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  17. 16 Disease and Epidemiology
    1. Introduction
    2. 16.1 The Language of Epidemiologists
    3. 16.2 Tracking Infectious Diseases
    4. 16.3 Modes of Disease Transmission
    5. 16.4 Global Public Health
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. Matching
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  18. 17 Innate Nonspecific Host Defenses
    1. Introduction
    2. 17.1 Physical Defenses
    3. 17.2 Chemical Defenses
    4. 17.3 Cellular Defenses
    5. 17.4 Pathogen Recognition and Phagocytosis
    6. 17.5 Inflammation and Fever
    7. Summary
    8. Review Questions
      1. Multiple Choice
      2. Matching
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  19. 18 Adaptive Specific Host Defenses
    1. Introduction
    2. 18.1 Overview of Specific Adaptive Immunity
    3. 18.2 Major Histocompatibility Complexes and Antigen-Presenting Cells
    4. 18.3 T Lymphocytes and Cellular Immunity
    5. 18.4 B Lymphocytes and Humoral Immunity
    6. 18.5 Vaccines
    7. Summary
    8. Review Questions
      1. Multiple Choice
      2. Matching
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  20. 19 Diseases of the Immune System
    1. Introduction
    2. 19.1 Hypersensitivities
    3. 19.2 Autoimmune Disorders
    4. 19.3 Organ Transplantation and Rejection
    5. 19.4 Immunodeficiency
    6. 19.5 Cancer Immunobiology and Immunotherapy
    7. Summary
    8. Review Questions
      1. Multiple Choice
      2. Matching
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  21. 20 Laboratory Analysis of the Immune Response
    1. Introduction
    2. 20.1 Polyclonal and Monoclonal Antibody Production
    3. 20.2 Detecting Antigen-Antibody Complexes
    4. 20.3 Agglutination Assays
    5. 20.4 EIAs and ELISAs
    6. 20.5 Fluorescent Antibody Techniques
    7. Summary
    8. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  22. 21 Skin and Eye Infections
    1. Introduction
    2. 21.1 Anatomy and Normal Microbiota of the Skin and Eyes
    3. 21.2 Bacterial Infections of the Skin and Eyes
    4. 21.3 Viral Infections of the Skin and Eyes
    5. 21.4 Mycoses of the Skin
    6. 21.5 Protozoan and Helminthic Infections of the Skin and Eyes
    7. Summary
    8. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  23. 22 Respiratory System Infections
    1. Introduction
    2. 22.1 Anatomy and Normal Microbiota of the Respiratory Tract
    3. 22.2 Bacterial Infections of the Respiratory Tract
    4. 22.3 Viral Infections of the Respiratory Tract
    5. 22.4 Respiratory Mycoses
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  24. 23 Urogenital System Infections
    1. Introduction
    2. 23.1 Anatomy and Normal Microbiota of the Urogenital Tract
    3. 23.2 Bacterial Infections of the Urinary System
    4. 23.3 Bacterial Infections of the Reproductive System
    5. 23.4 Viral Infections of the Reproductive System
    6. 23.5 Fungal Infections of the Reproductive System
    7. 23.6 Protozoan Infections of the Urogenital System
    8. Summary
    9. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  25. 24 Digestive System Infections
    1. Introduction
    2. 24.1 Anatomy and Normal Microbiota of the Digestive System
    3. 24.2 Microbial Diseases of the Mouth and Oral Cavity
    4. 24.3 Bacterial Infections of the Gastrointestinal Tract
    5. 24.4 Viral Infections of the Gastrointestinal Tract
    6. 24.5 Protozoan Infections of the Gastrointestinal Tract
    7. 24.6 Helminthic Infections of the Gastrointestinal Tract
    8. Summary
    9. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  26. 25 Circulatory and Lymphatic System Infections
    1. Introduction
    2. 25.1 Anatomy of the Circulatory and Lymphatic Systems
    3. 25.2 Bacterial Infections of the Circulatory and Lymphatic Systems
    4. 25.3 Viral Infections of the Circulatory and Lymphatic Systems
    5. 25.4 Parasitic Infections of the Circulatory and Lymphatic Systems
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  27. 26 Nervous System Infections
    1. Introduction
    2. 26.1 Anatomy of the Nervous System
    3. 26.2 Bacterial Diseases of the Nervous System
    4. 26.3 Acellular Diseases of the Nervous System
    5. 26.4 Fungal and Parasitic Diseases of the Nervous System
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. Matching
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  28. A | Fundamentals of Physics and Chemistry Important to Microbiology
  29. B | Mathematical Basics
  30. C | Metabolic Pathways
  31. D | Taxonomy of Clinically Relevant Microorganisms
  32. E | Glossary
  33. Answer Key
    1. Chapter 1
    2. Chapter 2
    3. Chapter 3
    4. Chapter 4
    5. Chapter 5
    6. Chapter 6
    7. Chapter 7
    8. Chapter 8
    9. Chapter 9
    10. Chapter 10
    11. Chapter 11
    12. Chapter 12
    13. Chapter 13
    14. Chapter 14
    15. Chapter 15
    16. Chapter 16
    17. Chapter 17
    18. Chapter 18
    19. Chapter 19
    20. Chapter 20
    21. Chapter 21
    22. Chapter 22
    23. Chapter 23
    24. Chapter 24
    25. Chapter 25
    26. Chapter 26
  34. Index

Learning Objectives

  • Compare direct and indirect agglutination
  • Identify various uses of hemagglutination in the diagnosis of disease
  • Explain how blood types are determined
  • Explain the steps used to cross-match blood to be used in a transfusion

In addition to causing precipitation of soluble molecules and flocculation of molecules in suspension, antibodies can also clump together cells or particles (e.g., antigen-coated latex beads) in a process called agglutination (Figure 18.9). Agglutination can be used as an indicator of the presence of antibodies against bacteria or red blood cells. Agglutination assays are usually quick and easy to perform on a glass slide or microtiter plate (Figure 20.15). Microtiter plates have an array of wells to hold small volumes of reagents and to observe reactions (e.g., agglutination) either visually or using a specially designed spectrophotometer. The wells come in many different sizes for assays involving different volumes of reagents.

A plastic plate about the size of a hand with wells in a grid. The top is labeled 1-12 and the side is labeled A-H.
Figure 20.15 Microtiter plates are used for conducting numerous reactions simultaneously in an array of wells. (credit: modification of work by “Microrao”/Wikimedia)

Agglutination of Bacteria and Viruses

The use of agglutination tests to identify streptococcal bacteria was developed in the 1920s by Rebecca Lancefield working with her colleagues A.R. Dochez and Oswald Avery.9 She used antibodies to identify M protein, a virulence factor on streptococci that is necessary for the bacteria’s ability to cause strep throat. Production of antibodies against M protein is crucial in mounting a protective response against the bacteria.

Lancefield used antisera to show that different strains of the same species of streptococci express different versions of M protein, which explains why children can come down with strep throat repeatedly. Lancefield classified beta-hemolytic streptococci into many groups based on antigenic differences in group-specific polysaccharides located in the bacterial cell wall. The strains are called serovars because they are differentiated using antisera. Identifying the serovars present in a disease outbreak is important because some serovars may cause more severe disease than others.

The method developed by Lancefield is a direct agglutination assay, since the bacterial cells themselves agglutinate. A similar strategy is more commonly used today when identifying serovars of bacteria and viruses; however, to improve visualization of the agglutination, the antibodies may be attached to inert latex beads. This technique is called an indirect agglutination assay (or latex fixation assay), because the agglutination of the beads is a marker for antibody binding to some other antigen (Figure 20.16). Indirect assays can be used to detect the presence of either antibodies or specific antigens.

Photo of 6 wells. Well 4 has blue spots. All the other wells have a clear blue color.
Figure 20.16 Antibodies against six different serovars of Group A strep were attached to latex beads. Each of the six antibody preparations was mixed with bacteria isolated from a patient. The tiny clumps seen in well 4 are indicative of agglutination, which is absent from all other wells. This indicates that the serovar associated with well 4 is present in the patient sample. (credit: modification of work by American Society for Microbiology)

To identify antibodies in a patient’s serum, the antigen of interest is attached to latex beads. When mixed with patient serum, the antibodies will bind the antigen, cross-linking the latex beads and causing the beads to agglutinate indirectly; this indicates the presence of the antibody (Figure 20.17). This technique is most often used when looking for IgM antibodies, because their structure provides maximum cross-linking. One widely used example of this assay is a test for rheumatoid factor (RF) to confirm a diagnosis of rheumatoid arthritis. RF is, in fact, the presence of IgM antibodies that bind to the patient’s own IgG. RF will agglutinate IgG-coated latex beads.

In the reverse test, soluble antigens can be detected in a patient’s serum by attaching specific antibodies (commonly mAbs) to the latex beads and mixing this complex with the serum (Figure 20.17).

Agglutination tests are widely used in underdeveloped countries that may lack appropriate facilities for culturing bacteria. For example, the Widal test, used for the diagnosis of typhoid fever, looks for agglutination of Salmonella enterica subspecies typhi in patient sera. The Widal test is rapid, inexpensive, and useful for monitoring the extent of an outbreak; however, it is not as accurate as tests that involve culturing of the bacteria. The Widal test frequently produces false positives in patients with previous infections with other subspecies of Salmonella, as well as false negatives in patients with hyperproteinemia or immune deficiencies.

In addition, agglutination tests are limited by the fact that patients generally do not produce detectable levels of antibody during the first week (or longer) of an infection. A patient is said to have undergone seroconversion when antibody levels reach the threshold for detection. Typically, seroconversion coincides with the onset of signs and symptoms of disease. However, in an HIV infection, for example, it generally takes 3 weeks for seroconversion to take place, and in some instances, it may take much longer.

Similar to techniques for the precipitin ring test and plaque assays, it is routine to prepare serial two-fold dilutions of the patient’s serum and determine the titer of agglutinating antibody present. Since antibody levels change over time in both primary and secondary immune responses, by checking samples over time, changes in antibody titer can be detected. For example, a comparison of the titer during the acute phase of an infection versus the titer from the convalescent phase will distinguish whether an infection is current or has occurred in the past. It is also possible to monitor how well the patient’s immune system is responding to the pathogen.

(a) A diagram of positive agglutination test for antibodies. Large circles (latex beads) have smaller circles (antigens) on their surface. IgM antibodies (6 Y shapes attached at their base) are bound to the antigens. B) A diagram of positive agglutination test for antigens. The latex beads have antigens on them. Smaller blue ovals (pathogens) have circles (antigens) on their surface. The antigens bind to the antibodies.
Figure 20.17 (a) Latex beads coated with an antigen will agglutinate when mixed with patient serum if the serum contains IgM antibodies against the antigen. (b) Latex beads coated with antibodies will agglutinate when mixed with patient serum if the serum contains antigens specific to the antibodies.

Check Your Understanding

  • How is agglutination used to distinguish serovars from each other?
  • In a latex bead assay to test for antibodies in a patient's serum, with what are the beads coated?
  • What has happened when a patient has undergone seroconversion?

Hemagglutination

Agglutination of red blood cells is called hemagglutination. One common assay that uses hemagglutination is the direct Coombs’ test, also called the direct antihuman globulin test (DAT), which generally looks for nonagglutinating antibodies. The test can also detect complement attached to red blood cells.

The Coombs’ test is often employed when a newborn has jaundice, yellowing of the skin caused by high blood concentrations of bilirubin, a product of the breakdown of hemoglobin in the blood. The Coombs’ test is used to determine whether the child’s red blood cells have been bound by the mother’s antibodies. These antibodies would activate complement, leading to red blood cell lysis and the subsequent jaundice. Other conditions that can cause positive direct Coombs’ tests include hemolytic transfusion reactions, autoimmune hemolytic anemia, infectious mononucleosis (caused by Epstein-Barr virus), syphilis, and Mycoplasma pneumonia. A positive direct Coombs’ test may also be seen in some cancers and as an allergic reaction to some drugs (e.g., penicillin).

The antibodies bound to red blood cells in these conditions are most often IgG, and because of the orientation of the antigen-binding sites on IgG and the comparatively large size of a red blood cell, it is unlikely that any visible agglutination will occur. However, the presence of IgG bound to red blood cells can be detected by adding Coombs’ reagent, an antiserum containing antihuman IgG antibodies (that may be combined with anti-complement) (Figure 20.18). The Coombs’ reagent links the IgG attached to neighboring red blood cells and thus promotes agglutination.

There is also an indirect Coombs’ test known as the indirect antiglobulin test (IAT). This screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient’s serum (Figure 20.18). IAT can be used to screen pregnant women for antibodies that may cause hemolytic disease of the newborn. It can also be used prior to giving blood transfusions. More detail on how the IAT is performed is discussed below.

A) Diagram of direct Coomb’s test. 1.  Blood sample from a patient with hemolytic anemia with antibodies attached to red blood cells. 2. Antibodies coat the patient’s red blood cells. 3. A few drops of Coomb’s reagent (containing anti-human antibodies) is mixed in with the patient’s blood sample. 4. Agglutination reaction (clumping) is visible after cross-linkage of antibodies. B) Diagram of indirect Coomb’s test. 1. Patient's serum containing antibodies is drawn. 2. Donor blood is added. 3. Patient's antibodies bind to donor's red blood cells. 4. A few drops of Coombs’ reagent are mixed with the sample. 5. Agglutination reaction is observed.
Figure 20.18 The steps in direct and indirect Coombs’ tests are shown in the illustration.

Antibodies that bind to red blood cells are not the only cause of hemagglutination. Some viruses also bind to red blood cells, and this binding can cause agglutination when the viruses cross-link the red blood cells. For example, influenza viruses have two different types of viral spikes called neuraminidase (N) and hemagglutinin (H), the latter named for its ability to agglutinate red blood cells (see Viruses). Thus, we can use red blood cells to detect the presence of influenza virus by direct hemagglutination assays (HA), in which the virus causes visible agglutination of red blood cells. The mumps and rubella viruses can also be detected using HA.

Most frequently, a serial dilution viral agglutination assay is used to measure the titer or estimate the amount of virus produced in cell culture or for vaccine production. A viral titer can be determined using a direct HA by making a serial dilution of the sample containing the virus, starting with a high concentration of sample that is then diluted in a series of wells. The highest dilution producing visible agglutination is the titer. The assay is carried out in a microtiter plate with V- or round-bottomed wells. In the presence of agglutinating viruses, the red blood cells and virus clump together and produce a diffuse mat over the bottom of the well. In the absence of virus, the red blood cells roll or sediment to the bottom of the well and form a dense pellet, which is why flat-bottomed wells cannot be used (Figure 20.19).

A modification of the HA assay can be used to determine the titer of antiviral antibodies. The presence of these antibodies in a patient’s serum or in a lab-produced antiserum will neutralize the virus and block it from agglutinating the red cells, making this a viral hemagglutination inhibition assay (HIA). In this assay, patient serum is mixed with a standardized amount of virus. After a short incubation, a standardized amount of red blood cells is added and hemagglutination is observed. The titer of the patient’s serum is the highest dilution that blocks agglutination (Figure 20.20).

A photo of nonagglutination shows a red stripe. A diagram shows that without virus the cells from a compact pellet at the bottom of the well. A photo of agglutination shows a red spot and pink surrounding area. A diagram shows that the red blood cells and viruses form a clump.
Figure 20.19 A viral suspension is mixed with a standardized amount of red blood cells. No agglutination of red blood cells is visible when the virus is absent, and the cells form a compact pellet at the bottom of the well. In the presence of virus, a diffuse pink precipitate forms in the well. (credit bottom: modification of work by American Society for Microbiology)
A drawing of a well plate. The label at the top reads: Dilution. The rows are labeled: 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128. The columns are labeled: Sample A, titer = 128 (this spans columns 1 and 2). Sample B, no neutralizing antibody (spans columns 3 and 4). Sample C, titer = 64 spans columns 5 and 6.). Columns 1 and 2 have red dots in all rows but the bottom one. Columns 3 and 4 have no red dots. Columns 5 and 6 have red dots in all rows but the bottom two.
Figure 20.20 In this HIA, serum containing antibodies to influenzavirus underwent serial two-fold dilutions in a microtiter plate. Red blood cells were then added to the wells. Agglutination only occurred in those wells where the antibodies were too dilute to neutralize the virus. The highest concentration at which agglutination occurs is the titer of the antibodies in the patient’s serum. In the case of this test, Sample A shows a titer of 128, and Sample C shows a titer of 64. (credit: modification of work by Evan Burkala)

Check Your Understanding

  • What is the mechanism by which viruses are detected in a hemagglutination assay?
  • Which hemagglutination result tells us the titer of virus in a sample?

Eye on Ethics

Animals in the Laboratory

Much of what we know today about the human immune system has been learned through research conducted using animals—primarily, mammals—as models. Besides research, mammals are also used for the production of most of the antibodies and other immune system components needed for immunodiagnostics. Vaccines, diagnostics, therapies, and translational medicine in general have all been developed through research with animal models.

Consider some of the common uses of laboratory animals for producing immune system components. Guinea pigs are used as a source of complement, and mice are the primary source of cells for making mAbs. These mAbs can be used in research and for therapeutic purposes. Antisera are raised in a variety of species, including horses, sheep, goats, and rabbits. When producing an antiserum, the animal will usually be injected at least twice, and adjuvants may be used to boost the antibody response. The larger animals used for making antisera will have blood harvested repeatedly over long periods of time, with little harm to the animals, but that is not usually the case for rabbits. Although we can obtain a few milliliters of blood from the ear veins of rabbits, we usually need larger volumes, which results in the deaths of the animals.

We also use animals for the study of disease. The only way to grow Treponema pallidum for the study of syphilis is in living animals. Many viruses can be grown in cell culture, but growth in cell culture tells us very little about how the immune system will respond to the virus. When working on a newly discovered disease, we still employ Koch’s postulates, which require causing disease in lab animals using pathogens from pure culture as a crucial step in proving that a particular microorganism is the cause of a disease. Studying the proliferation of bacteria and viruses in animal hosts, and how the host immune system responds, has been central to microbiological research for well over 100 years.

While the practice of using laboratory animals is essential to scientific research and medical diagnostics, many people strongly object to the exploitation of animals for human benefit. This ethical argument is not a new one—indeed, one of Charles Darwin's daughters was an active antivivisectionist (vivisection is the practice of cutting or dissecting a live animal to study it). Most scientists acknowledge that there should be limits on the extent to which animals can be exploited for research purposes. Ethical considerations have led the National Institutes of Health (NIH) to develop strict regulations on the types of research that may be performed. These regulations also include guidelines for the humane treatment of lab animals, setting standards for their housing, care, and euthanization. The NIH document “Guide for the Care and Use of Laboratory Animals” makes it clear that the use of animals in research is a privilege granted by society to researchers.

The NIH guidelines are based on the principle of the three R’s: replace, refine, and reduce. Researchers should strive to replace animal models with nonliving models, replace vertebrates with invertebrates whenever possible, or use computer-models when applicable. They should refine husbandry and experimental procedures to reduce pain and suffering, and use experimental designs and procedures that reduce the number of animals needed to obtain the desired information. To obtain funding, researchers must satisfy NIH reviewers that the research justifies the use of animals and that their use is in accordance with the guidelines.

At the local level, any facility that uses animals and receives federal funding must have an Institutional Animal Care and Use Committee (IACUC) that ensures that the NIH guidelines are being followed. The IACUC must include researchers, administrators, a veterinarian, and at least one person with no ties to the institution, that is, a concerned citizen. This committee also performs inspections of laboratories and protocols. For research involving human subjects, an Institutional Review Board (IRB) ensures that proper guidelines are followed.

Blood Typing and Cross-Matching

In addition to antibodies against bacteria and viruses to which they have previously been exposed, most individuals also carry antibodies against blood types other than their own. There are presently 33 immunologically important blood-type systems, many of which are restricted within various ethnic groups or rarely result in the production of antibodies. The most important and perhaps best known are the ABO and Rh blood groups (see Figure 19.4).

When units of blood are being considered for transfusion, pretransfusion blood testing must be performed. For the blood unit, commercially prepared antibodies against the A, B, and Rh antigens are mixed with red blood cells from the units to initially confirm that the blood type on the unit is accurate. Once a unit of blood has been requested for transfusion, it is vitally important to make sure the donor (unit of blood) and recipient (patient) are compatible for these crucial antigens. In addition to confirming the blood type of the unit, the patient’s blood type is also confirmed using the same commercially prepared antibodies to A, B, and Rh. For example, as shown in Figure 20.21, if the donor blood is A-positive, it will agglutinate with the anti-A antiserum and with the anti-Rh antiserum. If no agglutination is observed with any of the sera, then the blood type would be O-negative.

Following determination of the blood type, immediately prior to releasing the blood for transfusion, a cross-match is performed in which a small aliquot of the donor red blood cells are mixed with serum from the patient awaiting transfusion. If the patient does have antibodies against the donor red blood cells, hemagglutination will occur. To confirm any negative test results and check for sensitized red blood cells, Coombs’ reagent may be added to the mix to facilitate visualization of the antibody-red blood cell interaction.

Under some circumstances, a minor cross-match may be performed as well. In this assay, a small aliquot of donor serum is mixed with patient red blood cells. This allows the detection of agglutinizing antibodies in the donor serum. This test is rarely necessary because transfusions generally use packed red blood cells with most of the plasma removed by centrifugation.

Red blood cells have many other antigens in addition to ABO and Rh. While most people are unlikely to have antibodies against these antigens, women who have had multiple pregnancies or patients who have had multiple transfusions may have them because of repeated exposure. For this reason, an antibody screen test is used to determine if such antibodies are present. Patient serum is checked against commercially prepared, pooled, type O red blood cells that express these antigens. If agglutination occurs, the antigen to which the patient is responding must be identified and determined not to be present in the donor unit.

Three wells the first well (labeled anti-A) shows black spots and is labeled clumping. This is due to the agglutinated RBCs. The second well (labeled anti-B) looks smooth and has no clumping. The third well (labeled) anti-Rh shows clumping because it has spots.
Figure 20.21 This sample of a commercially produced “bedside” card enables quick typing of both a recipient’s and donor’s blood before transfusion. The card contains three reaction sites or wells. One is coated with an anti-A antibody, one with an anti-B antibody, and one with an anti-Rh antibody. Agglutination of red blood cells in a given site indicates a positive identification of the blood antigens: in this case, A and Rh antigens for blood type A-positive.

Check Your Understanding

  • If a patient's blood agglutinates with anti-B serum, what is the patient’s blood type?
  • What is a cross-match assay, and why is it performed?

Table 20.3 summarizes the various kinds of agglutination assays discussed in this section.

Mechanisms of Select Antibody-Antigen Assays
Type of Assay Mechanism Example
Agglutination Direct: Antibody is used to clump bacterial cells or other large structures Serotyping bacteria
Indirect: Latex beads are coupled with antigen or antibody to look for antibody or antigen, respectively, in patient serum Confirming the presence of rheumatoid factor (IgM-binding Ig) in patient serum
Hemagglutination Direct: Some bacteria and viruses cross-link red blood cells and clump them together Diagnosing influenza, mumps, and measles
Direct Coombs’ test (DAT): Detects nonagglutinating antibodies or complement proteins on red blood cells in vivo Checking for maternal antibodies binding to neonatal red blood cells
Indirect Coombs’ test (IAT): Screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient’s serum in vitro Performing pretransfusion blood testing
Viral hemagglutination inhibition: Uses antibodies from a patient to inhibit viral agglutination Diagnosing various viral diseases by the presence of patient antibodies against the virus
Blood typing and cross-matching: Detects ABO, Rh, and minor antigens in the blood Matches donor blood to recipient immune requirements
Table 20.3

Footnotes

  • 9 Lancefield, Rebecca C., “The Antigenic Complex of Streptococcus haemoliticus. I. Demonstration of a Type-Specific Substance in Extracts of Streptococcus haemolyticus,” The Journal of Experimental Medicine 47, no. 1 (1928): 91-103.
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