Serology Review�

Overview

• Study your notes and labs from the spring.
• Make sure you know the information related to the objectives.
• The certification exam will test over knowledge of immunology and serology, so need to know immunological theories as well as testing specific for conditions.
• The Serology final will also test over this.
• This review is BRIEF and covers the high points only.

Overview

• Only a few select serological tests are performed in clinical laboratories. Due to this fact serology is performed on campus to give everyone equal experience.
• A kit test is a kit test. As an MLT you must be able to:
• Read and accurately follow directions of package insert.
• State principle, sample requirements, limitations of the procedure and causes of false positives and negatives.
• Accurately perform test according to package insert or written instructions.
• Report out results in required format.

Traits of Immunogens (Antigens)

• Foreignness
• Ex. Plant protein more immunogenic than animal protein
• Size
• >10,000 daltons
• Complexity
• Heterogeneity in building blocks that comprise the antigen
• Proteins>polysaccharides>>lipids and nucleic acids

Haptens

• Small molecule that, by themselves, are NOT immunogenic.

​

• When coupled to a high molecular weight protein, the hapten becomes the antigenic determinant (epitope) for the antigen .

Definitions

• Chemotaxis
• the migration of cells to an inflammatory site following interaction with chemoattractants or chemokines.
• Ex. C5a of the complement system
• Opsonization
• The process by which plasma proteins are attached to a foreign substance and prepare it for phagocytosis
• Ex. IgG
• Epitope (antigenic determinant)
• Small part of the immunogen that is recognized by B or T cells.
• Can be as few as 6 amino acids

​

More Definitions

• Antibody
• Please tell me you can define antibody by now.
• Heterophile antigen
• Antigens that appear on the surface of tissues of several different species
• Heterophile antibody
• Antibodies that are generated in response to an antigen in one species, but are cross reactive with an antigen in another species.
• Example: Epstein-Barr virus antibody detected in Monospot test

​

​

​

Antibody Structure

IgG

Classes of Antibodies – Know Structure and Function

• IgG, IgD, and IgE: all monomers
• IgG: Gamma heavy chains

​

• IgD: Delta (Δ or δ) heavy chains

​

• IgE: Epsilon (Ε or ε) heavy chains

​

• IgA: Alpha -monomer or dimer

​

• IgM: Mu - pentamer

Comparison of Antibodies

Innate versus Acquired (Adaptive) Immunity

Innate versus Adaptive Mechanisms

Factors that determine Immune Response

• Dose
• High dose: Tolerance or immunological paralysis
• Intermediate: Best response
• Low dose: little stimulus OR induces tolerance
• Route
• Enteric administration : preferred route for intestinal parasite immunogens—frequently induces B cell tolerance rather than an immune response
• Parenteral administration
• Intradermal and subcutaneous routes stimulate the immune cells of lymph nodes , faster and more pronounced immune response.
• IV administration stimulates immune cells in spleen first, slower and less effective

​

• Adjuvants enhance the immune response
• Often oil and water mixture
• Aluminum potassium sulfate is the only one approved for human use

​

​

Cellular versus Humoral Immunity

Humoral Immunity

• Exposure to antigen causes production of specific antibodies by B cells.
• Life long
• Two types of responses – must know length of time for antibody production and antibody class produced
• Primary – first exposure, takes weeks to months, IgM then IgG, slow
• Secondary – second exposure to same antigen, IgG, fast – days to weeks

Primary versus Secondary Response

Complement

• Purpose
• Promote the inflammatory response by opsonization which enhances susceptibility of coated cells to phagocytosis.
• Alter biological membranes to cause direct cell lysis.
• Three pathways:
• Classical – activation caused by IgM (1 molecule minimum) or IgG (2 molecules minimum in close proximity) - know recognition, activation and membrane attack units.
• Alternative (properdin) - occurs independently of antibody, must know triggers
• Lectin - begins when mannan-binding protein (MBP) binds to the mannose groups of microbial carbohydrates.
• Must know the activation sequence of each
• Note: complement is destroyed by HEAT. If complement interferes with serological testing, it can be denatured by heating serum to 56C for 30 minutes.

Classical Pathway of Complement Activation

Specificity versus Sensitivity

• Specificity: How true is the test result?
• Ability of a test to correctly EXCLUDE individuals who do NOT have the given disease/condition
• How likely is the test to detect the absence of a characteristic in someone without the characteristic?
• Sensitivity: How small of an amount can be detected?
• Ability of a test to correctly identify individuals who HAVE a given disease/condition
• How likely is the test to detect the presence of a characteristic in someone with the characteristic?

Principle of Serological Tests

• Cause a reaction between antigen and antibody to produce a DETECTABLE reaction.
• One must be UNKNOWN.
• Look for antigen OR antibody.
• An antibody MAY be the antigen in the test system, i.e., RF is an IgM class antibody directed against IgG. In this test latex particles coated with IgG antibody, looking for antigen which in this case is the IgM RF present.
• MANY different test systems developed.

Factors affecting Ag/Ab Reactivity

Basic Immunologic Procedures

• Measurement by Light
• Precipitation
• Electrophoretic Techniques
• Agglutination Reactions – still popular
• Labeled reactions – very popular
• Molecular techniques – gaining in popularity especially for pathogens.

Nephelometry

• Measures turbidity of sample by passing light thru it, amount of light scatter is measured.
• Two types:
• Endpoint – reaction goes to completion
• Kinetic – light scatter measured at specific time. Reaction occurs at a steady rate and the timing of measurement can be done.

Precipitation

• Involves combining soluble antigen with soluble antibody to produce visible, INSOLUBLE complexes.
• Relative concentrations of antigen and antibody must be equal

​

Liquid Precipitation

Gel Precipitation: Radial Immunodiffusion (RID)

Double Gel Diffusion : Ouchterlony

Electrophoresis

• Process of separating proteins in a mixture utilizing their different net electrical charges
• Size and shape can cause frictional drag
• Types
• Moving Boundary
• Disc
• Capillary zone
• Immunoelectrophoresis

​

Agglutination

• Occurs in two stages:
• Sensitization – cannot be seen
• Lattice formation – visible
• Antigen or antibody can be coated onto or an integral part of a carrier particle:
• Latex particles
• Red blood cells--HEMAGGLUTINATION
• Charcoal
• Bacteria
• Agglutination indicates presence of substance being tested for.

​

Labeled Reactions

• One of the reactants labeled with a tag:
• I¹²⁵ – measure radioactivity
• Enzyme – color or intensity of color measured.
• Measure intensity of light emitted as a result of reaction.
• Fluorescence
• Review lecture guide for specifics.

​

Molecular

• Rapidly exploding field.
• Polymerase Chain Reaction (PCR) allows replication of genetic material specific to an infectious agent or malignancy.
• Probe is prepared which has target sequence.
• Use thermocycler to cause DNA to denature (separate) then cool to cause annealing to probe.
• Amplify the specific target.
• Becoming EXTREMELY popular.
• Review lecture notes for specifics.

C-Reactive Protein

• Non-specific protein that appears in serum as a response to inflammatory conditions
• Involved in opsonization and complement fixation
• Latex agglutination available
• Coated with Anti-CRP
• Conditions elevated
• Bacterial infection
• Viral infection
• Active rheumatic fever
• Active rheumatoid arthritis
• TB infections
• Malignancies
• Following surgeries

C-Reactive Protein

• ESR also used to gauge inflammation.
• CRP has the following advantages over the ESR:
• Rises quickly DURING inflammation.
• Decreases quickly once inflammation resolved.
• Not affected by anemia or abnormal serum proteins.

Syphilis

• Caused by spirochete Treponema pallidum
• Early: chancre: 90% seropositive within 3 weeks
• Secondary: 6-8 weeks 100% seropositive
• Latent: seropositive, symptoms absent
• Tertiary: Years later, new lesions any body part

​

Syphilis Lab Tests

• Direct Microscopic
• Darkfield
• Fluorescent Antibody
• Nontreponemal: detect antibody to cardiolipin called reagin
• VDRL: flocculation, prone to false positives
• Antigen composed of cardiolipin, cholesterol, lecithin
• RPR: modified VDRL with charcoal particles, more sensitive
• Trust: similar to RPR
• Treponemal: detect antibody to the spirochete or spirochete itself
• FTA-ABS: confirmatory test that uses slides fixed with Nichols strain of T. pallidum that will react with patient antibody
• EIA: not as sensitive as FTA
• DNA probe

Syphilis Testing

• REMEMBER if a non-treponemal antibody screening test is positive MUST do specific treponemal antibody test.
• RPR CANNOT be performed on CSF or cord blood.
• VDRL can be performed on CSF.
• Review lecture notes for causes of false positive and false negative reagin tests.

​

Syphilis Testing

• VDRL
• Must heat serum to 56C for 30 minutes prior to testing to inactivate complement which can cause a false positive.
• Antigen must be prepared daily.
• Test read microscopically.

• RPR
• Modified commercially prepared antigen attached to charcoal.
• Serum does not have to be heated.
• Plasma can be used.
• Read macroscopically.

Lyme Disease

• Causative agent: Borrelia burgdorferi
• Transmitted by Ixodes scapularis
• Bull’s eye rash

​

Lyme Disease Stages

• Stage 1
• Flu-like symptoms
• Rash at bite (maybe)
• Fatigue
• Headache
• Stage 2
• Paralysis of face/neurologic problems
• Carditis/arrythmia
• Meningits
• Stage 3
• Chronic joint and muscle pain
• Restless sleep

​

Lyme Disease Laboratory Testing

• Immunofluorescence assay (IFA)
• B. burgdorferi antigen on slide
• Cross-reactivity possible
• Enzyme immunoassay (EIA)
• Sonicated B. burgdorferi
• Western Blot most common confirmatory test
• PCR

Cold Agglutinins

• Transient Ab that appears in patients with Mycoplasma pneumoniae
• Anti-I specificity
• Reacts preferentially at 4C, agglutination reversed if heated at 37C.
• Must run RBC control
• Fourfold or more rise in titer is significant

Infectious Mononucleosis

• Causative agent Epstein-Barr Virus (EBV)
• Forssman antigen=heterophile antigen

​

• Paul-Bunnell Test
• Sheep RBCs are added to dilutions of patient serum
• Agg indicates presence
• Serum sickness Ab
• Forssman Ab
• IM antibody

​

• Monospot: Uses horse RBCs rather than sheep

​

Pregnancy Test

• Human Chorionic Gonadotropin (HCG): glycoprotein hormone produced in increased amounts in pregnant women and in some tumors
• Hydatidiform mole: rare mass that forms inside the uterus at the beginning of a pregnancy.
• Germ cell tumors in both men and women
• Radioimmunoassay (RIA) testing
• Quantitative
• Can determine gestational age
• Serum
• ELISA
• Qualitative
• Anti-HCG bound to membrane
• Can detect HCG 7-10 days after conception
• Serum
• Urine

Hepatitis

• Panel done to determine whether infection is current or past.
• Presence of IgM indicates acute infection.
• Presence of IgG indicates previous exposure, cannot determine when.
• Hepatitis B Surface Antigen (HBsAg) first marker to appear in HBV infection,

Hepatitis

Hepatitis B Demystified

Human Immunodeficiency Virus (HIV)

• HIV belongs to the genus Lentivirinae of the virus family Retroviridae
• HIV is a spherical particle with an inner core with 2 copies of single stranded RNA, surrounded by a protein capsid and an outer envelope of glycoproteins embedded in a lipid bilayer.

Testing for HIV

• ELISA screening test.
• Western blot is confirmatory test.
• Molecular testing used to follow HIV load and determine success of treatment.
• CD4+ counts are used to determine if the individual has progressed to AIDS.
• The Centers for Disease Control and Prevention considers HIV-infected persons who have CD4 counts below 200 cells/mm³ to have AIDS

ELISA Animations

HIV ELISA

• http://www.biology.arizona.edu/immunology/activities/elisa/technique.html

​

BASICS OF ALL ELISA tests

• http://www.sumanasinc.com/webcontent/animations/content/ELISA.html

​

HIV Testing

HIV Time Line

End of Review