What specimen are acceptable for the ASO?
Other infections may also lead to the production of non-
Antibodies from blood serum
What specimen are acceptable for the Strep A antigen assay?
Antigens of bacteria in throat swab
Description of the antigen that is being tested for in the Group
A strep antigen assay
Lateral flow immunoassay for the detection of Strep A
carbohydrate antigen in a throat swab. Antibody specific to
Strep A carbohydrate antigen is coated on the test line region
of the strip
What is the purpose of adding reagents 1 and 2 in the Group A
Strep antigen assay? (3)
To have enough liquid to move up the strip.
To get the specimen off the swab.
Separate thick specimen.
Internal control in Dipstick immunoassay test
On the C line to be able to know if your test ran properly
External control in Dipstick immunoassay test
(+) and (-) controls in the kits
ASO agglutination kit measures ASO antibodies from 200-3500
IU/mL why would you not want the kit to be more sensitive?
Prevent false positive
In agglutination reactions why is it important to run positive and
negative controls with each assay? (2)
Make sure the kit is still working properly.
Reagents are still good or not.
Why don't you rock the ASO card no more than 2 minutes?
You can get false positives
At what point do you proceed with the semi-quantitative ASO
titer?
With an initial positive
Etiological agent of syphilis?
Treponema pallidum
2 types of syphilis antibodies found in serum and their
specificities?
1) Treponeme
2) Non-treponeme
Patient's plasma sample when analyzed by the qualitative RPR
test was found to be positive. Does this mean he has the
disease? What further testing would you suggest?
treponemal antibodies, therefore this doesn't mean he has the
infection. You would run treponeme testing.
Why is the RPR assay used to follow the patient's response to
antibiotics and not the treponeme-specific assays?
Because IgM antibodies decrease
If a Monospot test is positive is there any other testing required
to determine if the patient has Infectious Mononucleosis?
No
Testing for heterophile antibodies was negative, what other
tests need to be done if any?
Serologic testing method 2 (for EBV-specific antibodies)
Why is the spleen often enlarged with an IM infection?
A high level of WBC (B cells) leads to this making the casing of
the spleen fragile
Most common etiologic agent for IM?
Epstein-Barr virus
Mono spot tests measure what?
Heterophile Ab specific for IM. IgM and horse RBCs
Is fluorescence immunoassay a direct or indirect assay?
Direct
What is the purpose of the 'fixing' step of direct immunoassay?
(2)
Kills infection agents.
Perforates cells so fluorescent Ab can enter and bind.
3 tests one can use to distinguish between HSV-1 and HSV-2?
PCR (molecular methods)
Agglutination assay
Direct or Indirect immunoassay (Sandwich ELISA)
How do primary infections of HSV-1 and HSV-2 occur?
HSV-1 = can be spread in saliva
HSV-2 = Sexual intercourse
Describe HSV reactivation? Also describe the location of HSV-
1 and HSV-2.
HSV-1 = Remains latent in facial sensory trigerminal neuronal
cells
HSV-2 = Remains latent in lumbar neurons around waist
(During reactivation the virus travels down the neuronal
ganglion and the virus infects and replicates in epithelial cells
causing the genital lesions or mouth sores)
What two patient populations are at risk for CMV?
Babies ANA testing by immunofluorescence is a direct or indirect
Immunocompromised patients immunoassay?

In anti-hepatitis B surface antigen (anti-HBs) enzyme Indirect immunoassay

immunoassay (EIA) what Ig class antibodies can this assay
capture? (3) What exactly is the fluorescenctly labeled reagent?

IgG Goat anti-human IgG conjugated to fluorescein isothiocyanate

IgM (FITC)
IgA
What components/specificities is an ANA assay designed to
How would you make a qualitative Anti-hepatitis B surface detect?
antigen (anti-HBs) enzyme immunoassay (EIA) quantitative?
Anti-nuclear Ab to various cell nuclear proteins
Have a second set of standards wells
What specimen type is used for an ANA assay?
If you failed to wash the wells completely or only performed 2-3
washes how would this affect your result? Patient serum

You would have false positives that could occur
If excess wash was left in the wells how would it affect the
results?
You would dilute out your conjugate
A plasma specimen has tested an anti-HBs value >10 mIU/mL.
What 2 biological situations could have led to the production of
this antibody?
Previous infection
Vaccination
Why do we start with a 1:80 dilution instead of using serum
without dilution? (2)
1) To increase specificity we need to dilute (gets rid of
nonspecific Ab)
2) 1:80 is when we can get to a titer that is clinically relevant
(there are Ab that could get diluted)
When are dilutions >1:80 of serum made and tested?
Day 2
Why are dilutions of >1:80 of serum made and tested?

How does the anti-Hepatitis B antibody profile in a person with Patterns can be made
an effective HBV vaccination differ from a person with a past
List the diseases on can, in part, diagnose with this assay? (5)
infection with the hepatitis B virus itself?
Systemic lupus
A person infected will have Ab to every aspect of the virus.
Rheumatoid arthritis
A vaccinated person will only have HBsAg antibodies (Ab)
Mixed connective tissue disease
Is the HIV immunoblot assay direct or indirect? Progressive systemic sclerosis
Vasculitis
Indirect labeled immunoassay. The anti-human IgG (2nd Ab)
that is labeled ANA workflow (3)

What is the blocking reagent used in HIV immunoblot assay? Day 1: 1:80 dilution
What is the purpose of blocking? Why would you suspect that Day 2: Titer
this undefined blocking reagent would actually function better Day 3: Confirm - specificity
than a single purified protein like BSA or casein?
Is immunohistochemistry a direct or indirect immunoassay?
Non-fat dry milk (NFDM 5%)
Indirect immunoassay
Binding of non-specific antibody sites.
Because it's nonspecific and therefore it will bind to anything. What is it about the reagents and assay design that makes an
immunohistochemistry assay extremely sensitive to low
The Mult-spot is an FCA approved confirmation assay for HIV.
amounts of antigen?(3)
Describe a test to tests used for HIV screening?
Blocking/peroxidase destruction
ETA screening for HIV Ags (p24)
Secondary labeled Ab
Why is the multi-spot a better test than screening test? HRP enzyme

More specific for HIV Ags Blocking/peroxidase destruction does what in

immunohistochemistry to make it extremely sensitive?
The protein binds all non-specific protein binding sites such
that the detection reagents (Ab) don't bind non-specifically
Secondary labeled Ab does what in immunohistochemistry to
make it extremely sensitive?
1) Measles, Mumps, Rubella (MMR)
2) Polio
3) Varicella (chicken pox/shingles)
How are Inactivated vaccines produced?

The antigen that we are looking for is in such low abundance
that a secondary Ab is needed to enhance sensitivity
What does HRP enzyme do in immunohistochemistry to make
it extremely sensitive?
When adequate attenuation of a live virus can't be achieved
inactivation of the virus by heat or chemicals (formaldehyde)
can be an alternative
Examples of Inactivated vaccines (4)

It is a highly active enzyme that has high sensitivity itself 1) Cholera

2) Polio
What is the purpose of antigen retrieval? 3) Hep A
4) Rabies
The purpose of the high heat is to reverse the formation of
cross links so that the epitopes are available for antibody How are toxoid (inactivated toxin) vaccines produced (5 steps)
binding and also so the reagents can enter the tissue
1) Grow bacteria in culture that make the toxin
What is the purpose of blocking? 2) Purify the toxins secreted
3) Add chemical (formaldehyde) for 20-24 days
Reduce background 4) Re-purify the 'killed' toxin
5) Vaccine
Vaccination
Examples of toxoid vaccines (2)
Deliberately introducing a 'dead or weakened microbe' or part
of a microbe into a person Tetanus vaccine
Diphteria vaccine
Purpose of a vaccine
Some bacteria have a __1__ who's __2__ coating makes it
To stimulate active immunity and create immune memory
difficult for a baby or young child's immature immune system to
(memory B and T cells) recognizing a specific antigen so that
see and respond. __2__ vaccines are __3__ immunogenic in
exposure to the active disease causing microorganism will
children __4__ and don't stimulate __5__ immunological
stimulate an already primed immune system to fight disease
memory
3 characteristics of a good vaccine
1) capsule (H. flu Hib, N. meningitidis, S. pneumoniae)
1. Produce protective immunity with only minimal side effects 2) polysaccaride
2. Be immunogenic enough to produce a strong and 3) poorly
measurable immune response 4) under 2 years old
3. Be stable during its shelf life, with potency remaining at a 5) long term
proper level
With __1__ vaccines we can now effectively protect children
What are the 5 types of vaccines? from bacteria with __2__

1) Live attenuated ( live organism with low virulence) 1) conjugated

2) Inactivated (organism has no virulence)
3) Inactivated toxins - "toxoids"
4) Conjugated vaccines - no virulence
5) Genetically engineered - contains no original infectious or
disease-causing agent
How are Live attenuated vaccines produced?
Produced by the selection of mutants induced at random
through serial passage of cells cultured in vitro (agent becomes
less virulent, but body still mounts an immune response
against it)
Examples of Live attenuated vaccines (3)
2) capsules
How are conjugated vaccines produced?
The process of attaching (linking) the polysaccharide antigen to
a protein carrier (e.g. diphtheria or tetanus) that the infant's
immune system already recognizes in order to provoke an
immune response
Examples of conjugated vaccines (3)
H. flu (Hib)
Meningitis
Pneumoncoccal diseases
How are genetically engineered vaccines produced (3 steps)
1) Yeast making HbsAg (using recombinant DNA techniques)
2) Purify the surface antigen
3) Vaccine
What is an adjuvant
Substance that enhances the immune response to the
vaccine's antigen
2 modes of action of an adjuvant
1) To carry the vaccine antigen and to slow its release
2) To provoke a local inflammatory response
What is the mechanism of action vaccines take to eliminate
bacterial infections?
Multiple antibodies which (neutralize, opsonize, complement
activate...)
What is the mechanism of action vaccines take to eliminate
viral infections? (2)
Multiple antibodies (neutralization essentially) which act before
the viruses penetrate the cell
Cellular mediated immunity (CD8+ T cells)
What is the mechanism of action vaccines take to eliminate
toxoid infections?
Antibodies are produced which bind to and neutralize the toxin
Series of events that lead to humoral immunity immunity
induced by vaccines?
1. B cells with the right receptor shape recognize a vaccine
antigen and bind to it
2. CD4+ T helper cell helps B cells
differentiate into clones
3. The B cells are activated to produce a clone of antibodies
with the same specificity
4. The B cells mature and become "plasma" cells (capable of
excreting 2000 molecules antibody/second) and "memory"
cells
5. If the "memory" cells encounter the antigen again they will
change into plasma cells and produce large
numbers of specific antibodies
6. The size, specificity and speed of the response will increase
with repeated exposure
Series of events that lead to cell-mediated immunity induced by
vaccines? (4)
1. Viral antigens stimulate a type of CD8+ T cells
2. These cytotoxic T cells kills virally infected cells
3. They can also activate macrophages causing destruction of
intracellular pathogens
4. Certain pathogen specific cytotoxic T cells persist as
memory cells and are able to respond quickly
and effectively if the individual is exposed to the pathogen
again
What is the purpose of a booster?
To boost your immune response
Varicella Zoster virus causes what diseases?
Chicken pox (varicella) and shingles (herpes zoster)
How varicella vaccine was made?
Took scraping from pustule from child with very mild chicken
pox disease, passaged it in culture on guinea pig cells and
then normal human diploid fibroblasts
What strain of VZV is used today?
Oka strain
The Oka strain is the ____ vaccine
Live attenuated
Varivax is __1__% effective. Zostervax contains __2__ the
dose found in Varivax. Zostervax is __3__% effective but
declines as your immune system does with advanced age.
Zostervax is recommended once you hit __4__
1) 80-90%
2) 14X
3)>70%
4) 60
Human Papillomavirus causes __1__
1) Cervical cancer
Human Papillomavirus vaccine is composed of what?
Recombinant L1 protein from the HPV16/18 strain
What are some safety concerns with the HPV vaccine?
1) Injection site reactions (pain, redness, swelling)
2) Systemic (fever, ache, soreness, swollen lymph nodes)
3) Severe (cerebrovascular diseases; cardiovascular diseases;
meningitis, encephalitis, and encephalopathy; Ramsay-Hunt
syndrome; or Bell's
palsy, autism)
Condition in which the body's own antigenic structure
stimulates an immune response and reacts with self Antigen in
a manner similar to the destruction of foreign Antigen.
Autoimmunity
When autoantibody can cause damage in multiple organ
systems it is called what?
Systemic autoimmune disease
When autoantibodies cause damage to a single organ
Organ-specific autoimmune disease
Group of autoantibodies directed against (attack) contents of
the cell nucleus
Anti-Nuclear Antibodies (ANA)
Two lab tests for Anti-Nuclear Antibodies (ANA)
ELISA
Fluorescent antinuclear antibody (FANA)
General procedure for Fluorescent Anti-Nuclear Antibody Test
(FANA) (5)
1) Nucleolar RNA
2) SLE
3) Scleroderma
Scleroderma is a systemic syndrome that literally means ____.
Hard skin
(Connective tissue can also be thicker: esophagus, intestines)
Centromere ANA pattern is antibodies against __1__. There
are __2__ dots in interphase. You can also see a __3__ patter
in mitotic cells. This pattern is suggestive of __4__

1. Add diluted serum to HEp-2 cells 1) centrioles

2. Incubate then wash 2) 46 dots (one for each chromosome)
3. Add anti-human IgG (FITC) 3) Zipper pattern in mitotic cells
4. Incubate then wash 4) Systemic sclerosis
5. Image under fluorescent microscope
Sclerosis is a __1__ disease. __2__ cell type is activated
Anti-Nuclear Antibody Test (FANA) patterns (4) causing __3__ cross links to form, affecting all __4__

1) Homogenous 1) Connective Tissue Disease

2) Speckled 2) Fibroblast
3) Nucleolar 3) Collagen
4) Centromere 4) Organs

The most common ANA pattern is __1__. Antibodies against What autoimmune disease has anti-Sm (Smith) antibodies and
__2__. what is the ANA pattern?

1) homogenous SLE
2) dsDNA speckled pattern

Homogenous ANA pattern is suggestive of __1__ can also be ANA confirmation tests (2)
__2__
1) Indirect Fluorescent Assay with a more specific substrate
1) SLE 2) ELISA with specific substrate
2) Rheumatoid Arthritis (RA)
In Indirect fluorescent assay you are using __1__ to confirm
Speckled ANA patter is antibodies against __1__. This pattern __2__ ANA pattern __3__ antibodies
is suggestive of numerous __2__ diseases (3)
1) Crithidia luciliae
1) Nuclear proteins 2) homogenous pattern
2) Systemic Autoimmune diseases 3) dsDNA antibodies
(SLE, Sjogren's Syndrome, Sclerosis)
In Rheumatoid Arthritis (RA) __1__ in joints bind __2__ and
Sjorgren's syndrome is an autoimmune disorder where your attracts __3__ and __4__
body attacks __1__ that produce __2__
1) Immune complexes
1) glands 2) Complement
2) moisture 3) PMNs
4) macrophages
Symptoms of Sjorgren's syndrome (5)
__1__ released from cells in joints cause damage to the
Dry mouth (leads to trouble talking, chewing, swallowing) cartilage in the joint. There becomes a balance between __2__
Dry eyes and __3__ that leads to continual inflammation and destruction
Dry peeling lips of __4__, __5__ and __6__
Dry or sore throat
Dryness of the skin 1) Cytokines
2) Proinflammatory cytokines
Nucleolar ANA pattern is antibodies against __1__ and this 3) Anti-inflammatory cytokines
ANA pattern is suggestive of __2__ or __3__ 4) Connective tissue
5) Cartilage
6) Bone
Rheumatoid Factor Antibody (RF) is most often of the __1__ 2-30 min
class and is directed against the __2__ portion of __3__. This
results in a __4__ hypersensitivity reaction Type I mediated by ____

1) IgM IgE
2) Fc
3) IgG Response time for Type II
4) Type III hypersensitivity (Immune complex)
5-8 hr
RA diagnosis used by physicians
Type II mediated by ____
- Serology (RF, ACPA)
Antibody-mediated cytotoxicity
- Joint involvement
(IgG and IgM with cells and tissues)
- Sedimentation rate
- Duration of symptoms Response time for Type III
RF latex agglutionation test: found in about __1__ percent of 2-8 hr
patients, meaning if you have a __2__ test it doesn't mean you
don't have RA and if you have a __3__ test it doesn't mean you Type III mediated by ____
have RA.
Mediated by immune complexes
1) 75%
2) negative Response time for Type IV
3) positive
24-72 hr
RF latex agglutination test has latex coated with __1__ on the
__2__ portion. Serum contains __3__ against __4__ Type IV mediated by ____

1) IgG Cell-mediated
2) Fc portion
3) IgM Allergens definition
4) IgG
Antigen that trigger allergic reactions
What test is a more reliable indicator of RA than the RF test?
Common allergens
Anti-citrullinated peptide EIA (ACP-EIA)
Poison plants (Ivy)
What is a hypersensitivity reaction? animal scratches
pollen
An exaggerated response to a harmless antigen latex
bee sting
A hypersensitivity reaction results in what three things? medication
nuts and shellfish
- Inflammation and injury to the tissue dust
- Disease mold
- Or even death animal dander
(involves either humoral or cell-mediated immunity)
Two hypersensitivity reactions that are allergies
Hypersensitivity reactions are dependent on __1__.
Type I (Immediate-type hypersensitivity)
1) host's response not the nature of antigen Type IV (Delayed-type hypersensitivity)

Sensitization Step 1 of Type I reaction

When a person's previous exposer to antigen developed an
immune response to the antigen
Antigen in hypersensitivity reaction can be __1__ or __2__
1) exogenous (environmental)
2) endogenous (cell surface or soluble)
Response time for Type I
Allergen is presented to CD4+ (Th2 subtype)
Step 2 of Type I reaction
CD4+ activation stimulates B cell production of IgE antibodies
specific to the antigen (allergen)
Step 2 produces a large amount of __1__ in response to a
small concentration of __2__
1) IgE 4) airborne allergens
2) antigen 5) foods

Step 3 of Type I reactions __1__ circulate in the blood and Symptoms of atopic dermatitis (2)
attach the __2__ end to __3__ of the tissues and __4__ in the
bloodstream. - Itchy, red skin rash
- Chronic lesions with thickened skin may develop
1) IgE (secondary bacterial infection may occur)
2) Fc
3) Mast cells Urticaria is commonly associated with ____
4) Basophils
latex (foods, drugs)
Step 3 of Type I reactions is called the ____ phase
Symptoms of urticaria (1)
Sensitization phase
Widespread, itchy white areas surrounded by redness
Clinical manifestation effects can be __1__ around the area of (erythema)
__2__. On rarer occasion if allergen enters bloodstream __3__ (called "wheal and flare" reaction)
issues can occur
Gastrointestinal reactions are usually associated with ____.
1) localized Symptoms (4)
2) allergen exposure (cat scratch)
3) systemic (bee sting) food allergy
Symptoms:
Allergic Rhinitis is the most common ____ disorder in US. Nausea
Vomiting
atopic (any IgE) disorder Abdominal pain
Diarrhea
Common symptoms of allergic Rhinitis (3)
Systemic Anaphylaxis (3)
Runny nose
sneezing - Potentially life-threatening whole-body allergic reaction
watery eyes - Can occur within seconds or minutes of exposure to
something you're allergic to
Common airborne allergens (4) - Can involve multiple organ systems

Pollens Systemic Anaphylaxis is commonly associated with (3)

Molds
Animal dander - Food (shellfish, peanuts, certain other nuts)
Dust mites - Antibiotics (penicillin)
- Venom (bees and wasps)
__1__ often occurs with allergic rhinitis. Is caused by some of
the same __2__ that cause allergic rhinitis. Often associated Severity of Systemic anaphylaxis
with __3__ or __4__
Ca be fatal without prompt treatment (injection with
1) Allergic asthma epinephrine)
2) allergens
3) pollution Testing for allergies In vivo methods
4) cigarette smoke
- Studying physiological reaction of an individual to a particular
Allergic asthma symptoms (3) allergen
- Involve direct skin testing which is the least expensive and
Coughing most specific type of testing
Wheezing
Shortness of breath Testing for allergies In vitro methods

Atopic Dermatitis is triggered by contact with __1__ such as Lab testing, evaluating levels of components of the immune
__2__ or __3__, __4__, __5__ response to an allergen - total IgE

1) irritants Two types of In Vitro allergy tests

2) soap
3) detergents 1) RadioImmunoSorbent Test (RIST)
2) RadioAllergoSorbent Test (RAST)
RadioImmunoSorbent Test (RIST) is quantitation of __1__ but 3) Neutrophils
does not identify __2__ 4) granules
5) chronic inflammation
1) total IgE 6) tissue destruction
2) Specific allergen
Type III testing
RadioAllergoSorbent Test (RAST) detects __1__. This test has
more __2__ value. Look for Immune complex (Ig) deposition in tissues with
Immunohistochemistry
1) allergen-specific IgE
2) clinical value Type IV hypersensitivity is __1__ and no __2__ are involved.
Differences between Type IV and the other types, Type IV is
Type II hypersensitivity reactions are caused by the reaction of mediated by __3__ and __4__
__1__ and __2__ with __3__ and __4__
1) delayed hypersensitivity (2-3 days)
1) IgG 2) no antibodies involved
2) IgM 3) CD4+ Th1 T cells
3) cells 4) CD8+ cytotoxic cells
4) tissues
Two phases of Type IV
Type II hypersensitivity reactions are __1__-dependent, __2__-
mediated, __3__ reactions Sensitization phase
Effector phase
1) antibody-dependent
2) Complement-mediated 3 steps in sensitization phase in Type IV
3) cytotoxic reactions
1) antigen contact
3 clinical manifestations of type II hypersensitivity 2) 1-2 weeks after initial antigen contact
3) Antigen processed and presented to antigen-specific Th
1) Transfusion reaction (person's immune system targets cells Th cells activated and proliferate; these cells are called
transfused cells from another person) delayed-type hypersensitivity T cells
2) Hemolytic disease of the newborn (HDN)(mother's Ab target
baby RBCs Ag) 5 steps in effector phase in Type IV
3) Hemolytic Anemias
1. Subsequent exposure to Ag
Type II testing (1) 2. Memory TDTh cells produce a variety of cytokines and
chemokines
Direct antiglobulin test (DAT) 3. Attract monocytes and PMNs to the site
- person's RBCs are sensitized in vivo (Ig on RBC) 4. Monocytes develop into activated tissue macrophages
- In lab mix their blood with anti-human globin IgG 5. Inflammatory response follows - results in tissue damage,
- Agglutination is positive granulomas, and/or necrosis

Type III hypersensitivity __1__ combines with __2__, immune Clinical manifestations of Type IV (2)
complexes are formed that __3__ out of the __4__. Normally
such complexes are cleared by __5__, but if the immune Contact dermatitis (skin reaction caused by contact with
system is overwhelmed, these complexes __6__ detergent, cosmetics, poison ivy. Allergens act as haptens and
bind skin protein activate specific memory T cells. Rxn hrs
1) Soluble antigen to days later)
2) soluble antibody Hypersensitivity pneumonitis (Inhalaltion of allergen, spores,
3) precipitate chemicals. most cases occupation, farmer's lung, pigeon
4) serum fancier's disease)
5) phagocytic cells
6) deposit in the tissues Testing for Type IV (2)

When Ab-Ag complexes deposit in tissues they bind __1__ - M. tuberculosis screening test
which it's activation results in __2__. __3__ migrate to site and - Allergen patch testing for allergic contact dermatitis
release __4__ causing __5__ and __6__

1) complement
2) histamine/cytokine activation