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109 Cards in this Set
- Front
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Directly measures binding of antigen to antibody in vitro.
antibody or the antigen labeled with something we can detect. Ex: fluorescent dyes, enzymes, radioisotope. |
Primary binding tests
Ex: fluorescent dyes, enzymes, radioisotope. |
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Measures the results of antigen-antibody interactions in vitro.
Ex: precipitation or agglutination of Ag-Ab complexes; cell lyses. |
Secondary binding tests:
Measures the results of antigen-antibody interactions in vitro. (e.g. precipitation, clumping, etc.) Usually less sensitive than primary binding tests, but may be easier to perform. |
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When the carrier is a RBC, the rxn is known as ?
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hemagglutination
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Hemagglutination indicates presence of ?
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Hemagglutination indicates presence of specific ANTIBODY in the patient’s serum
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* Anti-globulin test
* Diagnosis of Immune Mediated Hemolytic Anemia (IMHA) * Test for antibody on the surface of red blood cells (anti-erythrocyte Abs) * Look for agglutination of the red blood cells |
Coombs’ test
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Example of Serological reagents
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1. Complement:
A normal constituent of all fresh serum Ex:: Fresh, unheated guinea pig serum is the most efficient in hemolytic test. 2. Polyclonal/Monoc. antibody 3. Anti-globulins (Ab to Ab) |
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2 types of PRIMARY BINDING TESTS
(edit response) |
1. Direct FA (Detects ANTIGEN): e.g. testing for rabies Ag in brain tissue
2. Indirect FA (Detects ANTIBODY) e.g. Ab in patient serum |
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What does Coomb test for?
What is sign of postive Coomb's test? |
Imm.mediated hemolytic anemia; trying to detect "bad" Ab which attaches to RBC
Agglutination |
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An indirect test for detecting antibodies against specific viruses that can agglutinate RBCs.
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Hemagglutiination Inhibition (HI) Tests: indirect test
The mechanism is based on the inhibition of viral hemagglutination by the specific Abs found in the serum. |
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How does Ab block virus from causing agglutination?
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In the presence of specific Abs, the virus receptors are blocked, and the hemagglutination ability of the virus is inhibited
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What are all components added in HIT?
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The dilutions of a patient serum, a known virus and a defined amount of RBCs are combined:
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What is sign for positive Hemagglutiination Inhibition (HI) Test?
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No hemagglutination = Positive for antibody to the virus
See a pellet of RBCs that settle to the bottom after centrifugation of the micro-titer plate (for some reason if have hemagglutination/ negative inhibition, RBCs do not settle with centrifugation because of the lattice formation with virus-coated RBCs) |
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These are examples of ??
Precipitation tests: Radial Immunodiffusion Agglutination tests: Viral Hemagglutination tests Neutralization tests Complement fixation |
SECONDARY BINDING TESTS
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ELISA
Western Blot Immunohistochemistry Are examples of ? |
PRIMARY BINDING TESTS
more specifically...Immunoenzyme assays |
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Direct Fluorescent Antibody Tests
Indirect Fluorescent Antibody Tests Are examples of ? |
PRIMARY BINDING TESTS
more specifically.. Immunofluorescence assays |
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Types of Secondary binding tests: (4)
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1. Precipitation:
2. Agglutination: 3. Neutralization tests: 4. Complement fixation…Lysis: |
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Neutralization Tests Estimate the ability of what to do what?
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Estimate the ability of antibody to neutralize the biological activity of an antigen when mixed with it in vitro.
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Serum Neutralization Test:
Used to detect ? |
Serum Neutralization Test:
Used to detect ANTIBODIES in patient serum that neutralize viruses and/or bacterial toxins. similar to HIT but instead of blocking agglut.; in SNT, Ab is blocking cytopathic affects of toxin, etc. |
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Example of SN test?
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Example: The BVDV SN test:
To detect antibody to the Bovine Viral Diarrhea Virus (BVDV) > Use kidney cells and a particular strain of BVDV that can infect and lyse these kidney cells. |
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What would you expect to see if BVDV SN test positive?
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Incubate the cytopathic strain of BVDV with patient’s serum. Then add this to the kidney cells
If no cell lysis: Ab to BVDV present (positive)** IF cell lysis: NO Ab to BVDV present. |
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2 types of vaccines?
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MODIFIED LIVE VACCINES
KILLED VACCINES |
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First vaccine? Example of?
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used cow pox to protect people against small cox
> example of cross reactivity; epitopes shared by cow pox and small pox |
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MODIFIED LIVE VACCINES are also known as?
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Attenuated
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Which type vaccine has more epitopes/antigens?
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MODIFIED LIVE VACCINES
(killed only have surface proteins) |
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Which type vaccine requires adjuvant?
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KILLED VACCINES
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Vaccines are stimulating active or passive immunity?
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If giving antigen -> promoting active immunity response
if giving Ab -> promoting passive immune response (giving Ab from another animal) |
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The tetanus vaccine induces protection against ?
How? |
induces protection against toxin (tetanus); vaccinating with toxoid (modified toxin which is no longer toxic e.g. fixed in formaldehyde)
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tetanus vaccine induces what kind of immune response? what is stimulated? what is produced?
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Stimulating adaptive memory immune response, -> getting neutralizing Ab in memory B cells -> type 2 (Th2?)
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If give anti-toxin, what type of vaccine/immune response stimulating?
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anti-tetanus for example is neutralizing Ab, so inducing passive immunity
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Main cause of adverse rxn to vaccine?
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b/c of adjuvant
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When might passive immunity (neutralizing Ab) be useful?
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to provide *immediate* passive immunity to animal that has not been vaccinated before
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Disadvantage of TAT (anti-toxin)?
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serum sickness
>If TAT from horse given to another species, then immune-complexes may form which can cause a type III hypersensitivity reaction (recepient gen. Ab against horse Ab) |
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BCG for Tb is example what kind of vaccine?
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MODIFIED LIVE VACCINES
- M.Bovis bacteria lost its virulence (in humans) by being cultured in an artificial medium for years. |
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T/F
With MODIFIED LIVE VACCINES want attenuated to infect cells, intracellular antigen uptake, TH1 cell-mediated response |
True
MODIFIED LIVE VACCINES organisms that replicate inside cells. Infected cells process endogenous antigen and present antigen via MHC-I molecules. Thus MLV trigger a response dominated by CD8+ CTLs, a Th1 response. |
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T/F There is no vaccine available to protect cattle against M. bovis.
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True
M.Bovis related to M. tuberculosis |
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T/F Killed vaccine acts as exogenous antigen …and thus engulfed and processed by APCs and presented via MHC-I, stimulating a Th1-dominated response.
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False
Killed vaccine acts as exogenous antigen …and thus engulfed and processed by APCs and presented via <MHC-II>, stimulating a <Th2>-dominated response. |
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When might killed vaccine not be suitable?
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Killed vaccine may not be the best kind of response to some organisms (like many viruses and intracellular bacteria),
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Types of Adjuvants :
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Types of Adjuvants :
* Depot * Particulate * Immunostimulatory * Mixed |
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Most widely used type of adjuvant.
2 Example? Type response each promotes? (Th2 or Th1) |
Depot Adjuvants
Example: 1. Aluminum salts (Alum) <- Only adjuvant approved for human vaccines in US Produces strong Th2 responses 2. Water-in-oil (Freund’s incomplete adjuvant) Common in veterinary medicine, promotes Th1 response (it's true) |
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what adjuvant promote delivery of soluble antigens to APCs?
why are they helpful? |
Particulate Adjuvants
In general, APCs can trap and process particulate antigens more efficiently than soluble antigens. |
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Examples of Particulate Adjuvants:
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Examples:
Liposomes, micro particles. ~size of bacteria. Readily engulfed by the APCs. |
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What kind of adjuvant delays antigen removal ….protects antigens from rapid degradation …prolongs immune responses.?
What type of immune response is promoted here? |
Depot Adjuvants - delay degradation of antigen, keep around longer e.g. aluminum salts
~promote Th2 response; but this isn't always one we want |
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PAMPs
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molecules associated with groups of pathogens, that are recognized by cells of innate immune system; conserved within class of microbes
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Substances that stimulate cytokine production which in term promotes immune response.
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Immunostimulatory adjuvants
Usually complex microbial products that represent PAMPs. PAMPs very good at stimulating macrophages thr. MØ TLR; make cytokines that promote T-helper cell function! |
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Examples of Immunostimulatory Adjuvants:
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Examples: LPS; killed bacteria; Microbial DNA, (can even include cytokines themselves, or more likely DNA that codes for cytokines)
(LPS can kill bacteria themselves b/c have molecules on surface which stimulate macrophages to make cytokines) |
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Canine lepto vaccines are example MLV or killed v.?
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Killed Vaccine
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Which type vaccine contain better cross reactivity?
e.g. cow pox immunity protect from small pox |
MLV have more cross reactivity b/c process more antigens
KV are strain specific; |
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If the vaccine is a killed bacterium, it is called a ?
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bacterin.
FYI: None of the commercial canine lepto vaccines contain the MAIN Lepto serovars currently circulating in dogs in Grenada. |
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How do bacterins make up for lack of cross reactivity / strain specific nature of KV?
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Some bacterins may contain several different strains of the bacteria
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Autologous bacterins are ?
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Autologous bacterins are like custom made killed bact. vaccines for specific herd, provide very specific immunity
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What can cause cross links proteins and nucleic acids resulting in structural rigidity, thus may not mirror native epitopes
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Formaldehyde
cross linking bad b/c some epitopes are conformational; so recognition by TCR or BCR requires secondary and/or tertiary structure remain intact |
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Ethylene oxide
B-priopiolactone – used to make the killed rabies vaccine are examples of? |
Alkylating agents used to produce killed vaccines
~may cross-link nucleic acid chains halting replication, but maintaining surface proteins unchanged |
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Water-in-oil (Freund’s incomplete adjuvant) are what kind of antigen? promote what kind of defense?
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Depot Adjuvant
Common in veterinary medicine (only), promotes Th1 response |
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Name Mixed Adjuvant which causes painful inflammation as side effect?
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Freund’s complete adjuvant
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What are components of Freund’s complete adjuvant?
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An oil-based depot adjuvant mixed with killed Mycobacterium….
A depot plus immuno- stimulatory activity. Can’t be used in food animals (cattle in particular)…. Why? Might develop an immune response to the Mycobacterium proteins and thus test positive on a TB skin test |
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Why might BCG be useful in providing protection against tumor cells?
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has a lot of immunostimulatory molecules on surface which can promote nonspecific imm.resp.
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Which would be expected immune response to a killed vaccine?
1. Th1 lymphocytes activate macrophages and cytotoxic T cells 2. Antigenic peptides are presented by MHC class I molecules 3. Stimulate IFN-gamma production Th2 lymphocytes stimulate B lymphocytes to produce neutralizing antibodies |
Killed vaccines:
> Stimulate IFN-gamma production Th2 lymphocytes stimulate B lymphocytes to produce neutralizing antibodies |
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Would you expect to see a lot of CTL's with killed vaccines?
If so, when/how would you? |
unlikely as this is exogenous antigen, unless have cross reactivity which can't always rely on that happening (more info needed)
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Would MLV antigen ever be presented as MHC class 2?
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Yes, if/when some of modified live bacteria or viruses die, taken up as exogenous antigen and expressed as MHC II
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THESE ARE PROBLEMS ASSOCIATED WITH ?
1. Severe inflammation due to adjuvants -> Vaccine-induced feline sarcomas (e.g. cancer in scruff of neck where injected vaccine) 2. Increased need for multiple doses ….hypersensitivity reactions in some individuals 3. tend to induce a Th2-dominated response, but may need a Th1 response |
Killed vaccines
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MLK problems?
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1. MLV preparations may be contaminated with other organisms that are also alive and pathogenic.
2. residual virulence -> only vaccinate young animals, no pregnant Example: Brucella abortus vaccine used in cattle (although risk to human handlers, vet, etc.) |
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Example of MLV preparations contaminated with other organism?
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Example: Outbreak of bovine leukosis (due to the Bovine Leukemia Virus [BLV]) was a result of a batch of babesiosis vaccine containing whole calf blood inf. with BLV. Note: there is no vaccine to protect against BLV.
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Governing body which approves animal vaccines?
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USDA approves animal biologics. (Vaccines are biologics)
(FDA approves human vaccines and animal/human drugs) |
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USDA classification of genetically engineered vaccines
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Category I, II, III
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Which category vaccine (based on USDA classification) contain live organisms with gene deletions or modified genes which result in reduce virulence?
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Category II: contain live organisms with gene modifications
FYI: Similar to a MLV but the process of attenuation is via molecular genetic techniques. |
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Example of Category II: ?
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1. Pseudorabies virus vaccine.
~vaccine strain has gene removed just so can differentiate btw. field strain 2. Herpesvirus with Thymidine kinase (TK) removed. ~can infect but not replicate inside host cell |
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Inactivated (killed) recombinant organisms or proteins derived from recombinant organisms.
Proteins are purified and used as a protein vaccine (subunit vaccine). |
Category I:
Inactivated (killed) recombinant organisms or proteins derived from recombinant organisms. (cloning antigen in bacteria) |
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First commercially available category I veterinary vaccine?
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FeLV vaccine
Isolated DNA coding for the gp70 antigen of the virus. ~cloned in e.coli |
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Which category vaccine (based on USDA classification) contain live expression vectors into which genes from a pathogen have been inserted?
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Category III
The live expression vector replicates in the vaccinated animal. |
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Differences btw. Category I and III?
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In category III, pathogen produced inside cells (endogenous antigen) in order to promote a CMI response.
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Do category III vaccine require adjuvants?
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No
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What category vaccine?
Rabies vaccine for coyotes and raccoons One gene from the rabies virus was inserted into the vaccinia virus. The vaccinia virus is the vector. It is alive and replicates. |
Category III Example: rabies gene insert into non-path. virus; leads to endogenous antigen, CMI
Can this vaccine cause rabies? NO |
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Examples of core vaccines?
3 dogs, 1 both, 3 cats |
* Canine parvovirus, canine distemper, rabies, adenovirus 2
* Feline parvovirus (panleukopenia virus), Feline calicivirus, Feline herpes virus |
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Examples of Noncore vaccines – against rare diseases or mild diseases or untested vaccines
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Lyme’s disease, Giardia, canine adenovirus-1, cornavirus, leptospirosis
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T/F
USDA requires data on duration of immunity (DOI) for vaccine approval. |
False
Previously USDA did not require data on for DOI for vaccine approval. Only new ones now need this…. Not those that have been on the market and previously approved. |
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T/F
Manufactures are liability to the client for adverse effects. |
False; Manufactures have no liability to the client for adverse effects, YOU DO! Client consent!
Vaccine-associated feline sarcoma Hypersensitivities (Types I, II, III, IV) Autoimmune diseases (Antibodies to cell lysates) MLV not recommended for pregnant animals, can lead to abortion or weak neonatal syndromes in some cases |
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Vaccine schedules for Kittens and puppies ?
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Kittens and puppies - vaccinate frequently as maternal antibody is waning, start core vaccines at 6 - 8 weeks every 3 - 4 week intervals
~until 12 – 16 weeks of age |
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Vaccine schedules for Large animal neonates ?
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Large animal neonates – vaccinate at 6 months and at a year
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Vaccine schedules for adults?
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Adults - Annually? Every 3 years?
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What secondary binding test checks if an Ab can activate the classical complement pathway and the Ag is on a cell surface?
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Complement fixation lysis test
: If an Ab and Ag bind, can activate the classical complement pathway, then no cell lysis - get precipitate . |
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What is the zone of equivalence in a precipitation test?
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Approximately equal ratio of Ag and Ab
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What is the prozone in a precipitation test?
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Antibody excess relative to antigen
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What is the postzone in a precipitation test?
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Antigen excess relative to antibody
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What type of test is Coggin's test?
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* Double immunodiffusion, center well is filled with soluble antigen; other wells filled with patient serum
* trying to determine if patient serum contains antibody to the antigen we put in the center well |
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Can agglutination tests be used to determine titers?
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Yes
When the carrier particle in an agglutination test is an RBC, the reaction known as hemagglutination |
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In a hemagglutination test, do the viruses cause the RBCs to agglutinate?
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No, they just attach to them
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What does Coomb's Test test for?
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Looks for antibody on the surface of RBC
Diagnosis of Immune Mediated Hemolytic Anemia (IMHA) |
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What is used as an indicator in complement fixation test?
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Sensitized RBCs
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In a complement fixation test, what does an RBC pellet indicate?
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Positive, complement fixing antibodies to the Ag are present
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In a complement fixation test, what does RBC hemolysis indicate?
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Negative, no complement-fixing antibodies to the Ag are present
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Can a complement fixation test be used to determine titers?
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Yes
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When is acute serum collected?
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Early after onset of illness
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When is convalescent serum collected?
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14-21 days after onset of illness
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What is diagnostic of an infection when comparing acute and convalescent serum samples?
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A four-fold rise in titer from acute to convalescent
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What is a positive test that correctly identifies a positive animal?
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True positive
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What is a negative test that correctly identifies a negative animal?
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True negative
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What is a positive test that identifies a negative animal as a positive animal (wrong)
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False positive
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What is a negative test that identifies a positive animal as a negative animal?
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False negative
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What is the proportion (%) of DISEASED animals that correctly test positive with the test?
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Sensitivity
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What is the proportion (%) of non-diseased animals that correctly test negative with a test?
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Specificity
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Can you trust the positives or negatives with a highly sensitive test?
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Trust the negatives
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Can you trust the positives or negatives with a highly specific test?
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Trust the positives
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Under what circumstances do you want to use a very sensitive test?
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Rare disease, early diagnosis improves prognosis
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What are some advantages of active immunization compared with passive?
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Prolonged period of protection
Memory response after boosting |
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Under what circumstances do you want to use a very sensitive test?
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Rare disease, early diagnosis improves prognosis
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What are some advantages of active immunization compared with passive?
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Prolonged period of protection
Memory response after boosting |
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What is essential in a killed vaccine if long-term memory is to be established to soluble antigens?
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Adjuvants
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What is the reciprocal of the highest dilution of a serum sample giving a positive test result in a titration test?
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Titer
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Which one of paired antibody titer results is most compatible with calf having active infection w/infectious bovine rhinotracheitis virus?
Acute 1:2, Convalescent 1:8 Acute 1:4, Convalescent 1:128 Acute 1:2, Convalescent 1:2 Acute 1:16, Convalescent 1:8 |
A four-fold rise in titer from acute to convalescent is diagnostic of infection when comparing.
Acute 1:4, Convalescent 1:128 |
