What is the basis for a variety of immunologic or immunodiagnostic tests?
The antigen-antibody interaction is affected by?
Forces of attraction, concentration of the antigen and antibody
Serum dilutions are used to measure what?
the amount of antibody in the serum
How is the amount of antibody in a serum determined?
by testing increasing dilutions of the serum sample for antibody reactivity to the known antigen of interest
What is the reciprocal of the highest dilution of a serum sample that gives a reaction to the known antigen of interest in an immunological test?
What does a color change in an ELISA plate mean?
antibody was detected at that dilution
What directly measures binding of antigen to antibody in vitro? Either the antibody or the antigen needs to be labeled with something we can detect.
primary binding tests
What measures the results of antigen-antibody interactions in vitro. Results include precipitation or agglutination of Ag-Ab complexes; cell lyses.
secondary binding tests
What is the top fraction after blood sample clots (contains proteins, but does not contain any cells; nor any clotting factors)?
What are cells or tissues to probe with antibody when looking for antigen?
whole blood in anti-coagulant, tissue secretions on dead or euthanized animals or from biopsy
What is a normal constituent of all fresh serum (fresh, unheated guinea pig serum is the most efficient in hemolytic tests)?
What is it called when multiple B cell clones make antibodies to different epitopes of an antigen and many antibodies of different specificity to different epitopes are generated?
What is an antibody produced from a single B cell clone?
What is blood serum containing antibodies against specific antigens?
What are immunoenzyme assays?
ELISA, western blot, immunochemistry
What are primary binding tests?
immunofluorescence assays, immunoenzyme assays
What are secondary binding tests?
precipitations tests, agglutination tests, neutralization tests, complement fixation tests
What does a direct fluorescent antibody test detect?
What does an indirect fluorescent antibody test detect?
What test is used to identify specific protein antigens in a complex protein mixture; or to identify specific antibody to a known antigen?
What is the most widely used enzyme in IHC?
During what test are antibodies against a particular antigen conjugated to an enzyme and can be used to detect and locate that specific antigen in tissue sections?
What controls are used during a immunohistochemistry test?
use an irrelevant antibody, use a normal section of tissue to compare with the test section of tissue
What happens when antibodies combine with soluble antigens in solution or gel?
If antigens are particulate, then antibodies may make them clump or________
What tests estimate the ability of antibody to neutralize the biological activity of an antigen when mixed in vitro?
What is it called when an antibody can activate the classical complement pathway and the antigen is on a cell surface, then cell lysis may result.
complement fixation lysis
In a precipitation test, the amount of precipitate that develops is determined by what?
by the relative proportions of antibody to that antigen
Where is there an approximately equal ratio of Ag and Ab, promotes maximal Ag-Ab binding, best for cross-linking of multivalent antigens by their specific antibodies?
Zone of Equivalence
Where there is antibody excess relative to antigen and poor lattice formation occurs. Can't visualize.
Where there is antigen excess relative to antibody and poor lattice formation occurs. Can't visualize.
In double immunodiffusion, where the reactants meet in optimal proportions what forms?
zone of equivalence, an opaque white line of precipitate forms
In the double immunodiffusion test, if the patients serum contained antibody specific for EIAV antigen then what happens?
a line of precipitate will form where optimal proportions of antigen and antibody meet
What antibody class is more efficient at agglutination?
What does agglutination depend on?
the concentration of antibody relative to the antigen
When do agglutination reactions occur?
when particulate Ags with multiple epitopes on their surface bind with specific antibody molecules to these epitopes to form aggregates or clumps
When may antigens occur?
naturally on RBCs, bacterial cell surfaces, they may consist of commercially prepared antigen-coated inert carrier particles
What can be used as indicators to visualize agglutination?
RBCs, bacteria, or beads
When are agglutination tests used?
to detect unknown antigens found in a clinical sample, to identify bacteria by using known anti-sera to the bacteria, to detect or quantitate circulating agglutinating antibodies found in the patients serum by using known particulate antigens
When the carrier paticle is an RBC, the reaction is known as?
What is the test of choice for diagnosis of IMHA?
Hemagglutination inhibition tests: in the presence of specific Abs, the virus receptors are blocked, and the hemagglutination ability of the virus is inhibited, when there is no hemagglutination the result is?
Hemagglutination inhibition tests: in the absence of specific antibodies, the viruses cause hemagglutination, when there is hemagglutination the result is?
What tests estimates the ability of antibody to neutralize the biological activity of an antigen when mixed with it in vitro?
What test is used to search the presence of antibodies in patient serum that neutralize viruses and/ or bacterial toxins?
serum neutralization test
In what tests are sensitized RBCs used as an indicator and can activate complement which generates the MAC resulting in RBC membrane disruption and heoloysis?
Complement fixation test
When is acute serum collected vs. convalescent?
acute collected early after onset of illness, convalescent collected 14-21 days later
What rise in titer from acute to convalescent serum is diagnostic for an active infection?
What is the acute titer vs. convalescent titer?
acute is 1:2, convalescent is 1:64
What is a positive test result that correctly identifies a positive animal. it is a property of the test. It does not mean all the truely positive animals in a population as determined by a gold standard?
A negative test result that correctly identifies a negative animal. It is a property of the rest. it does not mean all the truly neg animals in a population as determined by a gold standard.
What is a POSITIVE test result that is incorrect, identified negative animal as possible?
What is a NEGATIVE test result that is incorrect, identifed a positive animal as negative?
What is a portion (%) of diseased animals that test positive with the test, want a test with high sensitivity when screening animals for a condition that is very important not to miss positive animals.
A test with _____ sensitivity, but low specificity is one that will identifiy almost all of the positive animals.
What is a portion of non-diseases animals that test negative, to rule out the disease. Want a test with high specificity to re-test any positives in a screening test.
With a highly sensitive test you can trust the?
With a highly specific test you can trust the?
What is a toxin that is modified (by formaldehyde for example)
What is an ideal vaccine?
safe, effective, inexpensive to produce, stable, easy to administer, provides long lasting, strong immunity, no side-effects
What are advantages of active immunization compared with passive?
prolonged period of protection, memory response after boosting
What needs to happen with a vaccine?
antigen must be delivered efficiently, T and B cells must be stimulated to generate large numbers of memory cells, Helper and effector T cells must be generated to several epitopes of the pathogen.
What type of vaccine is grown in conditions in the lab that decrease virulence, has lots of epitopes, no adjuvant needed, infected cells process endogenous antigen and present MHC-I molecules?
modified live vaccines
What vaccine acts as exogenous antigen and are engulfed and processed by APCs and presented via MHC-II, cannot cause disease, is strain specific?
What is a bacterin?
if the vaccine is a killed bacterium
What are substances that can enhance the body's response to the vaccine. Are essential if long-term memory is to be established to soluble antigens?
What protect antigens from rapid degradation and therefore prolonged immune responses.
What can promost delivery of souble antigens to APCs. These types of particles are about the size of bacteria and are readily endocytosed by the APCs?
What adjuvants are substances that can stimulate cells to make cytokines which in term promote an immune response, usually these adjuvants are complex microbial products that represent PAMPS, stimulate macrophages through TLR?
What is Freund's complete adjuvant: an oil based adjuvant mixed with killed mycobacterium, a depot plus immunostimulatory activity, can't be used in food animals.
What are examples of problems with killed vaccines?
severe inflammation due to the adjuvants, increased need for multiple doses may produce hypersensitivity, ten to produce a Th2 dominated immune response.
What are examples of problems with modified live vaccines?
MLV preparations may be contaminated with other organisms that are also alive and pathogenic, attenuated organisms used in live vaccines may possess residual virulence.
What causes abortion in cattle, need CIM for prevention and control of infection, vaccine is a MLV.
What licenses veterinary biologics (vaccines)?
What is category I of USDA classification of genetically engineered vet vaccines?
contain inactivated recominant organisms or proteins derived from recombinant organisms, proteins are purified and used as a protein vaccine
What is a potential problem of category I of USDA classification of genetically engineered vet vaccines?
the purified protein may not be a good immunogen because it may not be correctly folded like the native protein as it exists as part of the pathogen
What is the first available category I vet vaccine, used the DNA coding for the gp70 antigen of the virus. Was inserted into the E. coli plasmid.
What vaccine contains live organisms that contain gene deletions or modified genes which result in reduce virulence?
In category I, what is injected?
In category II what is injected?
the live expression vector is injected and the protein antigen of interest is made inside the cells infected by the expression vector
What category are vaccines that contain live expression vectors into which genes from a pathogen have been inserted?
What are live expression vectors?
viruses with large genomes where it is easier to insert extra genes, include pox viruses, adenoviruses, and herpesviruses.
True or false: the vaccinia virus vaccine consist of a live rinderpest virus which will replicate when the cow is vaccinated?
True or false: the vaccinia virus vaccine consists os a live vaccinia virus, which will replicate when the cow is vaccinated?
True or false: The vaccinia virus vaccine might, in some vaccinated cattle, cause a mild form of rinderpest?
True or false: Vaccinated cattle will produce an immune response to rinderpest protein antigens?
True or false: vaccinated cattle will produce an immune response to vaccinia protein antigens?
Through which pathway will the vaccine antigens of the vaccinia virus vaccine be processed?
What are vaccines that are essential in that thy protect against dangerous diseases, and failure to use places animal at significant risk?
What are vaccines uses against rare or mild diseases, or untested vaccines?
What is an example of duration of immunity?
rabies vaccine: some provide 3 year duration of immunity, others only 1 year
Why is MLV not recommended for pregnant animals?
can lead to abortion or weak neonatal syndromes in some cases
What is a condition characterized by a specific humoral and/or cell mediated immune response against consitituents of the body's own tissues.
What is a normal immune response to unusual or abnormal antigens?
previously hidden self-antigens that are released through tissue damage from "immune priviledged sites"
What are immune priviledged sites?
lens material in eye, spermatozoa in seminal vesicles
Antigens inside cells are not exposed to lymphocytes unless what?
released from the cells due to tissue damage
What contains antigens that cross-react with mammalian neurons and cardiac muscle?
What is molecular mimicry?
similar or shared epitopes between infectious agents, parasites. self-antigens may stimulate cross-reacting antibodies
What happens when IgM against-IgG, immune complexes stimulate macrophages to release proinflammatory cytokines. Neutrophils enter and release enzymes which cause cartilage damage.
What are abnormal immune responses to self-antigen?
loss of regulation (loss of tolerance)
What are the most important genes that influence naturally occuring autoimmune diseases?
What molecules regulate antigen processing and presentation, thus they influence resistance or susceptibility to many diseases?
Almost all autoimmune diseases in humans are linked to what?
specific MHC alleles
What are genetic defects that may contribute to autoimmune disease?
deleted or overexpressed genes, often that code for immune regulatory proteins, such as cytokines and cytokine receptors
What is an example of Type-I hypersensitivity?
milk allergy in cattle, the immune response is mediated through Th2 cells and IgE autoantibodies are produced, affected cows may develop acute anaphylaxis due to IgE arming their mast cells
What type of hypersensitivity has autoantibodies against cell surface antigens causing cell lysis with assistance from complement or CTLs. In some cases the autoantibody bind a cell surface receptor and alter its function instead of causing cell destruction?
What type of immune mediated cytopenia is autoantibodies against RBC?
immune mediated hemolytic anemia
What type of immune mediated cytopenia is autoantibodies against platelets?
immune mediated thrombocytopenia
What type of immune mediated cytopenia is autoantibodies against leukocytes?
immune mediated leukopenia
How do you diagnose IMT or IMHA?
identification of Ab on the surface of erythrocytes or platelets by coombs test or platelet specific test, diagnose by treating with immunosuppressive drugs and if anemia resolves then immune-mediated disease was present.
What breeds are predisposed to myasthenia gravis?
german shepherds, gold retrievers, labradors, dachshunds
How do you treat myasthenia gravis?
What involves both type-II and type-IV hypersensitivity reactions: cytotoxic T-cells attack thyroid epithelium and antibody is made against T3, T4, and thyroglobulin?
lymphocytic thyroiditis in dogs
What needs to be done to confirm that hypothyroidism is due to an autoimmune mechanism?
need a biopsy to show the characteristic lymphocytic infiltration and anti-thyroid antibodies must be detected in serum using an ELISA assay or indirect FA
How do you treat hypothyroism?
replacement therapy with synthetic T4
What is one of four skin diseases in the pemphigus complex of diseases. Ag-Ab interaction allows for activation of complement as part of the type-II response, this causes tissue-destruction.
What happens when autoantibodies form immune complexes with autoantigens which then can cause inflammation. Most significant in systemic lupus erythematosis, a disease in which many different autoantibodies are produced?
What is it called when self-reactive Th1 cells react against myelin basic protein. The myelin-reactive Th1-cells cause inflammation and demyelination?
What is a generalized autoimmune myositis occuring in large dogs such as german shepherds. involves autoreactive T-cells and autoantibodies. ~50% of affected dogs also have antibuclear antibodies or antibodies to sarcolemma or both?
How are autoimmune diseases treated?
immunosuppressive drugs such as glucocorticoids
What is some evidence that the immune system helps to protect against cancer?
patients with HIV-AIDS more likely to develop cancer, transplant patients are on immunosuppressive drugs and these people more likely to develop cancer.
Not all cancers are more likely to emerge in immunosuppressed individuals, what are examples of this?
in humans lung or breast cancer do not develop more frequently in immunodeficient people
How can immunity be acquired?
through immunization with killed tumor cells
What is immunity specific for?
the antigens of the original tumor used for immunization
Both the innate immunity and the adaptive immunity help to do what?
prevent the overgrowth of tumor cells
What is the main defense against tumor outgrowth is what system?
innate system through the action of NK cells and macrophages
What are NK cells regulated by?
Triggering of NK cell cytotoxicity results from what?
change in the balance between activating and inhibitory signals to the NK cells
Do NK cells express antigen-specific receptors?
How can NK cells bind to antibodies that is bound to a tumor antigen on surface of tumor cell, and can thus help with ADCC?
have Fc receptors for the Fc region of antibody
What molecules are not expressed by normal healthy cells, but by some tumor cells, virus-infected cells and cells undergoing other kinds of stress.
"stress" molecules, NK cells have receptors that can interact with molecules expressed by cells during stress
What is mediated through perforins and granzymes as well as through the death receptor pathway. Death by apoptosis.
NK cell killing
Macrophages activated by what are especially active against tumors?
Macrophages can be nonspecifically activated by what?
BCG. thus, BCG is given in some cases to stimulate macrophages to fight cancer
What is used to vaccinate humans against M. tuberculosis in some countries outside the US and to vaccinate some high-risk people in the US?
BCG, modified live 'avirulent' strain of Mycobacterium bovis
What is associated with infection by bovine papillomavirus, and is tumor of the skin epithelium and dermal fibroblasts?
How can tumors express proteins?
expression of altered or new proteins, over-express a normal protein, express a protein that is normally only expressed in the fetus
What are examples of tumors expressing altered or new proteins?
carcinogens induce mutations so that cells produce different proteins that are no longer recognized as self-proteins, viral infections can cause the infected cell to express non-self proteins
What is found in the neoplastic T lymphoid cells of cats infected with Feline Leukemia Virus?
FOCMA (feline oncovirogenic-associated cell membrane antigen)
What are examples of over-expressed self-proteins?
What are antigens normally found on the fetus, that are formed in the adult?
What are mechanisms of aquired immunity against tumors?
antigen-specific cytotoxic T-cells, Antibody + complement activation (leading to MAC and lysis of free tumor cells), and ADCC
What are mechanisms of tumor evasion of the immune system?
low immunogenicity, antigenic modulation or masking, tumor-induced immunosuppression
What are examples of strategies to induce immunity to tumors?
non-specific modulation of the immune response, monoclonal antibodies to target tumors, expand antigen-specific T-cells, vaccination with tumor antigens
What is it called when you remove some T-cells from patient, add IL-s invitro to expand their numbers, and infuse it back into patient. Some of these T-cells should be the tumor antigen-specific cells (only works if there are already antigen-specific T-cells)
Anti-tumor strategy by expanding antigen-specific T-cells
When you vaccinate with naked DNA which codes for tumor antigens or vaccinate with DNA-transected dendritic cells. The dentritic cells must come from the patient, why?
has to be self-MHC (T-cells can only see antigen produced by self MHC)
What type of immunodeficiency is a result of an inherited defect (may notice breed suseptibilities, especially in breeds with reduced genetic diversity)?
What type of immunodeficiency is a result of some other cause, such as an infection or exposure to a toxin?
What is an example of primary immunodeficiency?
canine cyclical neutropenia (grey collie syndrome)
What are symptoms of canine cyclical neutropenia?
dilution of skin pigmentation and of hair coat color, lighter and more of a silver-grey, eye lesions, leukocyte number fluctuation, increased susceptibility to bacterial and fungal infections, rarely live beyond 3 years
How can 'grey collie syndrome' be treated?
repeated use of antibiotics, drugs to stimulate the bone marrow
What are examples of primary immunodeficiencies affecting the acquired immune system?
severe combined immune deficiency (SCID) in horses dogs, and mice. Hypotrichosis with tympanic aplasia in cats and nude mice
Describe SCID in arabian foals?
complete inability of the adaptice immune system to respond. Absence of functional T and B lymphocytes
X-linked SCID is seen in what breeds of dogs?
basset hounds and welsh corgis, males only
What are signs of X-linked SCID in dogs?
recurring infections, poorly developed thymus, decreased T cells, IgG. No IgA, but normal quantities of IgM, die following vaccination with modified live distemper virus
Autosomal recessive SCID is seen in what breed of dogs?
Jack russell terriers
What is it called when kittens are born without a thymus (no T-lymphocyte development)? Seen in Birman cats with an autosomal recessive inheritance pattern.
Hypotrichosis with thymic aplasia in cats
What are some important viruses that destroy lymphoid tissues?
retroviruses, herpes, feline leukemia virus, canine distemper virus, feline parvovirus, canine parvovirus, bovine viral diarrhea virus