Immunoassays

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  • Created by: LBCW0502
  • Created on: 09-11-18 12:56
What is an immunoassay?
A test which uses antibody and antigen complexes as a means of generating a measurable result
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What is an antibody?
A protein produced by the body is response to invading foreign substances, Produced by the immune system. E.g. test indicating antibodies to cancer molecules indicates invading cancer cells
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What is an antigen?
A substance that the body is trying to eliminate/reduce by mounting an immune response.
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Outline the structure of antibodies
Immunoglobulins. IgG, most common, Fab region (contains variable region), Fc region (constant region). Light chains, heavy chains, disulfide bonds, bivalent
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What are the 5 types of antibodies?
IgG, IgA, IgM, IgD and IgE (types responsible for secretions of enzymes/tears, lactation etc.)
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What is the function of papain?
Enzyme breaks down antibody structure to form Fab and Fc fragments
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What is the function of pepsin?
Enzyme breaks down antibody structure to form F(ab)2 and Fab/Fc fragments
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What are polyclonal antibodies?
Recognise different antigenic determinants/epitopes derived from multiple B cell clones
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What are monoclonal antibodies?
Recognise a single antigenic epitope derived from single B cell clones - more suitable for formulation of drugs
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Outline how polyclonal antibodies are produced
Multiple injections of antigen solution in animal over time. Production of antibodies, blood collected, serum isolated from blood. Antiserum produced, rich in antibodies (recognise same antigen but different epitopes/multivalent)
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What does epitopes mean?
Polyclonal antibodies recognise the same antigen but can bind to different areas on the antigen (vary in specificity on the same antigen)
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Outline how monoclonal antibodies are produced (1)
Inject specific antigen into host. Isolate B cells from spleen of mouse. Fuse B cells with myeloma with PEG in culture and produce antibodies. Hybridomas forms (grow/produce antibodies, specific to epitope/antigen)
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Outline how monoclonal antibodies are produced (2)
Grow antibodies in HAT medium. B/myeloma cells not used die and hybridomas survive. Culture cells in individual wells of microtitre plate. Isolation, purification, clonal expansion of monoclonal Ab
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What is the advantage of monoclonal antibodies?
Hybridoma cell line producing monoclonal Ab is immortal/can produce same antibodies consistently
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What is cross-reactivity?
When a particular antibody is expected to bind to a specific antigen but can also bind to another antigen due to similar epitopes
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What is direct ELISA?
Antibody with attached enzyme bind to antigen on surface. Enzyme causes reaction with produces a colour. Antigen present (colour), antigen not present (no colour)
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What is indirect ELISA?
Antibody with another antibody/enzyme attached binds to antigen on surface. Enzyme causes reaction with produces a colour. Antigen present (colour), antigen not present (no colour)
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What is Sandwich ELISA? (1)
Capture Ab binds to antigen (attached to antibody which is also attached to an antibody with an enzyme).Enzyme causes reaction with produces a colour. Antigen present (colour), antigen not present (no colour)
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What is Sandwich ELISA? (2)
Capture Ab. BSA applied to gaps between capture Ab. Antigen binds to capture Ab, HRP-conjugated (detection Ab). Production of colour which can be measured using UV abs at 675 nm. Directly proportional
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What is competitive ELISA?
Antigen measured by ability to compete with labeled antigen. Unlabeled antigen blocks ability for labeled antigen to bind (Ab binding site occupied). Less labeled measured in assay means more unlabeled antigen present (inversely proportional)
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What is non-competitive ELISA?
Measured labeled analyte is directly proportional to amount of antigen present in sample - Sandwich ELISA
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Give an example of an application of direct ELISA
Pregnancy kit (detect levels of HCG) - assay for antigens
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Give an example of an application of indirect ELISA
HIV test kit (detect levels of HIV antibodies) - assay for antibodies
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What is heterogeneous ELISA?
Ab*, Ag and ]-Ab in reaction cuvette. Isolate and wash complex (remove unlabeled Ab*). Measure bound Ab-Ag complex
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What is homogeneous ELISA?
Ab*, Ag and ]-Ab in reaction cuvette. Measure bound Ab-Ag complex
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What is the difference between homogeneous and heterogeneous ELISA?
Wash step not required for homogeneous method (easier/faster)
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Describe features of Fluorescence Polarisation Immunoassay (1)
Homogeneous, competitive immunoassay, used to detect small analytes (toxins/drugs of abuse)
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Describe features of Fluorescence Polarisation Immunoassay (2)
Analyte - Ab, Ag, Ab-F - low levels of Ag-F bound to Ab - fast rotation giving lower polarised light signal
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Describe features of Fluorescence Polarisation Immunoassay (3)
No analyte - Ab, Ab-F - high levels of Ag-F bound to Ab - slow rotation giving higher polarised light signal
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Describe features of RIA
Ab/Ag labeled with radioactivity, used in competitive/non-competitive format. Replaced by EIA due to complications/disposal of materials. Used when high sensitivity is required. Heterogeneous/competitive
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What is RIA used for?
Blood bank screening in hepatitis virus, tracking human T-lymphtropic virus (TLV-1) leukaemia virus. Detection of narcotic drugs. Early cancer detection
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Describe features of immuno-electrophoresis (1)
Rocket immuno-electrophoresis. Ag applied to wells cut into gel. Agarose gel impregnated with Ab. Ag migrates to anode, complexing with Ab to form precipitation of complex. Longer peak/more antibodies present, more complex
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Describe features of immuno-electrophoresis (2)
Counter immuno-electrophoresis. Assay for antigens of Neisseria menigitidis in CSF. Ag migrates to anode, Ab migrates to cathode
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Describe features of immuno-agglutination
E.g. test for IgM antibodies (RA-F binds to IgG). Serum in patient/typhoid fever. Also used to assay CRP levels (infection)
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What are the pitfalls and problems with using immunoassays?
Lack of concordance. Inter-laboratory variation in biomarker assay results and/or inter-biomarker assay kit variation. Potentially lead to clinical misdiagnosis
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Give examples of lack of concordance in the results for immunoassays
Immunoassay of TSH (marker for hypo/hyper-thyroidism) - Ab might recognise different epitope. Patient misdiagnosed with cancer recurrence (they are still in remission) - immunoassay of CA 19-9, tumour marker for pancreatic/colon carcinoma
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What are the issues with anti-reagent antibodies and patient auto-bodies?
Sandwich immunoassay can give false + results (leads to unnecessary treatment) - detection of anti-reagent Ab. Sandwich immunoassay can give false negative results (treatment required/not given) - anti-analyte Ab bound to Ag not reporter Ab
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What is the high does Hook effect?
High dose of Ag gives falsely low signal leading to false negative result. Spot this by diluting sample and look for diluted sample assay result > concentrated sample assay result. Prozone effect (reduce accuracy/precision)
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Give examples of tests using immunoassays (1)
Measure endocrine hormones e.g. TSH, cortisol, FSH, LH, prolactin, oestrogen, progesterone, testosterone, hCG etc. Indicators of cardiovascular damage e.g. myoglobin, troponin I, creatinine kinase MB etc. Hepatitis. HIV, cancer tumour markers.
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Give examples of tests using immunoassays (2)
Congenital tests. Metabolic tests e.g. vitamin B12 etc. Specific proteins e.g. IgG, RF etc.
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Other cards in this set

Card 2

Front

What is an antibody?

Back

A protein produced by the body is response to invading foreign substances, Produced by the immune system. E.g. test indicating antibodies to cancer molecules indicates invading cancer cells

Card 3

Front

What is an antigen?

Back

Preview of the front of card 3

Card 4

Front

Outline the structure of antibodies

Back

Preview of the front of card 4

Card 5

Front

What are the 5 types of antibodies?

Back

Preview of the front of card 5
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