Term
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Definition
| The detection and measurement of antigen-antibody binding reaction. |
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Term
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Definition
| Enzyme immunoassays, immunofluorescence assays, radioimmunoassays, and dye-labeled immunochromatography |
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Term
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Definition
| Precipitation, agglutination, and complement fixation tests |
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Term
| Enzyme-Linked Immunosorbent Assay |
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Definition
ELISA
Sandwhich: most common, sensitive, and robust
Used to ID prostate-specific Ag (PSA) in seminal stains & amylase in saliva
Based on colorimetric or fluorometric signal
Is proportional to the amount of bound antigen |
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Term
| ELISA Enzyme Labels: Reporting enzymes labeling the antiglobulin |
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Definition
Alkaline phosphatase (AP)
Horseradish peroxides (HRP) |
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Term
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Definition
Direct asssay
Indirect assay
Sandwhich assay |
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Term
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Definition
| Antigen - Primary Antibody Conjugate - Enzyme - Substrate |
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Term
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Definition
| Antigen - Primary Antibody - Secondary Antibody Conjugate - Enzyme - Substrate |
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Term
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Definition
Capture Antibody - Antigen - Primary Antibody - Secondary Antibody Conjugate - Enzyme - Substrate
Pro: can wash away any nonspecific bonding = less background
You can run a control with no antigen in it and subtract that color from all the other wells. |
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Term
| ELISA Substrate: Detecting Alkaline Phosphatase |
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Definition
PNPP: p-Nitrophenyl Phosphate Disodium Salt
Yellow water-soluble product, absorbs light at 405 nm |
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Term
| ELISA Substrate: Detecting horseraddish peroxidase |
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Definition
ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] - diamonium salt)
Green product, absorbance at 410 & 650 nm
Least sensitive, slow color development, less background |
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Term
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Definition
OPD (o-phenylenediamine dihydrochloride)
Water-soluble
Yellow-orange product
Absorbance at 492 nm |
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Term
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Definition
TMB (3,3',5,5'-tetramethylbenzidine)
Blue color absorbs at 370 and 652 nm
Color then changes to yellow when sulfuric or phosphoric acid is added with max absorbance at 450 nm
Very sensitive & may yield high background if using too much protein or Ab
Fast color development |
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Term
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Definition
Light spectrum
At wavelengths, absorbs/refracts differently |
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Term
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Definition
UV
Taking something up to a higher energy / exciting it and watching the electrons fall back to a normal state |
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Term
| What do you need to produce aggregation / lattice formation? |
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Definition
Certain amount of binding sites
Equal amount of antigen & antibody. |
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Term
| Precipitation-based Assays |
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Definition
Based on the precipitation reaction
Used for species ID |
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Term
| Types of Precipitation-Based Assays |
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Definition
Single Immunodiffusion: Radial Immunodiffusion
Double Immunodiffusion: Ring Assay, Ouchterlony Assay
Electrophoretic Assay: Immunoelectrophoresis (IEP), Crossed Immunoelectrophoresis (CRIE), Rocket Immunoeletrophoresis, Crossed-Over Immunoelectrophoresis / Counter Immunoelectrophoresis |
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Term
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Definition
Passive diffusion method
Concentration gradient is established for anAg and/or Ab
Single & Double |
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Term
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Definition
Concentration gradient is established for EITHER Ag OR Ab
Amount for either Ag or Ab compared to standard curve
Radial Immunodiffusion |
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Term
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Definition
Concentration gradient established for Ag
Ab uniformly distributed in the gel matrix
Ag diffuses from wells in the matrix
Results: Precipitate ring around the well
Measure the ring and compare it to a standard curve to find the unknown concentrations |
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Term
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Definition
Concentration gradient for BOTH antibody AND antigen
Ring Assay
Ouchterlony |
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Term
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Definition
Performed in a test tube or capillary tube
Requires both positive and negative results
Positive = ring
Negative = no ring
No quantification |
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Term
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Definition
Performed in an agarose gel supported by a glass slide or polyester film
Wells are punched in agarose
Usually 6 wells cicling a central well |
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Term
| Secondary Binding Assays: Electrophoretic Methods |
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Definition
Diffusion combined with electrophoresis to enhance results
Separates molecules according to differences in their electrophoretic mobility
IEP, CRIE, Rocket Immunoelectrophoresis, Crossover Immunoelectrophoresis |
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Term
| Immunoelectrophoresis (IEP) |
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Definition
Separates Ag via electrophoresis before immunodiffusion
Positive = arc-shaped precipitate line
Compare known control to questioned sample |
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Term
| Crossed Immunoelectrophoresis (CRIE) |
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Definition
Called 2-D immunoelectrophoresis
More sensitive than IEP
Separates Ag via electrophoresis
Gel turned 90 degrees and separated by electrophoresis into Ab containing gel
Positive = arc-shaped precipitate line / arc area
Compare questioned sample to a known control |
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Term
| Rocket Immunoelectrophoresis |
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Definition
Ab containnig gel
Separates Ab via electrophoresis
"Rocket" height is proportional to [Ag]
Can quantify antigen concentration using a standard curve |
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Term
| Crossed-over Immunoelectrophoresis |
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Definition
AKA Counter Immunoelectrophoresis
Electrophoresis drives Ag & Ab toward eachother
Positive = precipitate line
Sped-up version of an ouchterlony. Used when diffusion would take too long (ie: Ab or Ag has a high MW) |
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Term
| Agglutination Based Assays: Use |
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Definition
Blood group typing
Species identificaiton |
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Term
| Agglutination Based Assays |
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Definition
Simple, more sensitive than precipitation techniques
Identify antigen or antibody
Direct-agglutionatin assay vs. passive agglutination assay vs agglutination inhibition assay |
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Term
| Direct Agglutination Assay |
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Definition
Antibody interacts with the antigens located on a cell surface
Hemagglutination reaction (ie: blood group typing) |
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Term
| Passive Agglutination Assay |
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Definition
Uses coated erythrocytes
Species identification |
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Term
| Agglutination Inhibition Assay |
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Definition
Indirect way to identify the Ag in question
Known Ag is added to Ag-cells + Ab
Known Ag competes for Ab and inhibits agglutination |
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Term
| Immunochromatographic Assays |
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Definition
Rapid, simple test
Usually NOT quantitiative (unlike ELISA)
Yes/No answer |
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Term
| Immunochromatoraphic Assays: Uses |
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Definition
Field screening for seminal & salivary stains
Species identification |
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Term
| Immunochromatographic Assays: Process |
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Definition
Analyte contains antigen
Along the plate are antibodies conjugated to nanoparticles for visualization
At the end, test line & control line: if + for antibodies, test line is colored. If -, the control line is colored. |
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Term
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Definition
| False negative result due to excess Ag |
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Term
| Direct Assay: Known vs. Unkown |
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Definition
The antigen is unknown
The antibody comes from a lab |
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Term
| Indirect Assay: Known vs. Unknown |
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Definition
Antigen from a lab
Antibody in question
Secondary antibody is MONOCLONAL because it recognizes the FC portion of another antibody |
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Term
| Sandwhich Assay: Known vs. Unknown |
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Definition
Also uses a MONOCLONAL antibody from a lab that seeks the FC region
Has to do with the affinity of the binding sites
Can test for antibody 1 or antibody 2 or antigen |
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