Chapter 8

Three important applications of PCR

1. Amplification of DNA 2. Identification of a target sequence 3. Synthesis of a labeled antisense probe

Using serum electrophoresis, protein can be separated into how many fractions?

5

Lateral Flow Immunoassay

A sample that contains antigen flows through a porous strip, and positive reactions are shown by the appearance of a colored band The element combined with an antibody to form a visible reaction in lateral flow immunoassays is gold

Direct immunofluorescent assay

A technique to identify a specific antigen using an antibody that has a fluorescent tag attached. Uses conjugated antibody to detect antigen-antibody reactions at a microscopic level

Flow (cell) cytometry

Allows the user to quantitate components or structural features of individual cells uses laser (Light amplification by stimulated emission of radiation)

The traditional PCR technique

Amplifies the target region of DNA

indicator electrode

An electrode whose potential is related to the logarithm of the activity of one or more species in contact with the electrode Is the main component of potentiometric techniques

pH electrode

An ion selective electrode is separated from blood by hydrogen sensitive glass. The potential difference across this glass is proportional to the difference in hydrogen concentration across it. universally used in the clinical laboratory

applications of Western blotting techniques

Called immunoblot, it is used to detect antibodies to subspecies of antigens.

The most important application of IEP of urine is

Demonstration of Bence Jones (BJ) protein

Electrophoresis technique

Different molecules have different velocities in an electrical field.

In this procedure performed on a tissue sample, a specific nucleic acid is detected in an organism by an antibody directly tagged with a fluorochrome (the fluorescent dye). Which procedure is this?

Direct immunofluorescence

In immunoelectrophoresis (IEP), proteins are differentiated by

Electrophoresis, Diffusion coefficient, and Antibody specificity

Photometry

Employs color to determine the concentrations of various substances Employs color variations to determine the concentrations of various substances Measures luminous intensity of light

The distinct advantages of molecular testing are

Faster turnaround time Smaller sample volumes required Increased specificity and sensitivity

Indirect immunofluorescent assay

Has antigen first exposed to unlabeled antibody, then labeled antibody

You need to perform an immunofluorescence assay that has improved sensitivity, uses an unlabeled antibody to detect antigen, and is suitable for detecting a wide variety of antibodies. Which assay should you use?

Indirect immunofluorescence

In which methodology does a negative reaction confirm specificity?

Inhibition immunofluorescence

reference electrode

Is an electrochemical half-cell that is used as a fixed reference for the cell potential measurements

Inhibition Immunofluorescent Assay

Is based on the fact that antibodies can act as antigens and react with antiimmunoglobulins A blocking test in which an antigen is first exposed to unlabeled antibody and then to labeled antibody and is finally washed and examined

In chromatography:

Mixtures of solutes dissolved in a common solvent are separated from one another by a differential distribution of the solutes between two phases.

Reflectance spectrophotometry

Objectively measures color by measuring the amount of light reflected back from a substance at selected wavelengths function to measure the concentration of glucose in blood by using dry-film technology

For the PCR reaction to take place, one must provide which of the following?

Oligonucleotide primers

Immunofixation Electrophoresis (IFE)

Separation of proteins based on the rate of migration of individual components in an electrical field. Electrophoresis of serum or urine. Double immunodiffusion following electrophoresis. Can test cerebrospinal fluid. best used in the workup of a monoclonal gammopathy

polymerase chain reaction (PCR) amplification technique

Strand Displacement Amplification Strand displacement amplification is a fully automated method that amplifies target nucleic acid without the use of a thermocycler. A double-strand DNA (dsDNA) fragment is created and becomes the target for exponential amplification. Transcription-Mediated Amplification Transcription-mediated amplification (TMA), another isothermal assay, targets either DNA or ribonucleic acid (RNA) but generates RNA as its amplified product. This method is currently being used to detect microorganisms (such as Mycobacterium tuberculosis). Nucleic Acid Sequence-Based Amplification Nucleic acid sequence-based amplification is similar to TMA, but only RNA is targeted for amplification. Applications of this technique are detection and quantitation of human immunodeficiency virus (HIV) and detection of CMV. Ligase Chain Reaction Nucleic Acid Amplification Oligonucleotide pairs hybridize to target sequences within the gene or the cryptic plasmid. The bound oligonucleotides are separated by a small gap at the target site. The enzyme DNA polymerase uses nucleotides in the ligase chain reaction (LCR) nucleic acid amplification reaction mixture to fill in this gap, creating a ligatable junction. Once the gap is filled, DNA ligase joins the oligonucleotide pairs to form a short, single-strand product that is complementary to the original target sequence. This product can itself serve as a target for hybridization and ligation of a second pair of oligonucleotides present in the LCR reaction mixture. Subsequent rounds of denaturation and ligation lead to the geometric accumulation of amplification product. The amplified products are detected by microparticle EIA.

In coulometry

The amount of current passing between two electrodes in an electrochemical cell is measured.

Immunofluorescence technique

Uses antibody labeled with fluorescein isothiocyanate (FITC)

Enzyme Immunoassay (EIA)

a blood test that can determine the presence of antibodies to HIV in the blood or saliva; a variant of this test is called enzyme-linked immunosorbent assay (ELISA) uses nonisotopic label

The units used to express the readings obtained by the electronic measuring device

absorbance (A) units (divided logarithmically) or percent transmittance (%T) units

Optical density

absorbed light

The primary use of IFE is:

characterization of monoclonal immunoglobulins

The blank solution

contains all the reagents used in the procedure, but it does not contain the unknown substance being measured.

Nephelometry

detects light scattering properties of antigen-antibody complexes -photodiode

3 basic immunofluorescent labeling techniques

direct indirect inhibition

Absorbance values

directly proportional to the concentration of a substance can be plotted on a linear graph to give a straight line

FISH technique

fluorescence in situ hybridization Molecular cytogenetic technique

The enzyme reverse transcriptase converts

mRNA to cDNA

Spectrophotometry (colorimetry)

measurement of the intensity of light at selected wavelengths

DNA polymerase catalyzes

primer extension

Chemiluminescence

process by which light is emitted as a product of a chemical reaction Has excellent sensitivity and dynamic range Does not require sample radiation

Percent transmittance

the amount of light that passes through a colored solution compared with the amount of light that passes through a blank or standard solution

Beer-Lambert Law (Beer's Law)

the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the log of the transmitted light. A = Ebc (amount of light absorbed by a solution = (molar absorptivity)(distance light travels through the solution in cm)(molar concentration of absorbing solute) the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light

Absorbance spectrophotometry

the concentration of an unknown sample is determined by measuring light absorption at a particular wavelength and comparing it to the absorption of light by a known standard solution, measured at the same time and wavelength. measure the concentration of hemoglobin in a solution

The optimal wavelength (Amax) for measuring absorbance is

the wavelength that is most absorbed by the compound in question provides maximum sensitivity for measurements

Immunoelectrophoresis (IEP)

two stage process to diagnose myeloma or humoral immunodeficiency states -> can detect which isotype is forming the paraprotein seen on serum electrophoresis, combination of electrophoretic separation of proteins and immunoprecipitation of proteins with either polyspecific or monospecific antisera; (2nd stage is rabbit anti-human serum added to cause precipitin lines to form along electrophoretic separation Diagnosis of monoclonal gammopathies is the most common application of immunoelectrophoresis (IEP)

Nucleic acid blotting

use a DNA sequence to find complementary DNA or RNA sequences in a gel via hybridization