Introduction to serologic methods (II)

Outline


Indicator labeled immunoassay

  

Enzyme Fluorescence Chemiluminescence

Molecular biological techniques

IndicatorIndicator-Labeled Immunoassays


(conjugate) AgAg-Ab

Fluorochromes  Enzyme  Radioactive Isotopes



Radioactive Isotopes: Molecules with unstable nuclei

He2+ e-

Radioisotope

EnzymeEnzyme-linked Approach
  

 

Enzymes are catalysts for chemical reactions. attachment Enzyme activity manifest as colored product or chemiluminescene. Enzymes function indirectly as indicator labels. Better than Radioactive isotopes long halfhalflife, no need of special precautions for handling.

Alkaline Phosphatase


Instrumentation depend on the substrate select for the assay. Has wide pH stability , stable for at least 1 year in 4

Horseradish peroxidase
Narrow pH optimum , stable for months at 4 . Many popular substrates are potentially Carcinogenic.

Enzyme immunoassay:EIA


AlkalineAlkaline-phosphatase ;ALP p-nitrophenyl phosphate(pNPP) 405nm horseradish peroxidase ;HRP o-phenylenediamine(OPD) 492nm

Immunofluorescence
Combines immunologic methods and histochemical methods  Use for detection of antigen & antibody  Two types of fluorescent antibody techniques: ---direct ---direct method ---indirect ---indirect method  Very high sensitivity & specificity


Direct immunofluorescent assay

Labeled Ab + Ag

*detect antigen

Indirect immunofluorescent assay

Ag + unlabeled Ab + labeled antihuman immunoglobulin

DIF & IIF

Fluorescence

Absorption & emision spectra of a fluorescent compound

Fluorescent label
 

fluorochrome, fluorophore, fluor In immunofluorescence, the term fluorochrome is used. When an Ab is labeled with a fluorochrome,it is called a conjugate The most commonly used fluorochromes are fluorescein, rhodamine, acridine, and phycoerythrin

Fluorochrome
FITC(green light): Adsorption :490nM Emition :517nM TMRITC(red light): Adsorption :550nM Emition :580nM

Fluorescence microscope with transmitted light

Heterogeneous vs homogeneous
 

heterogeneous:

 

e.g. ELISA

Heterogeneous FIA


: -----Indirect -----Indirect method -----Competitive -----Competitive method -----Sandwich -----Sandwich method -----Avidin -----Avidin biotin method

Enzyme-linked immunosorbent Enzymeassay(ELISA)

 

EnzymeEnzyme-linked

 

Immnosorbent

Competitive(limited-reagent) vs nonCompetitive(limitednoncompetitive


: /

Label

Sandwich assay

BiotinBiotin-Avidin system

Avidin(68kD) or streptavidin(60kD) biotin Streptavidin subunit Streptomyces avidinii , subunit , biotin

 

BiotinBiotin-labeled MHC FITC Avidin system to detect antigen specific T cell

Homogeneous immunoassay


AgAg-Ab reaction complex AgAg-Ab reaction

Enzyme-multiplied immunoassay Enzymetehcnique (EMIT)



Fluorescence polarization immunoassy

 

AgAg-F

Fluorescence Polarization Immunoassay (FPIA)

Chemiluminescence

Chemiluminescence (CL)
     

1~10% 10% Luminol, Acridinium esters, Peroxyoxalates Dioxetanes, Tris(2,2bipyridyl) ruthenium( ) Tris(2

Luminol

H2O2

Acridinium esters catalyst

Chemiluminescence immunoassays
  

( ) 3 Luminescence immunoassay (LIA) Luminescence enzyme immunoassay Luminescence cofactor immunoassay

SCIENCE IMAGING SYSTEMS -- Introduction to LAS-3000

Home work


Indicator labeled anti-human Ig antiobdy is antigenerally used in clinical diagnosis to detect patients antibody (secondary antibody). Please describe different approaches to produce


Rabbit polyclonal anti-human IgG specific antiantibody Mouse monoclonal anti-human IgM specific antiantibody

Molecular Blotting Techniques  Southern blot  Polymerase chain reaction (PCR)  Restriction fragment length polymorphism (RFLP)  Northern blot  Western blot: HIV

Molecular Biology Tools  Restriction endonucleases  Restriction nucleases or restriction enzymes  DNA methylated at restriction enzyme sites is protected

Hybridization
Hybridization of nucleic acids is based on the following complementary.

Primers and Probes

Primer are short strands of nucleotides complementary to a particular DNA sequence and used to initiate DNA replication  Probes are used to target a particular sequence of complementary RNA or DNA  Indicator labels to detect: Radioactive or fluorescent labels


Southern Blot: Detect DNA

Restriction fragment length polymorphism (RFLP)

Detection Genetic Mutations In Clinical Laboratories



Sickle cell disease: RFLP and Southern Blotting

Single point mutation in the globulin gene.  normal has 3 Mst cleavage sites, mutated has only 2 RFLP  Southern blotting


Polymerase chain reaction (PCR)

Denaturetion 90-95 Primer annealing 30-65

Primer extension 60-75 Repeat cycle

Northern Blot
Detect mRNA  Detect by radiolabeled , single-stranded singleDNA fragment  Not routinely used in clinical molecular diagnostics


Developmental Northern blotting. (A-E) Procedure for Northern blotting. (A) RNA is isolated from various tissues and is separated by size using gel electrophoresis. (B) The gel is then placed on a paper wick, which absorbs an ionic solution from a trough. (C) A filter that traps RNA is placed above the gel, and blotting paper is placed above that. Capillary action draws the solution through the gel, trapping the RNA on the filter. (D) The filter is incubated with radioactive single-stranded DNA complementary to the mRNA of interest. (E) After washing off unbound DNA, autoradiography localizes the mRNA in the samples that contain it. (F) Drawing of a developmental Northern blot showing the presence of Pax6 mRNA in the eye, brain, and pancreas of a mammalian embryo. (F after Ton et al. 1991.)

Western Blot
  

Detect protein (antibody) Separated by molecular weight Often used to confirm the specificity of antibodies detected by enzyme-linked enzymeimmunosorbent assay (ELISA) screen procedures

Western blot
Band pattern Interpretation Lane 1, HIV+ serum (positive control) Lane 2, HIV- serum (negative control) Lane A, Patient A Lane B, Patient B Lane C, Patient C

ELISA

Western blot

substrate

Water soluble

Water insoluble

In Situ Hybridization
In Situ : [


Labeled probes to bind target DNA in thin tissue section or cytologic smears  Useful for detecting abundant RNA species or viral DNA

Tissue in situ hybridization. The example shows the pattern of hybridization produced using a 35Slabeled F-myosin heavy chain antisense riboprobe against a transverse section of tissue from a 13 day embryonic mouse. The dark areas represent strong labeling, notably in the ventricles of the heart.

Microarrays


Affymetrix Genechip

Figure 2. The Use of Oligonucleotide Arrays. mRNA is extracted from cells and amplified through a process that labels the RNA for analysis. The sample is then applied to an array and any bound RNA stained.

Industry advances in multiplex and parallel analysis

 

Test tubes

Microwell plate

Microarray

Microsphere-based liquid array

Microspheres
 

 

 

Microspheres are dyed to create 100 distinct colors Each microsphere has spectral address based on red/infrared content Microspheres are suspendable Microspheres are coated with capture reagent (oligo or antibody) Sample is added to microspheres Analyte is captured to microspheres Fluorescent reporter tag added

Luminex system
  

Assays are read using a compact microsphere analyzer Analyzer samples well Lasers excite fluorescent dyes - red laser for bead classification and green laser for assay result Multiple readings for each microsphere set Software reports results in realreal-time Up to 9600 results read in 1 hour