ImmunologyChapter 6, Lecture 2

• Richard L. Myers, Ph.D.

• Department of Biology

• Southwest Missouri State

• Temple Hall 227

• Telephone: 417-836-5307

• Email: rlm967f@mail.smsu.edu

Agglutination reactions

• Antibody reacts with a “particulate” antigen– similar to a precipitation reaction– must watch antibody excess (prozone effect)– incomplete antibodies may block agglutination

• Hemagglutination– agglutination involving RBCs– used in blood typing– IgM is the most effective agglutinating Ab

Agglutination

• Bacterial agglutination– antibodies can be made to agglutinate bacteria– can determine the Ab (agglutination) titer– this aids in diagnosis of bacterial infection– an example is typhoid fever (S. typhi)

• Passive agglutination– simple and sensitive– can detect Ab as low as 0.001 g/ml

Agglutination

• Agglutination inhibition– very sensitive assay for antigen– best example of the pregnancy test

• uses antibody to HCG coated latex particles

– another example is detection of Ab to viruses that will hemagglutinate RBCs

• Ab interfere with hemagglutination

• example is testing for immunity to rubella virus

Radioimmunoassay• A very sensitive test (0.001 g/ml)

• Used to quantitate hormones, serum proteins, drugs, vitamins and other things

• Involves competitive binding– radiolabeled antigen and unlabeled antigen to a

high-affinity Ab– Ag usually labeled with 125I– Ab cannot distinguish between labeled and

unlabeled Ag

ELISA or EIA

• ELISA/EIA is similar to RIA but requires an enzyme rather than a radioisotope– can be indirect, sandwich or competitive

ELISA

• An enzyme conjugated to an antibody reacts with a colorless substrate to generate a colored product

Western blotting• Method for identification of specific protein

– Southern blotting identifies DNA– Northern blotting identifies mRNA

• Protein mixture electrophoresed on PAGE– with SDS, a dissociating agent

• Proteins then transferred to a membrane

• Proteins of interest are detected by flooding membrane with radiolabeled monoclonals

Immunofluorescence

• Antibody molecules are “tagged” with a fluorescent dye (fluorochrome)– fluorescein-labeled antibodies are popular– protein A from S. aureus freqeuntly used– another is biotin-avidin labeling

• Immunofluorescence used to identify cells– can be direct or indirect

FACS

• Fluorescence-activated cell sorter (FACS) used to identify and separate subpopulations of lymphocytes– process called flow cytometry

• It is possible to analyze three fluorochromes on a single stained sample

Assignment

• Begin reading Chapter 7, Organization and Expression of Ig Genes

• Review question 4, (pg 192)