Complement based techniques Complex protein system by which certain antibodies are capable of killing cells Proteins of the complex system are thermolabile Cascade of reactions Terminal component can kill cells by the formation of pores in the cell (especially bacteria and red bloodcells but under some circumstances also nucleated cells)

Complement system

Complement based techniques Technique is applied in traditional serological HLA-typing Antisera of persons who got immunized by circumstances against known HLA-antigens are used in a microcytotoxicity assay Purify the cells to be HLA typed from the blood sample of a patient (T cells for HLA class I and B cells for HLA class II)

HLA-typing Fluorescein diacetate colours living cells green, propidium jodide colours dead cells red Analyse by microscope

HLA-typing with complement techn Problems: –for some rare HLA-antigens  no antiserum availale  not detected –Cross-reactivity –In Class II typing: B-lymphocytes more sensitive to cross reactivity and non specific complement lysis –Less specific than analysis of MHC genes (LiPa strip, sequencing)

Line Immunoassay (LIA) For the detection of antibodies in patients against other specific antigens (microorganisms or other antigens) (compare with latex agglutination) The antigens are bound in strips on a nitrocellulose membrane Detection of bound antibodies with enzym-labeled anti- human Ig (compare with ELISA, see later)  substrate precipitation  visible strip Better specificity by simultaneous detection of several epitopes Several commercial kits available: HIV, Hep C, Human T-cell Lymphotrope Virus (HTLV)

LIA Antigen Antibody from serum of patient Alkaline phosphatase-labeled anti-human Ig BCIP/NBT (5-bromo-4-chloro- 3-indoyl-phosphate / nitro blue tetrazolium)

LIA

Western blotting Used for detection of the presence of antibodies in for eg virology (compare with LIA) –The antigens are produced/purified and separated on a SDS- PAGE gel according to their molecular weight  see figure Can also be used for the detection of certain proteins (antigens) present in a sample –Separate total protein extract on a SDS-PAGE gel  screen nitrocellulose membrane with specific (labeled) monoclonal antibodies or specific antiserum –eg. Test for the presence of prions in CJD and VCJD

Prion diagnosis in CJD and VCJD Fast test (1 day) on a small amount of brain tissue Proteinase K digest on SDS-PAGE  nitrocellulose membrane  screen with prion- specific Ab  3 bands of kDa (non-mono- di-gly-PrP res ) Normal PrP c  complete digestion by proteinase K

ELISA Enzyme Linked Immunosorbent Assay Can be used for the detection and quantitation of Ab’s Can also be used for the detection and quantitation of antigen (proteins) See figures Very common technique  many commercial kits are available

Flowcytometry Very common technique in medical diagnosis especially in the field of hematology Uses fluorescently labeled monoclonal antibodies to study the presence of certain specific markers on cells  indication of disease sate or identification of certain cell types