1

o immune-mediated hemolytic anemia. o Hemolytic disease of the newborn (ABO, Rh) 4 o identify antibodies present in a patient's serum prior before blood transfusion. o Blood transfusion preparation. o Antenatal antibody screening.

Aggregation of “particulate antigens” bound with agglutinins of more than one specificity. Example: coagglutination staphylococcus aureus protein A for serotyping strains of Streptococcus pneumoniae,

o Example: Influenza virus 9

o Heterophiles antibodies tests (Monospot test, Paul – Bunnell test): o agglutination of the horse RBCs by heterophile antibodies in patient's serum for rapid diagnosis of EBV infectious mononucleosis. o Cold agglutnin test: o Cold agglutinins are antibodies made by the immune system in response to infection having the ability to agglutinate RBC in Low temperature. E.g. Mycoplasma pneumoniae o Weil felix test: o it is agglutination test for the diagnosis of rickettsial infections. o based on the presence of antigenic “cross-reaction” between Rickettsia and certain serotypes of Proteus o VDRL: screening for syphilis caused by treponema pallidum. o Widal test: standard tube agglutination test for salmonella typhi. 10

oIt is a reaction in which the Ab interact with a free soluble Ag. oAt the optimum concentration,positive reaction form a precipitin line oIt is a reaction in which the Ab interact with a free soluble Ag. oAt the optimum concentration,positive reaction form a precipitin line

oThis is precipitation reaction to test identity between two antigens or between two antibodies

14

Ag Mg/ml Diameter /mm Sample Diameter Sample concentration oThis is precipitation reaction to test presence of entire subclass of immunoglobulin by applying specific antiglobulin forming a precipitation rings.

oThis is method to separate the serum by electrophoresis. oCreate a trench containing specific anti serum. oDiffusion of both serum and anti serum forming a precipitin lines oThis is method to separate the serum by electrophoresis. oCreate a trench containing specific anti serum. oDiffusion of both serum and anti serum forming a precipitin lines

o technique used to detect specific proteins in the given sample. o It uses ”gel electrophoresis” to separate proteins (or polypeptide) and are then transferred to a membrane. o (nitrocellulose) where they are stained with labled antibodies specific to the target protein.

Sample AB Test AgEnzym linked AbEnzyme substrate

oAntibodies containing fluorescent dye react with serum (either Ag or Specific Ab).. oPositive reaction is shown under fluorescent microscope. oAntibodies containing fluorescent dye react with serum (either Ag or Specific Ab).. oPositive reaction is shown under fluorescent microscope.

The toxin( hemolysin) sample (serum) Ab RBC lysed with the toxin oTesting the presence of the antitoxin in the serum. oIf they are present the toxin will be neutralized.RBCs not lysed. oIf there is no antitoxin the toxin will lyse the RBCs oTesting the presence of the antitoxin in the serum. oIf they are present the toxin will be neutralized.RBCs not lysed. oIf there is no antitoxin the toxin will lyse the RBCs

The toxin( hemolysin)sample (serum) Ab RBC lysed with the toxin

o Testing immunity of the patient against diphtheria. o 0.2 ml of the diphtheria toxin in the test forearm and 0.2 ml of inactivated diphtheria toxin (by heat ) in the control forearm.

oPreparing sensitized complement & two systems: oSystem1: oContaing serum sample & prepared antigen oSystem 2 (indicator system): ocontaining sensitized sheep RBCs & Ab to sheep RBCs. oIn +ve reaction the serum containing Ab will react with the Ag the complement will be consumed by system 1 (RBCs will not be lysed).. oIn –ve reaction where there is no Ab in the serum so the complement will consumed by system 2 (RBCs will be lysed). oPreparing sensitized complement & two systems: oSystem1: oContaing serum sample & prepared antigen oSystem 2 (indicator system): ocontaining sensitized sheep RBCs & Ab to sheep RBCs. oIn +ve reaction the serum containing Ab will react with the Ag the complement will be consumed by system 1 (RBCs will not be lysed).. oIn –ve reaction where there is no Ab in the serum so the complement will consumed by system 2 (RBCs will be lysed).

Sample ABTest AgcomplementAb to sheep RBCSheep RBC

29

oThis a solid phase reaction through which the serum antibody will be captured to the wells using capture Ag. oAn Antihuman globulin conjugate containing chromogenic enzyme will react with the sample Ab. oAdding enzyme substrate will produce colour on the well. oSo +ve test is manifested by presenece of colour, in –ve test there is no colour. oThis a solid phase reaction through which the serum antibody will be captured to the wells using capture Ag. oAn Antihuman globulin conjugate containing chromogenic enzyme will react with the sample Ab. oAdding enzyme substrate will produce colour on the well. oSo +ve test is manifested by presenece of colour, in –ve test there is no colour.

Sample ABAgEnzym linked AbEnzyme substrate

oThis a solid phase reaction through which the serum antibody will be captured to the wells using capture Ag. oThe conjugate here is directed to the Ag i.e. the sample and conjugate will compete each other. oAdding enzyme substrate will not produce colour on the well. oSo +ve reaction is manifested by no colour, -ve reaction there is a colour. oThis a solid phase reaction through which the serum antibody will be captured to the wells using capture Ag. oThe conjugate here is directed to the Ag i.e. the sample and conjugate will compete each other. oAdding enzyme substrate will not produce colour on the well. oSo +ve reaction is manifested by no colour, -ve reaction there is a colour.

Sample ABAgEnzym linked AbEnzyme substrate

oThis a solid phase reaction through which the serum antigen will be captured to the wells using capture Ab. oThe conjugate here is directed to the Ag i.e. the Ag will be sandwiched between capture & test Abs oAdding enzyme substrate will produce colour on the well. oSo +ve test is manifested by presenece of colour, in –ve test there is no colour. oThis a solid phase reaction through which the serum antigen will be captured to the wells using capture Ab. oThe conjugate here is directed to the Ag i.e. the Ag will be sandwiched between capture & test Abs oAdding enzyme substrate will produce colour on the well. oSo +ve test is manifested by presenece of colour, in –ve test there is no colour.

AB Sample Ag Enzym linked AbEnzyme substrate

o is a biophysical laser based technology employed in cell counting, cell marker detection, allowing synchronized analysis of the physical and chemical characteristics of up to thousands of particles per second. o Example: used in the diagnosis blood cancers, and CD4 cell counting in AIDS patients 37

o rapid and easy. o Antigen-antibody immune complexes flow through a region and encounter antibody against them, resulting in a visible coloured line o E.g Used in rapid diagnosis of Malaria 38