1 Labeled Immunoassay Enzyme Linked Immuno Sorben Assay
1. Labeled Immunoassay Enzyme Linked Immuno Sorben Assay ( ELISA ) Radio Immuno Assay ( RIA ) Immuno Flurescent Antibody Technique ( IFAT ) Electrochemilominsance ( ECL ) 2. Non Labeled Immunoassay. Agglutination tests Precipitation tests Fluccolation tests
Agglutination Reactions Ab + particulate Ag Agglutination (clumping) Direct agglutination Passive agglutination Agglutination inhibition n
Direct agglutination Example # 2 – Using bacteria n. (Ag) looking for Ab in serum
Indirect or Passive agglutination Results when inert particles are n coated with soluble Ags which may react with Ab. Particles include latex, rbc’s, charcoal, etc. Example – Ag attached to latex n particle (known) + serum looking for (unknown) Ab. If Ab present, you get visible agglutination.
Reverse passive agglutination Involves the adherence of Ab n to inert particles which can then be used to detect the presence of Ag. Example – latex particle coated n with Ab (known) + serum looking for (unknown) Ag. If Ag present, then you get visible agglutination. (CRP test)
Agglutination inhibition - pos
Agglutination inhibition - neg
ﻋﻮﺍﻣﻞ ﻣﻮﺛﺮ ﺩﺭ ﻭﺍکﻨﺸﻬﺎی آگﻠﻮﺗیﻨﺎﺳیﻮﻥ Y YY Y Y Amount of lattice LATTICE Dilution of antibodies Y Y Y Y excess antibody Y YY Y excess antigen
Direct Coombs’ Test
Differential Diagnosis 1. Anti-Ig. G Positive + Anti-C 3 Negative - n Antibodies to Rh protein, Drug induced warm AIHA (penicillin, methyldopa) 2. Anti-Ig. G Positive + Anti-C 3 Positive + n SLE, idiopathic warm AIHA, rarely drug associated 3. Anti-Ig. G Negative - Anti-C 3 Positive + n Cold agglutinin disease, Paroxysmal cold hemoglobinuria, rarely warm AIHA if low-affintiy Ig. G
Trouble Shooting y a s As l e n n o s Per t n e pm S e l p am i u q E d e t r o p e s R m e l b o pr s d o h t e M
ﺑﺮﺭﺳی کﻴﻔﻴﺖ کﻴﺖ ﻫﺎ TP 100* Sensitive = ------TP+FN TN 100*Specificity = -------TN+FP TP 100*Predictive valve of positive= -----TP+FP TN 100*Predictive valve of Negative= ------TN+FN
Variables that affect the quality of results The educational background and training of the laboratory personnel The condition of the specimens The controls used in the test runs Reagents Equipment The interpretation of the results The transcription of results The reporting of results § § § §
The Impact of Prevalence on Predictive Value DISEASE Positive TEST Negative Present Absent 45 5 5 a b c d Sens = 45/50 i. e. 90% Prevalence = 50% 45 Spec = 45/50 i. e. 90% Positive predictive value a = 45 = 90% a+b 50 Negative predictive value d = 45 = 90% c+d 50
Different of Cold Autoantibodies Ig Clonality DAT Hemolysis Primary cold Agglutinin disease Secondary cold autoantibodies Paroxysmal cold Hemoglobinuria Ig. M Monoclonal C 3 Chronic , mild Ig. M Ig. G Polyclonal C 3 Episodic, selflimtied: mild to severe P Mono/polyclonal C 3 Self-limited, mild to severe I, i Target RBC antigen I
(Cold Auto Antibodies (Ig. M directed against ‘Antigen I’ on red cells n Seen in n Elderly – Lymphomas – Infections - EBV, mycoplasma – DAT (Coombs) - + for C 3, negative for Ig. G n Treat - stay warm n
Warm AIHA - Treatment Folic acid supplementation Steroids Splenectomy Immunosuppressives n n Cyclophosphamide – Azathioprine – Intravenous immune globumin (IVIG) n Plasmapheresis n
Differential Diagnosis Anti-Ig. G Positive + Anti-C 3 Negative - . 1 Idiopathic Warm AIHA, Drug induced warm AIHA (penicillin, methyldopa) n Anti-Ig. G Positive + Anti-C 3 Positive + . 2 SLE, idiopathic warm AIHA, rarely drug associated n 3. Anti-Ig. G Negative - Anti-C 3 Positive + Cold agglutinin disease, Paroxysmal cold hemoglobinuria, rarely warm n AIHA if low-affintiy Ig. G
Antibody and antigen can form a lattice Y YY Y Y Amount of lattice LATTICE Dilution of antibodies Y Y Y Y excess antibody Y YY Y excess antigen
c. Applications These include detection of anti-rhesus factor (Rh) antibodies. Antibodies to the Rh factor generally do not agglutinate red blood cells. Thus, red cells from Rh+ children born to Rh- mothers, who have anti-Rh antibodies, may be coated with these antibodies. To check for this, a direct Coombs test is performed. To see if the mother has anti-Rh antibodies in her serum an Indirect Coombs test is performed.
patient red cells + AHG = agglutination
Flocculation tests for Ab detection are based on the n interaction of soluble Ag with Ab, which results in the formation of a precipitate of fine particles. (Ag consists (of lipid type particles (Examples VDRL & RPR’s. (See syphilis tests – to follow n
Concentration of Ag and Ab also affects agglutination reactions, both the first and second stages.
Affinity • Strength of the reaction between a single antigenic determinant and a single Ab combining site High Affinity Low Affinity Ab Ab Ag Ag Affinity = ∑ attractive and repulsive forces
Avidity • The overall strength of binding between an Ag with many determinants and multivalent Abs Keq = 104 Affinity 106 Avidity 1010 Avidity
Cross Reactivity • The ability of an individual Ab combining site to react with more than one antigenic determinant. • The ability of a population of Ab molecules to react with more than one Ag Cross reactions Anti-A Ab Ag A Ag B Ag C Shared epitope Similar epitope
Direct agglutination
- Slides: 65