J07
Titer dynamics, complement fixation test and neutralization tests
aVLLM0421c (spring 2017) Outline
● titer, antibody titer dynamics
● complement, complement fixation reaction
● neutralization tests
2/35 Direct vs. indirect testing
direct indirect
● looking for microbe or ● looking for antibodies (Ab) it's part/product (part ● Ab is not part nor product of could be some bacterial the microbe (product of antigen/product e.g. toxin) macroorganism, response to the microbe presence)
● causative agent is present now ● causative agent was present some time in the past
3/35 Indirect testing – overview
● precipitation
● carrier particle agglutination
● complement fixation reaction (CFR)
● neutralization tests
● methods with labeled antibodies: – immunofluorescence – ELISA – Western blot (immunoblot) – immunochromatographic methods
4/35 Interpretation
● identification of antigen: – positive result means the microbe is now present in the patient
● identification of antibodies: – positive result means the microbe was present in the patient (we do not know when exactly) – time estimation according to: ● titer and it's dynamics ● class of antibodies: IgM/IgG ● avidity of antibodies (at the beginning of the infection low-avidity Abs are present)
5/35 Interpretation of indirect serological testing
● acute infection: large amounts of antibodies, mainly IgM class, or both IgM and IgG class (1)
● after infection: small amounts of antibodies, IgG class only (immunological memory) (2)
● chronic infection: variable, according to the activity of infection, microbial species, etc.
(1)
(2)
6/35 Antibody titer
● the inverse of the greatest dilution (in a serial dilution) that still gives a positive result
● we don't prove the presence of causative agent, but the reaction of the immune system (!) → significant individual variability of the patients' immune system responses individual variability, compare low and high reactivity patients (i.e. the same level of titer of two patients in different phases of infection)
7/35 Antibody titer dynamics
● we can't rely on antibody titer level itself
● based on antibody titer dynamics
● production of Ab can take up to 10 days since the first contact with Ag → serological reactions often negative in the first days of the infection
● we can prove increase in titers between paired samples, and decrease (subacute infection)
● titer and it's dynamics doesn't correspond with clinical symptoms (!)
● the largest amounts of antibodies are often reached after clinical symptoms disappear
8/35 Antibody titer dynamics (2)
● 1 – 2: seroconversion
● 3 – 4: titer level rises
● 5 – 6: titer level drops
9/35 Diagnostics of present infection
● paired sera samples
● the first sample taken as soon as possible at the beginning of the infection
● the second taken after 10 or more days since the first sample
● paired sera: – the first sample stored in the refrigerator till the second sample is taken; then both samples are evaluated simultaneously – 4 fold increase in titers between paired samples or seroconversion is significant
10/35 Diagnostics of present infection (2)
● unpaired sera: – second sample is examined separately – significant is 8 fold increase (because of a common laboratory mistake; a common laboratory mistake in serological reactions is one dilution) – positive seroconversion requires the second sample to be at least one dilution higher than the first dilution used (e.g. first dilution 1:4, positive seroconversion requires the second sample to be at least 1:8)
11/35 Complement system
● part of the innate immune system
● system of plasma and membrane glycoproteins – the most important ones are C1–C9
● inactive precursors work in cascade, proteases in the system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages
● inactive precursors cleaved into smaller biologically active part (inflamation mediators C3a, C4a, C5a) and bigger part, which are enzymes with proteolytic activity (b fragments)
● leads to activation of the cell-killing membrane attack complex (C5b, C6, C7, C8, 13-18 C9) → forms transmembrane channel → cell lysis 12/35 Complement system (2)
● complement system activation pathways: – differences in activation the key C3 part – the most important is cleavage of C3 – classical (activation by complex Ag-Ab, phylogenetically youngest, Ab must be produced prior to the activation of the pathway) – lectin (homologous to the classical pathway, activation by mannose-binding lectins; lectins are carbohydrate-binding proteins) – alternative (activation by the surface of a pathogen, phylogenetically oldest, C3b binds to the surface of pathogens, leading to greater
internalization by phagocytic cells by opsonization13) /35 Complement fixation test (CFT)
● exploits complement system properties: – ability to bind only Ag-Ab complexes (can't bind to Ag or Ab alone) – leads to cell lysis
● complement binding to Ag-Ab complex is not visible → addition of indicator system
● indicator system (complex of Ag and Ab → complement can bind to this complex too) – Ag = sheep RBCs – Ab = rabbit anti-sheep-RBC antibodies (a.k.a. amboceptor)
– Abs are pre-bound to Ags in the CFT 14/35 Complement fixation test (CFT): positive and negative result
15/35 Complement fixation test (CFT): positive and negative result (2)
● POS = RBCs sedimentation (complement was bound to complex Ag-Ab of interest)
● NEG = hemolysis (complement wasn't bound to complex Ag-Ab of interest, it binds to indicator system → hemolysis of RBCs)
● can't use patients own complement system (variability among patients); complement system inactivation by heat (30 min/56 °C), Ab are more stable
16/35 False positive and negative CFT
● false negativity: – too much complement added → hemolysis of RBCs even when Ag-Ab complex of interest is present – prevention by complement control (titration of complement)
● false positivity: – some component of the patient's serum binds part of the complement system (or serum is chylous or contaminated) – anticomplementarity test = CFT without
laboratory antigen → should be always negative 17/35 Task 1: Antibody titer dynamics
● fill in the following table using your card
● 4 fold increase in titers between paired samples or seroconversion is significant
● 1 – 2: seroconversion
● 3 – 4: titer level rises
● 5 – 6: titer level drops
18/35 Taks 2: Examination of the indicator system function
● determine which three components are necessary to the proper function of the indicator system
● amboceptor = rabbit anti-sheep-RBC antibodies
19/35 Taks 2: Examination of the indicator system function
● determine which three components are necessary to the proper function of the indicator system
● amboceptor = rabbit anti-sheep-RBC antibodies
1) no hemolysis 2) no hemolysis 3) hemolysis 4) no hemolysis
20/35 Task 3: Detection of Ab (respratory diseases)
● a patient with long term respiratory problems, a few clinical signs, the most probable diagnosis: atypical pneumonia
● atypical pneumonia may be caused by many respiratory viruses, but also several bacteria (Mycoplasma, Chlamydia)
● mycoplasmal/chlamydial aetiology → antibiotics can be used
● viral aetiology → antibiotics would have no effect
21/35 Task 3: Detection of Ab (respratory diseases) (2)
● whole microplate belongs to one patient
● six respiratory diseases, pair sera for each (I+II)
● first column is anticomplementarity test
● CFT starts from the second well with the dilution 1:5
● 5 viruses (influenza A, influenza B, parainfluenza, adenovirus, RS virus), plus one bacterial species Mycoplasma pneumoniae
22/35 Task 3: Detection of Ab (respratory diseases) (3)
● read the titres of CFT in one individual patient
● pay attention to anticomplementarity test in the first column
● draw your result, fill in all dilution values, also fill in titre values in positive cases, fill in increase/decrease level and try to interpret the results
23/35 Task 3: Detection of Ab (respratory diseases) (4)
● influenza A: both titers 1:5 (patient ill in the past, but this is not acute disease)
● influenza B
● parainfluenza no antibodies, i.e. patient ● adenovirus never suffered from this diseases ● RS virus
● Mycoplasma pneumoniae: – titer increase from 1:10 to 1:160 (16 fold increase, ≥4 fold increase significant) – suspected Mycoplasma pneumoniae infection
24/35 Task 4: Detection of Ab (tick-born encephalitis virus)
● three patients with suspected tick-born encephalitis, all have neurological symptoms and were bitten by a tick
● first row is positive control
● 2. and 3. row first patient (Khaled)
● 4. and 5. row second patient (Laura)
● 6. and 7. row third patient (Maria)
● evaluate similarly to the previous task, pay attention to anticomplementarity test in the first column
● first dilution is 1 : 4 (continues: 1 : 8, 1 : 16,
1 : 32, ...) 25/35 Task 5: Detection of Ab (toxoplasmosis)
● read CFT titres in the sera of patients suspect of Toxoplasma gondii infection
● Toxoplasma gondii is tissue parasitic diseases, definitive hosts are domestic cats
● seronegativity = pacient never suffered from this disease of the infection is going on too shortly too detect antibodies
● seropositivity will be further examined (another blood sample, e.g. ELISA for detection of class of Abs)
● first row is positive control, first column is anticomplementarity test, don't examine antibody titer dynamics
26/35 ● conclude as positive, negative, or anticomplementary Neutralisation tests
● serological methods, in which antibodies prevent antigen to manifest it's effect (mainly viruses and bacterial toxins) – virus netralisation test (VNT): ● Ab neutralise virus infectivity ● cell culture inoculated with virus and anti-virus Abs will stay intact (virus has no effect on cells) – hemagglutination inhibition test (HIT) ● virus can't agglutinate RBCs in presence of Abs (not a case of hemolysis!) – ASO = prove of antistreptolysin O (Abs capable
of causing autoimmune response) 27/35 Anti-streptolysin O (ASO)
● during infection Abs against streptococci, including Abs against streptolysin O (streptococcal toxin)
● if level of these Abs rises after infection they are used for diagnosis and follow up of rheumatic fever and acute glomerulonephritis (cross-reaction with human antigens, mainly collagen)
● doesn't serve for diagnosis of present infection, serves for diagnosis of late effects after streptococcal infection (autoimmune response)
● prove of Abs → indirect serological method
28/35 Task 6: ASO
● neutralisation of hemolysis
● streptolysin O under normal circumstances (without Abs) causes RBCs hemolysis NEG = hemolysis
● presence of Abs (antistreptolysin O) neutralise streptolysin O → RBCs stay intact and sedimentate POS = RBCs sediment
● titer above 200 I.U. → risk of developing autoimmune response (rheumatic fever and acute glomerulonephritis)
Well 1 2 3 4 5 6 7 8 9 10 11 12
Units 100 120 150 180 225 270 337 405 506 607 759 911
Risk none threshold great 29/35 Task 6: ASO (2)
● first row is positive control
● other rows are patients samples
● dilution values are in the protocol
30/35 Task 6: ASO (3)
● positive control is positive
● high risk in patients 3 and 4 (506, and 337 I. U.)
● threshold values in patient 1 (225 I. U.) and patient 7 (180 I. U.)
● in patients 2 and 6 no risk (titers are low)
● patient 5 has never suffered from streptococcal disease (previous diagnosis of streptococcal infection was wrong, most probable is previous viral infection)
31/35 Task 7: Hemagglutination inhibition test (HIT)
● several patients with suspected tick-born encefalitis
● previously tested by CFT
● HIT serves as indepented test to validate results of CFT (there is different set of patients in this task, therefore no correlation with task 4)
● virus can't agglutinate RBCs in presence of Abs (not a case of hemolysis!) – POS = sedimentation of RBCs (Abs prevent agglutination of RBCs) – NEG = hemagglutination (Abs not present or too low level to prevent agglutination of RBCs)
32/35 Task 7: Hemagglutination inhibition test (HIT) (2)
33/35 Task 7: Hemagglutination inhibition test (HIT) (3)
● read and draw results, fill in missing fields
● positive control in the first row
● dilution 1 : 5 in the first column, continues (1 : 10, 1 : 20, 1 : 40 etc.)
● make a clinical conclusion (acute infection/only memory cells/etc.)
● antigen control – antigen (virus) without Abs agglutinate RBCs
● RBCs control – RBCs don't agglutinate because of other unknown reason
34/35 Task 7: Hemagglutination inhibition test (HIT) (4)
● results: – one patient with acute infection – two patients only memory cells (past infection) – one patient has never been infected (no Abs)
35/35