Immunology & Serology PDF

IMMUNOLOGY & SEROLOGY IMMUNOASSAY FOUR: OTHER  The latent period required for the SECONDARY IMMUNOASSAYS development of rheumatic fever symptoms...

IMMUNOLOGY & SEROLOGY IMMUNOASSAY FOUR: OTHER  The latent period required for the SECONDARY IMMUNOASSAYS development of rheumatic fever symptoms coincides with the time required for the NEUTRALIZATION TES production of high titers of anti- streptococcal antibodies Principle of neutralization tests rests on the ability of an appropriate antibody to diminish or abolish  This may suggest that these antibodies may some biologic activity of the antigen other than its be involved in the etiology of rheumatic antigenicity: fever  Toxicity or infectivity for cells or laboratory  These antibodies are primarily detected animals against the antigens of the infecting GAS, and secondarily, against the cardiac tissue  Enzymatic activity because of its structural similarity to the GAS Neutralization tests are usually restricted to toxins, viruses, and enzyme-like Ag but can be performed  Only a small percentage of patients with with most infectious agents GAS infection develop rheumatic fever (2- 3%); however, once it has been initiated, the Specific toxin-neutralizing immunoglobulins are incidence of recurrence after a subsequent known as antitoxins streptococcal infection increase to 50% Toxins treated with formaldehyde are converted to  As a result of previous contact with toxoids which are used in immunization to induce streptolysin O, healthy people may have formation of antitoxins titers as high as 125 Todd units These antitoxins can be used to identify the  Therefore, a single titer on a patient is of corresponding toxin by the capacity of the antitoxin little diagnostic value unless it is extremely to protect laboratory animals against the lethal high activity of that specific toxin but not of other toxins  Most patients with RHD have ASO titers of This is applied in the identification of certain about 500 Todd units viruses isolated from patients  A fourfold rise in titer between paired sera With a stock of known viruses, antibodies in a taken 2-3 weeks apart is indicative of a patient’s serum (acute and convalescent) can also current or recent streptococcal infection be defined  A drop of titer from 250 to 125 Todd units Antistreptolysin O Test (ASO) over a longer period of time is indicative of  Used to detect antibodies that appear in a recovery patient’s serum as a result of an antecedent  One type of ASO test involves inhibition of infection with group A streptococci hemolysis by streptolysin O with the  Important laboratory procedure showing antitoxin antistreptolysin O neutralization reaction  Streptolysin O (SLO) can cause nonimmune  Group A streptococci release several lysis of RBC if unopposed by important harmful agents: antistreptolysin O (ASO)  Hyaluronidase  If the patient’s serum contained ASO, these will combine with the reagent SLO (antigen),  Erythrogenic toxin neutralizing the hemolytic activity of SLO  Streptolysin S (SLS) – not immunogenic;  Commercial kits are available that contains oxygen-stable SLO, special buffer, 5% rabbit or human  Streptolysin O (SLO) – immunogenic; group O RBC, and an ASO reference oxygen-labile standard Degones, C.  IMMUNOLOGY & SEROLOGY  Care must be taken to avoid agitation Controls and Standards (aeration) of SLO because it oxidizes readily  RBC control should not lyse and loses its hemolytic property  Streptolysin control should produce  The dried, powdered form of SLO must not complete lysis be rehydrated unless it is to be immediately added to the test system  ASO reference standard is a lyophilized antibody of known ASO titer  When ASO reference standard is reconstituted with buffer according to directions, it may represent a 1:100 dilution; therefore, it must be run in parallel with the unknown in the 1:100 dilution series  The antistreptolysin O test is the most common serological test for the detection of group A streptococcal (GAS) infection Antistreptolysin O Test (ASO)  However, there are various agglutination tests for ASO available – e.g. ASO latex Sample Test Procedure agglutination test  Tubes are gently shaken to mix serum and buffer  Add 0.5 mL of reagent streptolysin O (SLO) to each tube  Gently shake and incubate at 37°C for 15 minutes – during this incubation, ASO when present in the serum will combine with the reagent SLO and neutralize it so that it cannot lyse the RBC  RBC suspension is added; tubes are gently COMPLEMENT FIXATION TES shaken, and incubated again for 45 minutes – during this second incubation, any free Complement itself can actually be used as a SLO can attach to RBC and cause them to reagent in the test known as complement fixation lyse Because complement fixation occurs after the  Tubes are centrifuged and read for degree binding of antigen and antibody, uptake of of hemolysis complement can be used as an indicator of the presence of either specific antigen or antibody  End-point titer is the reciprocal of the greatest dilution showing no hemolysis This technique has been used in the detection of viral, fungal, and rickettsial antibodies  Each unit of titer is called a Todd unit, named for E.W. Todd who pioneered the Only IgM and IgG can activate the complement test in 1933 system  Todd units only refer to ASO, not to any Activation of complement system by immune other kind of antitoxin complexes results in generation of factors capable of disrupting cell membranes  Absence of hemolysis means a positive test while hemolysis means a negative test If the immune complexes were generated on erythrocyte surfaces, then the erythrocyte cell membranes will be lysed – i.e., hemolysis Degones, C.  IMMUNOLOGY & SEROLOGY This reaction is used to measure serum antibody  Complement can be non-serologically levels inactivated by: Complement fixation test (CFT) involves a two-  Vigorous agitation stage process:  Chemicals and enzymes  Test system with antigen and antibody, one  Yeasts or bacterial cells of which is unknown  Tissue extracts  Indicator system consisting of sheep RBC coated with hemolysin (also known as  Hemolysis amboceptor), which will lyse in the presence of complement  All glassware used must be chemically and bacteriologically clean Test System Indicator System  To destroy any complement present, patient’s serum must be heated at 56°C for  Required to detect whether or not 30 minutes prior to testing complement has been bound by interaction between patient’s serum (containing  Then, dilutions of patient’s serum are antibody) and the Ag added combined with known Ag and a measured amount of normal guinea pig serum  Usually consists of sensitized sheep RBC – i.e. anti-sheep RBC attached to sheep RBC  Guinea pig serum shall provide a source of complement  Anti-sheep RBC are commonly produced by injecting rabbits with washed SRBC  Guinea pig serum is most commonly used because its complements have  If an antibody can activate complement and high hemolytic activity cause immune lysis of the SRBC, it is termed hemolysin (also known as  Guinea pig complement can be used amboceptor) for testing because complements are not species-specific  In this case, anti-SRBC is the amboceptor  If patient antibody is present, it will combine  In the absence of complement, the with the reagent antigen, and complement undiluted amboceptor behaves as an SRBC will be bound hemagglutinin  The amount of free complement remaining  At high dilutions (low concentrations), the is measured by adding and indicator system only demonstrable serological effect of the amboceptor is fixation of complement Precautionary Measures  In the presence of complement, the  Patient’s serum should be heat-inactivated amboceptor behaves as a hemolysin prior to each test since some degree of complement reactivation may temporarily  Anti-SRBC is a stable antibody under regained within a day following heat refrigeration inactivation Indicator System  Complement will rapidly lose its hemolytic Interpretation activity:  Lysis of sensitized SRBC, seen as the  Within 2 hours at room temperature development of a transparent red solution,  Over 3 or 4 days under refrigeration is a negative result, since it indicates that complement was not fixed and that  One month if stored at -40°C antibody was, therefore, absent from the patient’s serum  Longer periods if lyophilized Degones, C.  IMMUNOLOGY & SEROLOGY  Absence of lysis, seen as a cloudy red suspension, is a positive result, since it indicates presence of antibodies directed against the reagent Ag, resulting to Ag-Ab interaction which consumed the complement; therefore, little or no complement was available to lyse the sensitized SRBC  End-titer may be considered to be the highest dilution of serum in which no more than 50% of SRBC are lysed  Prior to performing CFT, both amboceptor and complement must be titrated to determine their respective “units” – i.e., the smallest amount of each reactants that interact to completely lyse the quantity of SRBC that will be used in the CFT (standardization step)  Addition of the correct amount of complement is critical:  Too little complement results in incomplete lysis  Excessive complement is not completely fixed by immune complexes and therefore will lead to false negative results  Most important disadvantage of this test is its complexity, particularly with regard to the standardization and preparation of the reagents required  Best known example of CFT is the Wasserman test for the detection of syphilitic reagin Degones, C.