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Antigen-Antibody Interactions: an analysis

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Antigen-Antibody Interactions: an analysis Introduction Common to all antibodies is the specificity they exhibit in binding to epitopes present on the surface of antigen in which ultimately a lattice formation results due to cross-linking. The present experiment exploits this interaction and is physically observed as agglutination (when antigen is present on a cell) or precipitation (antigen free-floating in solution) (Elek et al., 1964). Presently, the experiment uses both qualitative and quantitative analyses in order to ascertain the levels of antibodies in sera; such information, for example, is useful in assessing the presence of a current infection in a patient. More specifically, during Part A, presence of agglutination, due to the interaction between Salmonella H (flagellar) and O (somatic) antigens and the appropriate antibodies, was used as a qualitative measure in order to assess whether the patient is currently infected with Salmonella typhi or paratyphi A strains, a process known as the Widal test (Parry et al., 1999). Part B employed a quantitative analysis to determine the level of precipitation upon interaction between bovine serum albumin (BSA) antigen and antiBSA to allow the determination of the concentration of antibody in an original antiserum sample. Materials and Method As per BIOL3141 Infection and Immunity Laboratory Manual 2011 pg 19 -26. ...read more.


Bearing this in mind, an analysis of the observed results ensues. S. typhi (group D serotype) expresses O antigen 9 and 12; conversely, S. paratyphi A (group A) possesses O antigen 12 (Carlsson et al., 1972). As both organisms contain O antigen 12 the possibility of cross reactions arises; O antibody of group D serotype can bind to O antigens present on group A and D organisms. Olopoenia et al., 1999 reports that cross-reactions may occur frequently, lessening considerably the diagnostic specificity of the Widal test. The effect here is not clear as only a single test was performed. However, this may explain the observed no increase in titre (yet presence of agglutination) in tubes subject to S. paratyphi A. Another explanation concerning the agglutination in these tubes: small amount of S. paratyphi A antibodies maybe present in sera as a result of immunisation or previous infection. Antigen/antibody interactions of BSA There are three distinct elements in Graph 1. The first, excess antiBSA, resulting in complexes that are small, with limited bridging. The second region, equivalence, represents an optimum ratio of antiBSA to BSA antigen, resulting in a lattice formation and consequently precipitation. In the third region, excess BSA antigen, there is a reduced supply of bridging antibody molecules in relation to BSA antigen (Janeway et al., 2007). ...read more.


1 2 3 4 5 6 7 8 9 10 Precipitation 4 3 2 0 0 0 0 0 0 0 Dilution 1/10 1/20 1/40 1/80 1/160 1/320 1/640 1/1280 1/2560 - Titre 10 20 40 80 160 320 640 1280 2560 - Serum B Tube No. 1 2 3 4 5 6 7 8 9 10 Precipitation 4 4 3 3 3 2 0 0 0 0 Dilution 1/10 1/20 1/40 1/80 1/160 1/320 1/640 1/1280 1/2560 - Titre 10 20 40 80 160 320 640 1280 2560 - Graph and Calculations Amount of antigen at equivalence point: 103 �g (in 1 mL of NaOH). 1 mg/mL solution of the BSA antigen has an OD280 of 0.660. Thus the absorbance due to antigen is 0.103 mg/mL * 0.660 = 0.068. The absorbance due to antibody is then the total absorbance minus the optical density due to the antigen added at equivalence. That is total absorbance at equivalence is 0.68; the absorbance due to antibody is this total absorbance minus the absorbance due to antigen. Therefore 0.68 - 0.068 = 0.612 The mass of antibody is then calculated using the knowledge that 1 mg/mL solution of antibody equates to an absorbance of 1.4. Thus an absorbance of 0.612 gives the concentration of antibody at equivalence point to be 0.44 mg/mL. Considering the original antibody was present in 0.5 mL and was diluted 1 in 2, the antibody present in the original antiserum sample was: 0.88 mg/mL. ...read more.

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