Antigen-Antibody Interactions: an analysis

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. Results Part A. The agglutination technique employed presently is the test tube method. Agglutinations were visualised as white particulate matter clumped together at the bottom of the test tubes. The scoring method, from 0 to 4, used presently is that adapted from Olopoenia et al. , 1999, where 0 is recorded for no agglutination and 4 for 100% agglutination. Olopoenia et al. 1999 indicates that the smallest quantity of serum that exhibits a score of 2 is considered to be the titre.

Agglutination was observed only in tubes 1-3 for both serum samples A and B (a titre of 20 for both samples was recorded; see Table 1 in Appendix) when incubated with S. paratyphi A. However, inoculation with S. typhi yielded agglutination in tubes 1-3 inclusive for serum sample A (a titre of 40), whilst tubes up to and including six were positive for agglutination for serum sample B, yielding a titre of 320 (see Table 2 in Appendix). The control tube (10) was absent of agglutination in both scenarios. According to Sansone et al. , 1972 a four-fold increase in titre is evidence to support a current infection.

As no change in agglutination is observed between serum sample A and B for S. paratyphi A, this indicates no infection. However, an eight-fold increase in agglutination levels between serum sample A and B for S. typhi is evidence for a current infection. Part B. The optical density of the precipitate solutions was measured at 280 nm; OD280 provides an indication to the total protein content. Graph 1 (in Appendix) shows the relationship between absorbance at OD280 and the amount of BSA antigen.

The amount of antigen at equivalence point is 103 g (in 1 mL of NaOH); through a series of calculations shown in the Appendix the concentration of antibody is determined as 0. 88 mg/mL. Discussion Interpretation of the Widal Test Olopoenia et al. 1999 suggests that the Widal test is subject to many controversies and depending on the setting of its use, may produce inaccurate results. Indeed presently only a single Widal agglutination test was performed; Hoffman et al. , 1986 indicated that a single Widal test is not diagnostically significanct, producing unreliable results.

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).

Vincent et al. , 1970 reports a lag period being evident when the reaction proceeds slowly as is the case during the antigen excess zone, however no such observation is made presently. This apparent discrepancy is attributable to an increased concentration of reactants employed here. This leads to decrease in the inter-particle space and subsequently increases the number of collisions, removing the lag period. Hawkins 1964 reports that the optical properties exhibited by precipitates formed in the various regions of the precipitin curve are not identical.

For example, an analysis of Graph 1 shows that the precipitates of the antigen excess region show more turbidity than precipitates of the antibody excess region. This is due to differences in the physical properties of the precipitates. In the antibody excess zone, there is reduced compactness in the packing of these bulky complexes; more light is transmitted through the sample resulting in a lower absorbance reading. Conversely, in the antigen excess zone, the complexes contain a relatively lower proportion of antibody and are able to pack more tightly; less light is transmitted resulting in a higher absorbance reading.

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