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Background: Different antibodies were produced during primary and secondary antibody responses. The types of antibodies produced and their relative quantities during immunisation were studied in this experiment. Methods: Mice sera were observed for haemagglutination after first immunisation and second exposure to antigen. Results: A mean of 0, 4.5, 1.5 and 7.5 wells with haemagglutination for non-dithiothreitol (non-DTT) treated wells, and 0, 0, 1.5 and 7.5 wells with haemagglutination for DTT-treated wells, before immunisation (day 0), day 7, 14 and 21 (7 days after second exposure) respectively. Conclusion: IgM was predominant during primary, and IgG was predominant during secondary antibody response.
Keywords: Adaptive, Haemagglutination, Antibodies, Immunisation, Mouse, Titre
The main role of the immune system is to protect the host from infection. The immune system is comprised of two systems: the innate and the adaptive immunities. Innate immunity includes skin and mucosal membrane, the natural killer (NK) cells, macrophages, dendritic cells, granulocytes and certain cytokines (Hughes & Mestas 2004). In innate immunity is non-specific to antigens (Hughes & Mestas 2004).
In order to allow maximum protection of the host, it is significant to comprehend the important link between the innate and the adaptive part of the immune system. The adaptive immunity is capable of recognising specific antigens through antigen presentation (Janeway & Medzhitov 1998). It involves T cells and B cells (Janeway & Medzhitov 1998). T cells have specific T-cell receptors (TCR) present on the cell surface that help to detect intracellular infected cells, caused by viruses, intracellular bacteria and parasites, in the host (Janeway & Medzhitov 1998). Like T-cells, B cells have specific receptors on the cell surface, and when activated, will develop into plasma cells to produce antibodies (Janeway & Medzhitov 1998). B cells and their antibodies are important in fighting against extracellular bacterial and parasitic infections (Janeway & Medzhitov 1998).
Haemhaemagglutination occurs when immunocomplex, formed by the interaction of antigen coated blood and antibody, becomes insoluble and precipitates. As such, observation of haemagglutination can be used as an indication for the presence of antibody responding to an antigen (Mariano 1964). Haemagglutination assay has been widely used as a method for quantification of antibody, bacteria or viruses in the blood (Killian ML 2008).
Primary and secondary exposures to antigen usually have different antibody responses in a host. Memory cells were formed from the plasma cells after the first exposure to antigen (Janeway & Medzhitov 1998). These memory cells will recognise the antigen, and produce a stronger response upon future exposure to the same antigen. Such property of secondary antibody response is important in the field of vaccination. Understanding antibody response can contribute to the studies of vaccination including the development of human immunodeficiency virus (HIV) and dengue vaccinations (Keefer et al. 2011; Webster et al. 2009).
Mouse has been used frequently as an in vivo study species for human biology because of the many similarities between a mouse and a human (Hughes & Mestas 2004). In this experiment, sheep red blood cells (SRBC) were coated with heat-killed Staphylococcus aureus to determine the types of antibodies produced and their relative quantities during primary and secondary antibody responses.
Materials and Methods
Staphylococcus aureus was inoculated into a saline solution from a blood agar plate culture. The concentration of Staphylococcus aureus in the saline suspension was determined by dilution in saline and counted using a Helber chamber (BDH, Sydney, Australia). The suspension of Staphylococcus aureus was subjected to 600C for 5 minutes in a water bath. It was then cooled to room temperature. The heating and cooling was repeated for another 2 cycles. A blood agar plate was inoculated with the heat killed Staphylococcus aureus suspension, and incubated to check for any growth.
Blood smears of 6 mice, on 6 different slides, were stained for 7 minutes with Geimsa stain. Equal volume of rinse buffer was added to dilute for one minute. After washing off the stain with rinse buffer, the slides were examined under the microscope to determine the whether the mice were immunocompetent. The percentage of lymphocytes, monocytes and neutrophils were obtained for the 6 slides by counting.
After which, the mice were immunised with the heat killed Staphylococcus aureus and a second immunisation was given after 14 days of the initial immunisation. Blood samples were collected before the immunisation (day 0), day 7, day 14 (before second exposure to antigen), and day 21 (7 days after second exposure). The blood sera (supernatant) of the respective mice and day intervals was obtained by centrifuging at 2000 rpm for 10 minutes, using Hettich T4000 (Melbourne, Australia).
A serial doubling dilutions of each serum, starting at 1:2, was prepared across 8 rows of 10 wells in a 96-well microtitre tray. Each well was added 50Î¼L of the serum. SRBC coated with Staphylococcus aureus particles were washed 3 times and suspended at 1% v/v in saline. The suspension was divided into 2 portions, with one of the portions treated with dithiothreitol (DTT). An equal amount of the non-DTT-treated suspension was added to the first 4 rows of the wells. The next 4 rows of wells were added with DTT-treated suspension. The process was repeated for all the 6 mice. The microtitre trays were mixed gently and incubated for one hour at 370C. Haemagglutination titres were observed.
The percentage distribution of lymphocytes, monocytes and neutrophils (Table 1; Figure 1) in every mouse was within acceptable range according to the reference table provided by the demonstrator. After the heat-kill of Staphylococcus aureus, there was no growth upon inoculation onto the blood agar plate after incubation. The concentration of Staphylococcus aureus was 109cells/mL in the saline suspension.
Non-DTT-treated wells with haemagglutination showed the presence of IgG and/or IgM in the blood serum. There was no haemagglutination in day 0. The mean number of wells with haemagglutination was 4.5 at day 7. There was a decrease to a mean number of 1.5 wells with haemagglutination at day 14. At day 21, the mean number of wells with haemagglutination increased to 7.5 (Table 2).
Since the presence of DTT prevented haemagglutination of IgM with antigens, the haemagglutination of the DTT-treated wells indicated the presence of IgG only. There was no haemagglutination in the negative control wells at day 0. There was also no haemagglutination at day 7. The mean number of wells with haemagglutination was 1.5 at day 14 and increased to 7.5 at day 21 (Table 3).
Presence of Antibodies during Immunisation
The presence of IgM was obtained by subtracting the mean number of wells with haemagglutination in the DTT-treated rows (IgG) from the non-DTT-treated rows (IgM and IgG) (Table 4). The haemagglutination titres were the Log2 of the number of wells with haemagglutination. The results showed the presence of IgM at day 7 after immunisation, but not present at day 14. There was also no haemagglutination at day 21. Unlike IgM, IgG was not present at day 7. However, the amount of IgG increased at day 14 and shapely increased at day 21 (Table 5; Figure 2).
The initial microscopic observation of the mice blood was to make sure they were immunocompetent. The Staphylococcus aureus was required to be killed to prevent it from causing disease to the mice.
During the initial exposure to the coated SRBC, IgM was the first antibody to be produced. At day 14, the level of IgM fell due to the fall in the stimulating antigen level (Gilbert & Perlmutter 1984). Class switching was involved in the production and maturation of IgG (Gilbert & Perlmutter 1984). Memory cells were formed by plasma cells, and most of them had undergone class switching which will produce IgG (Janeway & Medzhitov 1998). The affinity of these antibodies and the antigen was low during the primary antibody response (Batista & Harwood 2010).
However, during the secondary antibody response (exposure of antigen at day 14), most of the antibodies produced were IgG. At the second exposure, the memory cells were activated and differentiated into plasma cells (Janeway & Medzhitov 1998). Since the most memory cells were class switched to IgG, the antibody produced was IgG. The binding affinity of IgG to the SRBC in the secondary response was much higher than the first response, thus helping the host to eliminate the pathogen fast (Alexander et al. 1987). Having higher binding affinity of IgG to antigen also increased the sensitivity of antigen detection thus resulting in higher haemagglutination titre too (Alexander et al. 1987). Although there might have presence of IgM, however, IgM may not get detected due to more IgG binding to the antigen (Batista & Harwood 2010).
In conclusion, both IgM and IgG were produced by the mouse upon immunisation. IgM was the predominant antibody produced during primary antibody response, and IgG was the predominant antibody produced during secondary antibody response. During secondary antibody response, the total level of antibodies was much higher than during the primary response.
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