Posted at 11.27.2018
Practical Record: Heamagglutination
Many important assays are based mostly around an antibodies capability to its antigen and cause a change in its physical point out which is generally visible by vision. One example of the is agglutination, this often occurs when the antibody binds to the antigen which is displayed on the top of a big particle like a pathogenic bacterium. However everything we are looking at in this experiment is the power of antibodies to bind to the antigens viewed on the surface of red bloodstream cells (RBC), this is recognized as haemagglutination. With this experiment we added antibody to sheep red blood vessels skin cells, if agglutination occurs then the observation will be that the red bloodstream cells will form a continuous layer across the base of the microtitre dish well. If no agglutination occurs then the observation will be a little clump at the bottom of the V-shaped well due to the settling of the cells. The main goal of this test is to learn the result that immunization has on the focus of antibodies present and whether immunization has a good affect helping the disease fighting capability produce antibodies to protect against pathogens. I expect that the more the sera has been immunised then your more agglutination will appear, as increased immunization leads to a higher focus of antibody created so when dilution is completed the concentration will stay higher for longer allowing agglutination to still occur. (239 words)
Materials: - V-bottomed Microtitre plate
- Phosphate buffered saline (PBS)
- 1% Sheep red bloodstream cells (SRBC)
- 3 Serum samples:-
Sera 1: Pre-immunization
Sera 2: Rabbit anti sheep red blood vessels cell antisera 6 days after one immunization with SRBC
Sera 3: Rabbit anti sheep red bloodstream cell antisera seven days after the third of three separate immunizations with SRBC. Each immunization was carried out 7 days aside.
50l of PBS was added, using a Gilson P200 versatile pipette, to all he wells in four rows (A, B, C & D) of an V-bottomed microtitre plate. Then another 50l of PBS was added to well A1, 50l of serum 1 was put into well B1, 50l of serum 2 was put into well C1 and 50l of serum 3 was added to well D1. A1, B1, C1 & D1 were then twice diluted over the microtitre plate. Following this was completed 50l of 1% SRBC was put into every well in the four rows used and they were kept at room temperature for one hour before observations could be produced. (180 words)
Fig. 1 shows my results, the original PBS and serum combine is the furthest well to the left with each two times dilution another well to the right. You can view that in some wells, mainly the first two rows, there may be a tiny red clump at the bottom of the V-bottomed microtitre plate displaying that no agglutination has happened, but others have a red carpet-like covering exhibiting that agglutination has occurred. (75 words)
Our results show some wells that contain been through agglutination as there's a red carpet-like covering however others have a small red clump at the bottom of the microtitre well. The results from the first two rows show no agglutination has happened in any of the wells as there is certainly a small circular clump at the bottom of the well. Within the first three wells of the 3rd row there is a red carpet-like covering, this shows that agglutination had took place. The reason it has agglutinated is due to the actual fact the serum added to well C1 had been immunized with SRBC 6 times before we had used it, this possessed allowed the immunoglobulins to get approved it's 'lag stage' (around 6 days and nights) and reach the primary stage where Immunoglobulin Mu (IgM) is produced allowing binding so agglutination occurs. No more wells have agglutinated due to the dilution, decreasing the focus by 4 collapse each well. 6 in 7 wells on the fourth row have agglutinated, more wells haven't agglutinated due to the fact that the serum put into well D1 was immunized three times over 21 days prior to utilize. This means the 'lag period' has been surpassed and the supplementary response has happened, by this stage memory skin cells are produced so antibodies get produced faster and in an increased amount. (222 words) (Talk about about the anomaly on 4th row)
The results are as I would have expected; the first row only is made up of PBS and the second row contained sera 1 which was pre-immunisation, this supposed it included no antisera, signifying no antibodies were present to produce immunoglobulins. The results from the next two rows are also in accordance from what I would've expected as the first few rows have agglutinated with an increase of agglutination happening in the final row credited to a higher attentiveness of antibodies present. Overall I believe the experiment spent some time working as expected, displaying that immunisation has an enormous effect on the awareness of antibodies present and that it is crucial in helping the disease fighting capability to combat off invasive pathogens. (116 words)
Sera 1 possessed a titre of 0 as no agglutination occurred so no antibody was present. Sera 2 acquired a titre of 8 as agglutination previous took place in well 3 which experienced a dilution of 1 1:16. Sera 3 had a titre of 2048 as agglutination last took place in well 7 which got a dilution of just one 1:4096.
The different serum samples have different antibody titres due to the immune system response of the rabbit. The reason serum one has no antibody titre is due to the fact it was never immunised, signifying it never had any antisera so immunoglobulins weren't present and in a position to create antibodies. The titre of serum two is higher as it was immunised 6 days and nights prior to make use of, this means antisera exists and therefore immunoglobulins are present so antibodies can be created as the 'lag phase', of around 6 days and nights, has been surpassed. The titre of serum 3 is the best due to the fact it's been immunised 3 times over 21 days prior to utilize. This means that not only the 'lag phase' has been surpassed but the primary and secondary response of the immune system has been triggered meaning that much more antibody is created when the prospective antigen is discovered. (154 words)
The main isotypes of serum 2 and 3 will be Immunoglobulin Mu (IgM), as this is what is created during the primary immune system response and is very good at creating agglutination, and Immunoglobulin Gamma (IgG), as this is of the best awareness in the bloodstream and also offers the longest half-life, of around 21 days.
A laboratory test which could be completed to confirm the isotypes of anti-SRBC antibody in the serums would be Immunofluorescence. The primary antibody recognises an antigen and binds to a particular region known as the epitope. The fastened fluorochrome can be detected by fluorescent microscopy. Direct Immunofluorescence has certain advantages within the secondary (indirect) method. Direct attachment of the messenger to the antibody reduces the amount of steps, conserving time and minimizing non-specific background signal. This limits the opportunity of cross-reactivity and potential flaws along the way. However, as the number of fluorescent molecules able to bind to the principal antibody is limited, direct immunofluorescence is a lot less sensitive and frequently results in false negatives. Indirect uses two antibodies; the primary antibody binds the target molecule and the supplementary antibody, which holds the fluorochrome, recognises the primary antibody and binds to it. However, multiple extra antibodies have the ability to bind to an individual key antibody, this causes sign amplification by increasing the amount of fluorochrome molecules per antigen. This is more complex and time consuming than the direct procedure, but permits more overall flexibility as different secondary antibodies and diagnosis techniques can be used for the principal antibody. (200 words)
A) The pathogenesis of Hashimoto's thyroiditis is sophisticated and includes numerous factors. A lack of tolerance to normal thyroid cells can result in the production of antibodies against thyroid cells, this triggers the thyroid gland to be damaged. The major histocompatibility complex (MHC) antigen delivering cells, including dendritic cells and macrophages, invade the thyroid gland. These present autoantigen components of the thyroid to the immune system. Thyroglobulin is the main protein stated in the thyroid, it is considered to have around 40 different epitopes, and play a essential role in the pathogenesis of the condition. The formation of auto-reactive cells, directed up against the thyroid, could derive from flaws in central or peripheral tolerance. Lack of immune tolerance is associated with genetically decided immune defects or having less regulatory T-cells which impose the suppressive function. That is followed by development, clonal expansion, and maturation of T-lymphocytes and B-lymphocytes in the lymph nodes. A stage of autoimmunity the follows, characterized by uncontrolled development of self-reactive cells and autoantibodies in response to the antigens. This initially occurs in the lymph nodes but as the disease progresses the process moves to the thyroid where the development of lymphoid tissue follows. The B-lymphocytes produce antithyroglobulin (TGAB) and antithyroid peroxidase (ATPO) antibodies which are directed against thyroid cells. The auto-reactive T-cells infiltrate the thyroid and mediate destruction through cytotoxicity. Macrophages activated in this technique produce cytokines which, along with antibodies, cause apoptosis. Finally, caspases induce enzymes which are directly mixed up in destruction of thyroid gland. In a standard thyroid, development of new skin cells and destruction of old skin cells is controlled around a constant, however this control is lost during the course of the disease. The severity and clinical results are determined by the rate of apoptosis. Symptoms are similar to an underactive thyroid gland, which are very general and can include: fatigue, putting on weight, constipation, dry epidermis, depression. The swollen thyroid gland could also cause a lump to form in the throat, known as agoitre. (331 words) B) In anagglutinationtestserumis put into atest tube which includes a particularantigen. In the event the antibodies an antigens agglutinate and formimmune complexes, then the test is positive. However, if way too many antibodies can be found which have the ability to bind to the antigen, then your epitopes become layered by antibodies meaning few or no antibodies are able to bind to several antigen. This means the antibodies are unable to bridge between antigens, which in turn causes no agglutination that occurs, hence the test is interpreted as negative. However, in this case, the effect is a false negative. The area of high antibody concentrations within which no agglutination was able to occur is called theprozone. To conquer the result of prozone the attention of the agglutinin, in this case the antibodies, must be lowered. This dilution means you will see fewer antibodies present which allows the antibodies to bind to one or even more antigens and allow agglutination to occur. (166 words)