An enzyme linked immunosorbent assay ELISA was used to quantify IgM IgE Essay

An enzyme linked immunosorbent assay (ELISA) was used to quantify IgM, IgE and IgG, as shown in figures 1 and 2. The optical density measured was the result of a colour change during the assay. This was due to antigens binding to the immobilised antibody coating the ELISA plate wells. This allowed horseradish peroxidase (HRP) conjugated secondary antibodies to also bind to the antigens, which when the chromogenic TMB substrate was added caused a reaction resulting in a colour change (1). As there was no statistically significant difference between the patient and control IgM, IgE and IgG antibody concentrations, this suggests that patient D does not have abnormalities in any of the three immunoglobulins.

This rules out hyper IgM syndrome, which is associated with increased IgM and decreased IgA, IgG and IgE (2). The results also eliminate X-linked agammaglobulinemia, which is due to the decreased production of B cells, causing reduced levels of all isotypes of immunoglobulins (3). In addition, Omenn’s syndrome is associated with increased serum IgE levels; as the IgE level in patient D was comparable to the control, this also cannot be the diagnosis (4).

Don't use plagiarized sources. Get Your Custom Essay on
An enzyme linked immunosorbent assay ELISA was used to quantify IgM IgE Essay
From as low as $9/Page
Order Essay

Omenn’s syndrome results in death within the first year of life, which due to the age of patient D again rules out this disorder (5). Oxidative Burst Oxidative burst, stimulated by PMA, allowed ROS to be produced by neutrophils during phagocytosis (6). Neutrophils incubated with PMA stimulated oxidation of the DHR probe by hydrogen peroxide. Non-fluorescent DHR was oxidised to the fluorescent form of rhodamine, which allowed detection of ROS via flow cytometry (7). On the other hand, PBS did not stimulate oxidative burst which resulted in fewer ROS being produced. The results in figure 3 suggest that the neutrophils of patient D responded normally to the negative control of PBS and the positive control of PMA. This eliminates disorders such as chronic granulomatosis disease, in which neutrophils are unable to carry out oxidative burst and fail to produce ROS; this would result in reduced ROS production when treated with PMA (7). CD62L Shedding Assay Shedding of CD62L was examined using PBS, PMA and LPS. The reduced signals shown in figure 4 for PMA and LPS results indicate CD62L shedding. This was due to the recognition of danger signals by toll-like receptors (TLR’s). MyD88 is an adaptor required for TLR signalling, which leads to activation of the NF-єB pathway (8). When T cells are activated, CD62L is shed. On the other hand, neutrophils treated with the negative control of PBS showed a lack of shedding, due to no danger signal recognition.MyD88 deficiency is associated with a lack of selectin shed; if the signal is low, this signifies CD62L shedding. If the signal is high, this indicates little CD62L shedding and possible MyD88 deficiency (9). The similarity in results between the patient and control samples with each treatment infers that the patient D does not have MyD88 deficiency. Neutrophil Migration This experiment mimicked chemotaxis; CXCL8 is a chemokine which plays a role in neutrophil activation and transmigration. It interacts with glycosaminoglycans (GAG’s) located on vascular endothelium. Binding to GAG’s allows CXCL8 to present to G protein-coupled receptors on neutrophils (10). This forms a chemotactic gradient for neutrophils to follow. Cell surface adhesion molecules including integrins and selectins allow for neutrophil rolling, adhesion and transendothelial migration to the infection site (11). In this experiment, the chemotactic filter represented the endothelium. The neutrophils migrated through the filter towards CXCL8, which was in the lower chamber of the wells (12). The similarity between patient and control neutrophil migration, shown in figure 5, indicates that patient D has efficient neutrophil migration. This rules out leukocyte adhesion deficiency, in which a mutation in CD18 subunits of І2 integrins results in reduced trandendothelial migration and defective neutrophil chemotaxis (13). Macrophage Phagocytosis The latex beads used in the experiment were labelled with fluorescein isothiocyanate, which allowed the quantification of macrophage phagocytosis using flow cytometry. The higher the signal indicated more C3b binding to CR1 on macrophages, allowing opsonisation. This allowed more beads to be engulfed into the phagosome (14). Furthermore, figure 6 shows that temperature affected phagocytosis. Macrophage incubation at 4°C reduced engulfment of latex beads into the phagosome. Therefore, the beads will have been removed with the supernatant. At normal body temperature of 37°C, many beads were engulfed by macrophages, indicating more phagocytosis. This resulted in a higher fluorescence being measured for both the patient and control (15). The results generated exclude Ch©diak-Higashi syndrome. This disorder would cause reduced phagocytosis in the patient results compared to the control. This is due to a mutation in the lysosomal trafficking regulator protein, which leads to abnormal lysosome production and degranulation (16).Classical Component Activity (CH50)The CH50 assay detects the activation of the classical complement pathway by testing the ability of serum complement components to lyse antibody-coated RBC’s (17). Activation of the classical complement pathway consists of antigen/antibody complexes binding C1q. This cleaves C2 and C4 to form C4b2a, a C3 convertase. This then cleaves C3 to C3a and C3b, allowing C3b to bind C4b2a and form a C5 convertase. The C5 convertase allows MAC formation, which inserts into cell membranes and creates pores leading to lysis (18). The optical change measured in the CH50 assay was the absorbance of haemoglobin released into the supernatant as a result of haemolysis. Complement activity was determined using the degree of lysis in each of the serum dilutions (17). The percentage of complement-dependent haemolysis was calculated in relation to lysis in pure water (100% lysis). Water causes haemolysis due to the osmotic gradient allowing a large volume of water to enter the cells. The volume is too large for the cells to hold, leading to lysis (19). If a component of the classical pathway was absent or present in reduced numbers, the CH50 results would be decreased (17). Due to the similarity in patient and control percentage lysis shown in figure 7, this suggests that patient D does not have a deficiency in classical complement components. Alternative Complement Activity (AP50)This assay procedure was similar to the CH50 assay, however EGTA and Mg2+ were added to the serum (20). EGTA and Mg2+ were used to inhibit the lectin and classical pathways, allowing for the measurement of the alternative pathway (21). Activation of the alternative complement pathway begins with C3b binding factor B. Factor B is cleaved by factor D to C3bBb, which is a C3 convertase. This activation process is enhanced by properdin. As with the classical pathway, the C3 convertase results in a cascade of events leading to MAC formation (22). The results in figure 8 show a large difference between the amount of lysis in the control and patient serum. This suggests that the patient has a problem with the alternative pathway of complement activation, such as properdin deficiency which would result in a low AP50 result. In conclusion, the investigations have eliminated all possible disorders apart from one. The remaining disorder, along with the clinical history, suggest a diagnosis of properdin deficiency for patient D. Properdin DeficiencyProperdin is a positive regulator for the activation of the alternative complement pathway. It works by stabilising C3 and C5 convertases, allowing for the initiation of the alternative pathway. This results in pro-inflammatory cytokine production and the maturation of antigen presenting cells (23). Furthermore, properdin is able to directly bind to microrganisms such as Neisseria gonorrhoeae and directly deposit C3 on the microbial surface, resulting in opsonisation (24).Properdin deficiency leads to a reduction in alternative pathway activity; individuals are susceptible to infection with Neisseria meningitidis and gonorrhoeae (25). The clinical history describes the patient being diagnosed with meningitis. The other symptoms including petechial rash, are also associated with meningitis infection. The family history of N. meningitidis and N.gonorrhoeae infection also indicates properdin deficiency (26).Reduced properdin can also result in changes in CD4+ T cells and neutrophils. This can manifest to inflammation and joint alterations, leading to immune complex-induced arthritis. The patient did develop reactive polyarthritis and had previously suffered from arthralgia (24). Furthermore, properdin deficiency is an inherited X-linked disease; as the patient is male this supports the diagnosis (26).Currently, management of properdin deficiency involves treating manifestations of the disorder. Antibiotics including Cefuroxime are prescribed to treat bacterial meningitis. This was administered to the patient, effectively clearing the infection. Also, it is advisable to administer meningococcal vaccinations to properdin deficient young males (27). Purified properdin can also be administered, to restore the alternative pathway (21). Another alternative treatment is immunosuppressive therapy (28). Research into the cause of properdin deficiency in the alternative pathway is in process, in order to develop more effect treatments.

How to place an order?

Take a few steps to place an order on our site:

  • Fill out the form and state the deadline.
  • Calculate the price of your order and pay for it with your credit card.
  • When the order is placed, we select a suitable writer to complete it based on your requirements.
  • Stay in contact with the writer and discuss vital details of research.
  • Download a preview of the research paper. Satisfied with the outcome? Press “Approve.”

Feel secure when using our service

It's important for every customer to feel safe. Thus, at The Homework Writings, we take care of your security.

Financial security You can safely pay for your order using secure payment systems.
Personal security Any personal information about our customers is private. No other person can get access to it.
Academic security To deliver no-plagiarism samples, we use a specially-designed software to check every finished paper.
Web security This website is protected from illegal breaks. We constantly update our privacy management.

Get assistance with placing your order. Clarify any questions about our services. Contact our support team. They are available 24\7.

Still thinking about where to hire experienced authors and how to boost your grades? Place your order on our website and get help with any paper you need. We’ll meet your expectations.

Order now Get a quote