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Detecting Autoantibodies in Human Sera Samples using ELISA

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Introduction

Autoimmunity is a series of immune responses that is made against an organisms own cells and tissues due to inability to recognise own cells and tissues as self (Mandal, 2014). Diseases can arise as a result of autoimmunity. This includes lupus (SLE). Lupus (SLE) arises because of immunological mechanisms. With tolerance to antigens is lost and production of autoreactive lymphocytes the process of autoantibody is produced. Continuous production of autoantibodies from autoantibody producing cells results in formation of immune complexes. (Bolland and Ravetch, 2000). There are many factors which influence the susceptibility and development of lupus (SLE). These include hormonal, environmental, and genetic factors (Lisnevskaia et al, 2014). Genes involved in lupus (SLE) include MHC loci, tumor necrosis factor alpha, components of the complement factor and the mannose binding protein (Tsao and Grossman, 2001). Environmental triggers have influence on expression for lupus (SLE) such as vitamin D deficiency. Vitamin D has an important role in order for the immune system to function properly because receptors of vitamin D are found in the cells of the immune system such as T lymphocytes, monocytes and dendritic cells. Also reduced vitamin D intake due to photosensitivity is associated with lupus (SLE). Thus, deficiency in vitamin D has a major consequence for the immune system and can create autoimmune diseases (Albishri et al, 2015). Hormones have a role in acting as chemical messengers in the immune response (Csaba, 2014). These chemical signals produced from hormones are disrupted especially between the brain and target cells which is an important factor in lupus (SLE) (Pick, n.d.). Because of this disrupted balance of hormone production certain hormones are more prevalent which cause lupus (SLE). High estrogen concentrations have been linked to lupus (SLE) due to it causing autoimmunity and with patients having a fast conversion of androgens to estrogens. Patients with joint pains are linked with lupus (SLE) and also have a high concentration of estrogen (Lupusinternational.com, n.d.). Diagnosis of lupus (SLE) include the lupus band test which detects for the presence of antinuclear antibodies. This is done using immunofluorescence. By looking at the florescence pattern the type of antibody can be detected. For a person to be positive for lupus (SLE) IgG and other complement depositions will be found at the dermoepidermal junction. To be specific there will be a bandlike deposit along the epidermal basement membrane due to the presence of IgG. Also a bandlike deposit will be present in the nucleus of the epidermal cells. A high concentration of anti-dsDNA antibody from titers also shows the presence of SLE due to anti-dsDNA antibody having a high specificity for SLE (Gill et al, 2003). Diagnosis can also be made using the SLICC criteria. For a patient to have SLE, at least four criterions need to be met including one clinical criterion (Petri et al, 2012). There is currently no cure for SLE but a number of treatments are available. Prognosis for SLE has improved significantly since the 1950s with people diagnosed it living for less than five years. Now ninety percent of people with SLE live over ten years. The effect of SLE is more evident in men and children than in women. Causes of early death has been due to failure of organs and infections. Because of improved survival rate other factors have come in to play for the death of SLE patients. Cardiovascular disease is one factor and it is important to prevent this from being developed (Doria et al, 2006).    

The ELISA test is a diagnostic test used to measure the concentration of certain antibodies or antigens present in a sample from a disease patient. ELISA is unique due to the separation of specific and non-specific interactions during serial binding to the multiwell plate. At the end of ELISA, a coloured product is produced that is associated with the amount of antibody or antigen present in the solution sample (Bio-Rad, n.d.). The first step of ELISA is coating, where a layer of antigen or antibody is adsorbed to the wells on the plate. After coating, blocking and detection are the next steps. Several washes are needed between each ELISA step to remove unbound materials. During this process excess liquid is removed in order to prevent dilution of the solutions added in the next stage (Bio-Rad, n.d.). For detection of SLE in the patient, the patient's serum sample undergoes the ELISA test to detect the concentration of anti-dsDNA-antibodies which is specific for patients with SLE. A high concentration of anti-dsDNA-antibodies will indicate that the patient has SLE (Wigand et al, 1997).

The aim of this experiment is to measure the concentration of anti-dsDNA-antibody present in both of the serum samples using the ELISA test by binding to the complimentary antigen double stranded DNA in the wells. The samples come from a female patient known to be suffering from SLE. Sample A was obtained when she was feeling relatively well and sample B was collected on the day of the practical. By comparing the yellow colour intensity at the end of the ELISA test for both samples and compared to the controls and using the standard curve the concentration of anti-dsDNA antibodies can be obtained and correlated to the relevant SLE prognosis level. An assay result above the laboratory reference range for the anti-dsDNA-antibody at a particular prognosis level will show that the patient is positive for SLE and the level of SLE prognosis. Based on the level of SLE prognosis suitable treatments will be given to the patient.

Results

On each strip the first three wells were labelled the positive controls, the next three labels were measured the negative controls and the remaining wells were labelled sample A and B (three for each sample). In the first stage 50µl of purified antigen was added to each well of the microplate strip. The strip was incubated for two minutes at room temperature to allow time for the antigen to bind to each plastic well. A layer of antigens was present in each well once incubation had finished. After incubation the wells were washed using a wash buffer to remove excess liquid. In stage three 100µl of blocking buffer was added into each well and incubated for two minutes to remove unbound sites. The wells were washed again to remove excess liquid. In the next step 50µl of the positive controls, negative controls and the test autosera samples were loaded into the relevant wells. The strip was then incubated for 10 minutes at room temperature. After incubation for 10 minutes the wells were washed to remove the unbound antibodies. Once the wash was done 50µl of secondary antibody was added to the wells. Then the wells were incubated for 5 minutes at room temperature. The washing procedure was repeated again to remove any unbound secondary antibodies. In stage nine 50µl of the HRP enzyme substrate was added to the wells. The strip was incubated for 5 minutes at room temperature. This allowed sufficient time for the HRP enzyme which is conjugated to secondary antibodies to metabolise the TBT substrate. The metabolisation of the TBT substrate produced a blue-coloured product. Each well turned blue fairly quickly during the incubation and the final strip is shown in figure 1. The intensity for the positive control was six, negative control was zero, and sample A and sample B was five.

Figure 1. The micro plate strip showing the blue-coloured product after the enzyme substrate was added and then incubated for 5 minutes.

For the final stage of the ELISA test the reaction was stopped by adding 50µl of stop solution, (10% (v/v) phosphoric acid/ddH2O) into the wells. The blue solution turned yellow on addition of the stop solution. This is seen in figure 2. The intensity for the positive control was six, negative control was zero, sample A was one and sample B was two.

Figure 2. The micro plate strip showing the yellow-coloured product after the addition of the stop buffer to the blue-coloured product.

Absorbance measurements were obtained using a plate reader for the controls and samples. The absorbance relates to the concentration of anti-dsDNA antibodies present in the samples. The data is shown in table 1.

Table 1. The absorbance data for the controls and samples.

+ive controls

-ive controls

Sample 1

Sample 2

1

2

3

Avg

1

2

3

Avg

1

2

3

Avg

1

2

3

Avg

0.660

0.717

0.655

0.677

0.063

0.053

0.084

0.067

0.139

0.139

0.141

0.140

0.287

0.255

0.236

0.259

Discussion

The antigen that coated the wells of the microplate strip was double stranded DNA. Two epitopes were present. During the reaction when the control and the autosera samples are loaded, the antibodies present are being detected which is complementary to the antigens coated in the wells. The antibodies need to be diluted using a blocking buffer for prevention of non-specific binding of proteins in the antiserum on the well specifically the solid phase. The antibodies in the serum will bind to the complementary antigens during incubation. Any unbound antibodies are removed by washing. After this, secondary antibodies are added in order to detect the primary antibodies. During incubation the secondary antibodies binds to the primary antibodies (Vlab.amrita.edu, 2011).

Looking at figure 1, in the positive control samples, the intensity of the blue coloured product was six due to a known amount of anti-dsDNA antibodies present in the sample. This is used to show the procedure is working. The negative control had a blue colour intensity of zero due to no antibodies present in the sample. The intensities of both sample A and B were similar on the scale of five. From figure 2, looking at the positive control sample the intensity of the yellow coloured product is five due to the high amount of known antibodies present which a patient with SLE should have. The mean absorbance value from table 1 for sample 1 is lower than sample 2 which correlates to the colour intensity which is lower than sample 2. This means that sample 1 is from the patient when she was feeling relatively well due to a very low amount of anti-dsDNA antibodies present. Sample 2 has a higher absorbance value than sample 1 with a colour intensity which is also higher at two. Because of this result sample 2 comes from the patient when she was feeling unwell. Also this level of intensity shows that the patient has a low level for SLE because of low level detection.

The experiment was successful because the results obtained were precise and accurate. The only issue during the experiment was that the intensity of the blue-coloured product was the same for both sample A and B when the enzyme substrate was added.

Sample 1 had the lowest concentration of anti-DNA antibodies whereas sample 2 had the higher concentration of anti-dsDNA antibodies. This is because of the colour intensity of the final product where sample 1 is low and sample 2 is higher.

The mean absorbance value for sample A is 0.14. The laboratory reference range value for sample A is -0.02. Based on the laboratory reference value this means that when the patient was feeling relatively well she was negative towards SLE. The mean absorbance for sample B is 0.26. The laboratory reference value for sample B is 0.13. The absorbance value is higher than the reference value meaning it is positive for a disease prognosis level which is a low level. This means that the patient is mainly disease free but with periods where low disease activity occurs.

ELISA is a procedure used to measure the concentration of antigen present in the sample. The estimate of the analyte concentration is as a result from the construction of a standard curve. The standard curve is constructed from the making of several serial dilutions of a known concentration of the analyte across the range of concentrations close to the expected unknown concentration. The unknown samples concentration is derived by interpolation which needs a standard curve which has been properly generated (Natarajan and Remick, 2008).

As the intensity yellow colour in the end result has a value of only two we can say that the patient has a very low level of anti-dsDNA present which means the disease is likely to be calm but with a few periods of low disease activity (Kirkbride, 2015). These low disease activities include cutaneous manifestations, musculoskeletal manifestations and serositis which can be treated with nonsteroidal anti-inflammatory drugs (NSAIDS) or immunosuppression medications which have a low potency on top of the already taken hydroxychloroquine and corticosteroids (Mosca et al, 2001).

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