Humans have evolved an array of pattern recognition receptors including TLRs, RLRs, NLRs-567127 – My Assignment Help : Samples & Case Study Review Sample

There are two (2) questions, each of equal weighting.

QUESTION 1 (maximum word limit is 800 words)

Humans have evolved an array of pattern recognition receptors including TLRs, RLRs, NLRs and CLRs. Many of these are highly conserved across species. Describe, from an immunological perspective, why so many different pattern recognition receptors are required, why they are so highly conserved across species and how they function collaboratively to activate innate immune responses. Provide specific examples.

QUESTION 2 (out of 15 marks)

There has been an outbreak of Measles in parts of Sydney. Patients have been brought to the hospital with distinct signs and symptoms. Blood samples are collected at the hospital sent for analysis. To assess infection an ELISA was performed to look at the levels of IgG and IgM in the blood. The patient samples were not diluted to determine OD measurements (i.e. the patient OD values given are from neat/undiluted patient samples). As per the guidelines for the ELISA, a serum concentration of IgG or IgM < 0.9ug/mL is negative and a value of >1.1ug/mL is positive.

Diagnosis is made based on the table below;

	IgG	IgM
Uninfected/Unvaccinated	Negative	Negative
Vaccinated	Positive	Negative
Early infection	Negative	Positive
Current/recent infection	Positive	Positive

Results from ELISA:

IgG ELISA

µg/ml of standard or patient sample	OD
4	0.592	0.611
2	0.312	0.31
1	0.215	0.195
0.5	0.153	0.149
0.25	0.103	0.108
0.125	0.091	0.088
0	0.073	0.081
Patient 1	0.128	0.133
Patient 2	0.223	0.215
Patient 3	0.337	0.338
Patient 4	0.119	0.12

IgM ELISA

µg/ml of standard or patient sample	OD
4	0.823	0.807
2	0.428	0.436
1	0.23	0.261
0.5	0.146	0.161
0.25	0.113	0.144
0.125	0.083	0.093
0	0.074	0.077
Patient 1	0.482	0.456
Patient 2	0.078	0.081
Patient 3	0.397	0.419
Patient 4	0.105	0.11

Construct 2 standard curves from the above OD values by performing the following steps: (i) average the OD values for each of the concentrations of the standard protein, (ii) normalise the averaged OD values using the average of the 0 µg/ml samples (create a table to show your average and normalisation values), (iii) determine if any of the protein standard concentrations should not be used to construct the standard curve (provide an explanation why you have or have not removed any protein standards from the construction of the standard curve), and (iv) construct the standard curves. (4 marks)

Tables:

IgG ELISA

µg/ml of standard or patient sample	OD		Average OD	Normalized OD
4	0.592	0.611	0.6015	0.5245
2	0.312	0.31	0.311	0.234
1	0.215	0.195	0.205	0.128
0.5	0.153	0.149	0.151	0.074
0.25	0.103	0.108	0.1055	0.0285
0.125	0.091	0.088	0.0895	0.0125
0	0.073	0.081	0.077	0
Patient 1	0.128	0.133	0.1305
Patient 2	0.223	0.215	0.219
Patient 3	0.337	0.338	0.3375
Patient 4	0.119	0.12	0.1195

IgM ELISA

µg/ml of standard or patient sample	OD		Average OD	Normalized OD
4	0.823	0.807	0.815	0.7395
2	0.428	0.436	0.432	0.3565
1	0.23	0.261	0.2455	0.17
0.5	0.146	0.161	0.1535	0.078
0.25	0.113	0.144	0.1285	0.053
0.125	0.083	0.093	0.088	0.0125
0	0.074	0.077	0.0755	0
Patient 1	0.482	0.456	0.469
Patient 2	0.078	0.081	0.0795
Patient 3	0.397	0.419	0.408
Patient 4	0.105	0.11	0.1075

Comments:

It is understood that to accurately compute the concentrations of antibody (target protein) in patient samples using the ELISA results, the only the linear region of the plot is to be considered. For this purpose, the protein standard concentrations, marked in green in the above charts, should not be used to construct the standard curve as they distort the linear relationships.

Revised Standard Curves are presented below:

As expected, the R² value (measuring the goodness of linear fit) for both the models has increased to an extent. The revised regression equations will be used for further analysis.

Determine the concentration of antibody in your patient samples. (2 marks)

For IgG ELISA

The target antibody concentration of patient samples (x-values) is measured by substituting the average ODs (y-values) of each patient separately in the below regression equation:

Following table summarizes the results:

Sample	OD	Antibody concentration (µg/ml)
Patient 1	0.1305	1.0129
Patient 2	0.219	1.6844
Patient 3	0.3375	2.5835
Patient 4	0.1195	0.9294

IgM ELISA

The target antibody concentration of patient samples (x-values) is measured by substituting the average ODs (y-values) of each patient separately in the below regression equation:

Following table summarizes the results:

Sample	OD	Antibody concentration (µg/ml)
Patient 1	0.469	2.574
Patient 2	0.0795	0.4844
Patient 3	0.408	2.2468
Patient 4	0.1075	0.6347

Are the OD values of all the patient samples appropriate to use to calculate the antibody concentration? Why/why not? If there are samples that are not ideal for calculation, what would you do in the future to be able to calculate the antibody concentrations? Can you still provide a diagnosis based on these OD values from the patient samples? Why/why not? (5 marks)

The OD values of all the patient samples are approximately appropriate to use as the samples have an absorbance value within the range of the standard curve. However, if the OD of the sample surpasses the upper limit of the standard curve, these samples should be diluted before proceeding with the regression equation to obtain an accurate result. As for these samples, the concentration obtained from the standard curve must be multiplied by the dilution factor when analysing the results.

Provide a diagnosis of the 4 patients by completing the table below (4 marks):

Patient #	Positive or negative result for:		Diagnosis
Patient #	IgG	IgM	Diagnosis
1	Positive	Positive	Current/recent infection
2	Positive	Negative	Vaccinated
3	Positive	Positive	Current/recent infection
4	Positive	Negative	Vaccinated

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