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Changes and Clinical Significance of the Levels of Serum Under-O-glycosylated IgA1 in IgA Nephropathy.
China (IFMSA-China) - China Medical University, Shenyang
Second Department of Nephrology
Prof. Dr.Liu Dajun
Type of Research Project
- Clinical Project with Laboratory work
What is the background of the project?
IgA nephropathy (IgAN), the most common primary glomerulonephritis all over the world, which frequently leads to end-stage renal disease and kidney transplantation. Serum under-O-glycosylated IgA1 in IgAN patients may deposit more preferentially in glomeruli than heavily-O-glycosylated IgA1. Therefore, this project, a clinical research, aims to investigate the value of under-O-glycosylated IgA1 in the diagnosis of IgA nephropathy and explore its relationship with the clinic pathological features of the patients.
What is the aim of the project?
This project aims to investigate the value of under-O-glycosylated IgA1 in the diagnosis of IgA nephropathy and explore its relationship with the clinic pathological features of the patients.
What techniques and methods are used?
We will train the student to collect the patient’s data and analyze these findings. The serum samples were collected from, Shengjing Hospital of China Medical Unerversity, which included 40 IgA nephropathy patients (group A), 20 other primary glomerulonephritis patients (group B) and 20 healthy persons (group C). Serum and urine levels of GD-IgA1 were detected in all the samples. The 24-hour urinary protein and serum creatinine were measured in group A and B, the eGFR (Glomerular filtration rate) calculated. Recorded the pathological grade of renal biopsy samples and Lee’s classification. Study the value of serum and urine Gd-IgA1 level in diagnosing IgA nephropathy by drawing ROC (Receiver operating characteristic) curve and calculating the area under the curve. ELISA (Enzyme linked immunosorbent assay) method: 1.Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4℃. 2.Aspirate each well and wash with at least 300μl wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels. 3.Block plates by adding 300 μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1 hour. 4.Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition. 5.Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at room temperature. 6.Repeat the aspiration/wash as in step 2 of plate preparation. 7.Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1 hour at room temperature. 8.Repeat the aspiration/wash as in step 2 of plate preparation. 9.Add 200 μL of substrate solution to each well. Incubate for 20 minutes at room temperature ( if substrate solution is not as requested, the incubation time should be optimized ). Avoid placing the plate in direct light. 10.Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing. 11.Determine the optical density of each well immediately, using a microplate reader set to 450 nm. 12.Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each. 13.Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. 14.To determine the concentration of the unknowns, find the unknowns' mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. 15.Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration
What is the role of the student?
- The student will mainly observe
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
Depends on which state the projects currently in. The common skills that the students already earned in their university years. To help the ongoing research, with their best knowledge and dedication to the topic. 1 search relevant documents of IgAN 2 learn ELISA protocol 3 Collect IgAN cases 4 collect specimen 5Test the specimen together 6Statistical analysis the data 7Discuss the project with colleague 8Writing papers 9Report the results at department meeting 10Visit the renal pathology laboratory
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
No theoretical teaching provided
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- No specific outcome is expected
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Master the General introduction to internal medicine
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months)
- Tortajada Agustin;Gutierrez Eduardo;Pickering Matthew C;Praga Terente Manuel;Medjeral-Thomas Nicholas. The role of complement in IgA nephropathy.[J]. Molecular immunology;Tortajada Agustin;Gutierrez Eduardo;Pickering Matthew C;Praga Terente Manuel;Medjeral-Thomas Nicholas. The role of complement in IgA nephropathy.[J]. Molecular immunology;2019 Oct;114:123-132. doi: 10.1016
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