STERILITY TESTING OF PHARMACEUTICALS ppt by DR.C.P.PRINCE
ELISA results interpretation [Autosaved].pptx
1. دهؤكيا ثوليتةكنيكى انكوياز
ـنيةـقالت ـوكـهد ـةعجام
Duhok Polytechnic University
Lab 7: ELISA results interpretation
4th year students
Practical Immunopathology
Dr. Amir Hani Raziq
Ph D genetic engineering and biotechnology
Duhok Polytechnic University
Shekhan Technical College of Health
Dept. of Medical Laboratory Technology
2. ELISAs can be designed to yields three different types of results, quantitative,
qualitative and semi-quantitative.
Quantitative
• The precise amount of analyte in a test sample can be determined by comparing
its values to those generated from a serial dilution of a known, purified analyte
(a Standard Curve).
3. 1) Procedure and generation of a standard curve (Non-competitive ELISA)
Thyroid peroxidase (TPO) is a large (105 kDa) membrane-bound glycoprotein
of thyroid gland. It is the major enzyme involved in multiple steps of thyroid
hormone synthesis.
The presence of autoantibodies to TPO and thyroglobulin (Tg) is an
established tool for diagnosing chronic autoimmune thyroiditis as well as for the
differential diagnosis of hypothyroidism including its subclinical or latent type.
Autoantibodies to Tg and TPO are important for ruling out autoimmune thyroid
diseases.
Example: Anti-thyroid peroxidase antibody
4. over 98% of thyroiditis patients display autoantibodies to either one or both of these
antigens.
Anti-TPO antibodies are found in 71-97% of patients with Grave’s disease and 91-99% of
patients with Hashimoto`s thyroiditis. Negative tests for anti-Tg and anti-TPO can
virtually exclude an autoimmune thyroiditis.
These autoantibodies do also occur in patients with adenocarcinoma of the thyroid or
hyperthyroidism and are found in healthy individuals at low levels.
7. b. In many situations, calibrators are provided as a stock concentrated
solution that needs to be serially diluted to obtain several calibrators with
sequentially reduced concentrations.
Example
640 ng/L 320 ng/L 160 ng/L 80 ng/L 40 ng/L 20 ng/L
1 ml 1 ml 1 ml 1 ml 1 ml
1 ml 1 ml 1 ml 1 ml 1 ml
Generation of calibrators for the assessment of Human Interleukin
17(IL-17) in human serum
9. 1. Pipette into the designated wells above, 100 μl
of either:
a. Calibrators (CAL.A to CAL.F) for
QUANTITATIVE or
b. Cut-off Calibrator (CC) for QUALITATIVE
interpretation.
and 100 μl of each of the following:
Negative control (NC) and Positive control (PC),
and
Patients diluted serum (P1, P2...)
Assay procedure
2. Incubate for 30 minutes at 20-32°C
3. Wash 3x with 300 μl washing buffer
10. 4.Pipette 100 μl conjugate into each well.
5.Incubate for 30 minutes at 20-32°C.
6. Wash 3x with 300 μl washing buffer.
11. 7. Pipette 100 μl TMB substrate into each
well.
8. Incubate for 30 minutes at 20-32°C.
Protect from intense light.
9. Pipette 100 μl stop solution into each well.
12. 10.Incubate 5 minutes minimum.
11. Read absorbance at 450 nm
(recommended 450/620 nm) within 30
minutes.
13. Generation of standard curve
The concentrations of the calibrators (provided by the
manufacturer) will be plotted versus their correspondent
ODs. In this case, we will use excel software.
By the aid of the standard curve, we will be able to measure
the concentration of the analyte in each sample.
15. 2. Procedure and generation of a standard curve (Competitive ELISA)
Example: calibrators of an ELISA kits used to measure serum protein x
Absorbance (nm) Concentration (ng/ml)
2.942 0
1.806 5
0.758 10
0.372 25
0.213 50
0.145 100
17. y = -0.0004x2 + 0.1113x + 0.2018
R² = 0.9982
0
1
2
3
4
5
6
7
8
0 20 40 60 80 100 120
1/abs
Concentration (ng/ml)
Standard Curve
standard curve (Competitive ELISA) generated by plotting the
concentration versus/absorption to get better fit for optimum results
interpretation.
conc 1/abs
0.00 0.34
5.00 0.55
10.00 1.32
25.00 2.69
50.00 4.69
100 6.90
18. Qualitative
Qualitative interpretation read the optical density of the cut-off calibrator and the
patient samples. Compare patient´s OD with the OD of the cut-off calibrator. All
samples with higher ODs are considered positive, samples with lower ODs are
considered negative.
Negative: OD patient < 0.8 x OD cut-off
Equivocal: 0.8 x OD cut-off ≤ OD patient ≤ 1.2 x OD cut-off
Positive: OD patient > 1.2 x OD cut-off
19. If a cut-off value is not provided, it may be obtained by the following formula:
Cut-off value = Ś (negative control)+3SD
Example: if a negative control triplets are
negative control 1 = 0.011
negative control 2 = 0.008
negative control 3 = 0.044
Ś = (0.011+0.008+0.044)/3= 0.021
Standard deviation= 0.02
Cut-off = 0.021+3 x 0.02= 0.081
