This section includes two subtitles that outlined all materials used in the methodology of this study:
Clear 96 wells of flat plate (ELISA micro-titer plates) from NUNC brand products
Single and 8 multichannel pipettes with disposable plastic tips (Thermo scientific and Titertek)
Assorted graduated cylinders
Tubes (5ml volume)
Plastic bottles 100ml value, from Thermo scientific brand.
Pipettes 25ml value
Big bottle 100ml value
The table below outlined all chemicals and reagents used in this study. Names, batch or lot numbers and expiry day are important to guarantee the validation of company products.
Luminometer or plate reader: analyze the results by detecting the substrate at 405nanometer.
2.2.1 Calibration of pipettes/initial enzyme linked Immunosorbent assay test:
a) Check the validation of used pipettes with accepted volumes. By calibrating three different powers ten times for a single pipette and for a multichannel, one of each head in a cycle for three different powers too.
b) To ensure that enzyme linked Immunosorbent assay reader is working with good quality to get conventional results. Add 75µL of Dye (yellow food color) to 12mL of H2O then get 100µL into each well in a micro-titer plate. This gives 98.40% accurate positive result. This means the ELISA reader is valid monitoring equipment.
2.2.2 Sandwich enzyme linked Immunosorbent assay principle:
The aim of this study is measuring the quantity of prothrombin antigens between two layers of antibodies. A biotinylated detection antibodies and strepravidin-HRP indirect detection system by used reagents and TMB substrate.
Firstly, add 150µL of capture anti-human prothrombin dilution into every well in micro-titer plates and allowed for incubation for one week. Secondly, blocked the plates and left 30minutes of incubation. Next, add 100µL of diluted samples and controls after labeled the micro-titer plates. After 90minutes incubation, add 100µL of peroxidase conjugated anti-prothrombin dilutions, followed by another incubation period of 90-120 minutes. After every incubations period, plates must be washed to dispose of the excess dilutions of antibodies and antigens. Finally, it determined by using TMB for color reactions in the dark and killed by adding HCL. After the process, the plates are run in the ELISA reader.
2- In substrate step and getting color: after adding TMB leaves the plates in the dark for 15-30 minutes.
3- Both of block and of wash buffers should be fresh reagents.
The table below describes the process of making dilutions, and how much of diluents had been used in these experiments.
Table 2: The table highlights preparation of the dilutions by using coating buffer and sample of plasma starting from lowest concentration to highest with total diluents value of 1ml.
The anti-human prothrombin used was a purified immunoglobulin fraction of rabbit antiserum. This anti-factor II is used to develop enzyme linked Immunosorbent assay for prothombin. The diversity in concentrations leads to variation in quantity of optical density, which absorbed at 405 nm. In addition, this study used plasma samples of normal patients and comparing it with that of standard human plasma.
The above graph shows the differences in standard deviation calculations, between Optical Density and concentrations of Standard Human Plasma and Normal Patient Plasma. Blank =0 (no plasma), 1/1000=0.001, 1/500=0.002, 1/250=0.004, 1/120=0.008, 1/60=0.017, 1/30=0.033, 1/15=0.067 and 1/5=0.2.
Regarding to table 3 and 4, both present that the standard deviation was increased to a highest point in 1/125 dilution of plasma (0.008 %). After that increase, the standard readings were decreasing when concentrations of plasma were directly increased. That is clearly shown in figure 4, the graph line for optical density reached up to 0.455 for standard plasma and 0.179 for normal plasma, then fall with the continuation of concentration increment.
Table5: The optical density for concentrations diverse of normal patient plasma in 1/1000 M of captures Abs and two different dilutions of tag Abs.
Figure5: The correlation between Optical Density and concentrations of Normal Patient Plasma in two different dilutions of tag antibodies. Blank =0 (no p
Figure6: The variations between Optical Density and concentrations of Normal Patient Plasma in two different dilutions of tag antibodies. The concentrations are: Blank =0 (no plasma), 1/40=0.002, 1/20=0.005, 1/10=0.1, 1/5=0.2 and 1/2.5=0.4.
Only those samples of normal patient plasma have shown the compression in the optical density and increasing concentrations between 0.002 % and 0.001 % of peroxidase-conjugated anti-prothrombin. In graph 5, the high concentration of the tag, which is 0.002%, is directly proportional to raised concentrations of samples. Conversely, the low concentration of tag, that is 0.001%, varies inversely with the high concentration samples. However, the second chart (figure 6) shows a lack of variation between the two curves, where they complied with values is close to the stability by somewhat. That came after the increasing in concentrations gradually after 0.005%.
The read results depend on the speed of the device used to monitor the amount of protein or prothrombin antigens binding with antibodies, specifically for prothrombin, in the two layers surrounding it. The amount of the speed of absorption used in the experiments was 405 nm. This was used to measure the amount of prothrombin in plasma samples of healthy person (as a normal patient) and number of patients with heart and blood vessels conditions, as well as the standard control.
This study had the results few weeks after the start of the experiment by making particular modifications in methodology. The privileged aspect of the study was that which could be link to collection of some evidences. Those evidences are as confirmation about how the study started to the completion. According to the above results in figure 4, both diluted solutions 1 and 2 gave readings almost equal in the speed of absorption at 405nm. At low concentrations of plasma, the readings raise with higher the concentration until it reached 0.455 of 0.004% of standard human plasma, and 0.373 in 0.002% of normal patient plasma. After those concentrations, the readings began to decline again and reached to the lowest reading at higher concentrations (0.2%) of both plasmas. The parallel curves shown by the chart shows that there are no major differences between the result studied plasma concentrations. In addition, the possibility error rate between them would be minimal. Based upon that factor, it could be seen that the curve readings of Sample 2 is true and tied to the correct natural curve, which is the comparable standard human plasma curve.