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Salivary Secretory IgA Indirect Enzyme Immunoassay Kit

Document ID: DQ106
Relevant to following products: Manta WorkOut
Document description: How to setup: Salivary Secretory IgA Indirect Enzyme Immunoassay Kit



How to setup: Salivary Secretory IgA Indirect Enzyme Immunoassay Kit

Salivary Secretory IgA Indirect Enzyme Immunoassay Kit

Select Create a New Protocol

Select Generic Wizard.

On the Data Acquisition step of the wizard press the Create button to setup the measurements to perform with your instrument (i.e. 450nm measurements)

On the Microplate Layout step of the wizard press the Create button to setup the microplate layout:

When the microplate layout editor is displayed press Edit Types to setup the sample types. Press Edit and then press Add, create a sample type called NSB and change its Sample Type from User to Blank and also select a colour for this new type and press OK. Add further sample types, called Zero, Cont1 and Cont2 select appropriate colours. After adding all these types press OK and on the Microplate Layout Groups dialogue, select to include the Standard type and scroll down to the end of the list and tick your new sample types. On returning to the Editor these new types will appear in the top right list. Now press the Fill Settings button and select the bottom right thumbnail image to select to fill the plate in this orientation. Now you can setup the microplate layout, so first select the Standard group and use the mouse to select wells A1 to F6. (If you make a mistake then press the Undo button to go back a step). Continuing setting up the microplate layout as specified in the application notes. I.e. Zero group in G1-G2, NSB is H1-H2, Cont1 in A3-A4, Cont 2 in B3-B4 and Unknowns in C3-H4. When finished press Save and Close to return to the Wizard.

Continue through the Wizard accepting the default options and on completion press Finish to run the test. Press the Start button (play icon) to start the measurements. The Generic Wizard sets up some of the analysis for us.

After measurements are completed select the Edit Transforms button, the %CV calculation is not required so select the %CV entry and press Remove.

Next press Create to create a new transform and select Matrix Expression, under the Expression tab enter:

x/Zero1

Under the Output Matrix, enter the name B/B0

Next press Create to create a new transform and select Standard Curve Fit. Under the Concentrations tab enter the Concentrations of the standards:

600
200
66.7
22.2
7.4
2.5
(You can copy and paste these values in from here)

Under the Method tab select Four Parameter fit as the Fit Method. Under the Axes tab, select a Logarithmic X axis and select to specify titles, μg/mL and B/B0

Press Create to create another new transform and select Factor and enter 5 as the Factor, under the Output Matrix tab enter SIgA μg/mL

Press OK and then press the Recalculate button (which will flash briefly). If necessary make any other changes to the protocol. Finally select File | Save Protocol and press OK to enter a filename to store this protocol with all of these settings. This protocol can be launched from the Organiser for subsequent runs.

To summarise the calculations added from the application notes:

1. Compute the average Optical Density (OD) for all duplicate wells.

This is done automatically for you by – the averages appear in the table of the report.

2. Subtract the average OD for the NSB wells from the average OD of the zero, standards, and unknowns.

Because your NSB group is defined as a blank group, the blank correction transform is added by the wizard for this subtraction (the software subtracts on a well by well basis and the results are averaged as a last step).

3. Calculate the percent bound (B/BO) for each standard and unknown by dividing the average OD (B) by the average OD for the zero (BO).

This is the Matrix Expression transform we have added.

4. Determine the concentrations of the unknowns by interpolation using software capable of logistics. We recommend using a 4 parameter sigmoid minus curve fit.

This is the Standard Curve fit transform.

5. Multiply obtain the final concentrations of saliva samples by 5 to g/mL. of SIgA.

This is the Factor transform.

Example files available for download here.





Related articles:
DQ032: How do I use the changes I have made to the result analysis in future runs of the assay?
How To: Extract a version of the protocol for use in the future…

DQ002: Is it possible to convert a protocol that imports data (offline) to a measurement protocol (online)?
How To: Convert an offline protocol to online…





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