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Grade A. Conventional recovery testing for the detection of TgAb interference is not sufficiently accurate for predicting assay interference with modern highly sensitive Tg assays.
The enhancement factor is an often-cited property of a SERS tag but, as a relative measure of the effect of the nanoparticle on the scattering intensity of an adsorbed compound, this value is of limited use in predicting assay performance.
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Instead of relying solely on assay development experience and intuition to improve assay sensitivity, this systematic approach takes advantage of a predictive mathematical model generated through response surface methods that defines a specific path towards greater predicted assay sensitivity.
We found that the Illumina Infinium® design score accurately predicted assay success (Table 2) despite a few SNP drop outs (<3%) during assay manufacture which were independent of design score.
However, the practical consequence is not severe, because a reduced conversion rate from predicted assay to typable SNP is overcome merely by testing more SNPs.
We found qPCR was a reliable method for measuring the amount of input DNA to use, and for predicting downstream assay success.
We selected two assays as they generated a strong and specific amplicon of the predicted size (Assay 1 = 153 bp; Assay 3 = 71 bp).
PCR evaluations of these assays, using a temperature gradient (annealing step) and agarose gel electrophoresis, revealed that two of the four assays were suitable for further analysis, in that they appeared to generate a strong and specific amplicon of the predicted size (Assay 1 is 153 bp; Assay 3 is 71 bp).
Eight kinds of rules to predict Salmonella/microsome assay were constructed, and currently results of the assay on aliphatic and heterocyclic compounds can be predicted as accurately as +90%.
The predicted optimum assay condition consisted of methanol and potassium dihydrogen phosphate buffer (pH 3.2; 25 mM, 0.5% Triethylamine) in a proportion of 60 40% v/v, respectively, as the mobile phase at a flow rate of 1.2 mL min−1.
A designability score calculated for each SNP by Illumina was higher than 0.6, and this predicted high assay conversion rates.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com