【Megazyme】The Science Behind Megazyme Assay Kits

Our enzymatic assay kits are designed to be accessible for any user, including those without a specialised scientific background.

A full understanding of the principles behind our products is not required in order to generate meaningful, accurate results at your winery. However, for the interested user, the underlying chemistry behind our enzymatic assay kits is described below.

▍▏Enzymatic Assays Based on NAD+/NADH/ NADP+/NADPH

Megazyme assay kits usually involve enzymes that either directly, or indirectly (via other enzymes), produce or consume a compound called NADH (or NADPH).

NADH, although invisible to the human eye, absorbs light strongly at a wavelength of 340 nm (extinction coefficient [ε] = 6300 M-1 cm-1). This means that any changes in the quantity of available NADH can be detected using a spectrophotometer.

Figure 3 depicts the various types of enzymatic reactions that are employed in many enzymatic assay kits, either consuming or producing NADH (Reaction 1). As can be seen from Figure 3, it is sometimes necessary to include an additional reaction in order to obtain quantitative results (Reaction 2).

Reaction 2 is catalysed by the enzyme diaphorase, in the presence of a compound called INT, which converts the NADH (or NADPH) produced in the first reaction into a red coloured compound called INT-formazan.

The resulting change in absorbance at 492 nm can be used to quantify the analyte of interest, as discussed in the Beginner’s Guide in the previous pages.

▍▏Enzymatic Assays Based on GOPOD Determination

A number of our test procedures are based on the ability to quantify glucose using Megazyme’s own Glucose Oxidase/Peroxidase system (GOPOD).

The GOPOD method can be applied directly to measure glucose in a sample or to measure analytes that can be stoichiometrically converted to glucose.

The principle of the GOPOD system is shown in Figure 4. In Reaction 1, glucose is converted to glucono-δ-lactone by glucose oxidase with the production of H2 O2 . In Reaction 2, H2O2 is used by peroxidase to form a quinoneimine that absorbs at 510 nm.

The resulting change in absorbance at 510 nm can be used to quantify the analyte of interest as discussed previously.

The Megazyme range offers complete laboratory solutions, by supplying enzymatic assay kits and the associated equipment required to perform the analyses.

The Megazyme range caters to wine businesses of all sizes, from small vineyards with minimal scientific equipment to specialised, high-throughput analytical laboratories carrying out thousands of tests per day.

▍▏Laboratory Equipment

Enzymatic assays can be carried out on three main types of analyser: the manual spectrophotometer (described above), the microplate reader, and the auto-analyser. If you wish to find out more about which analyser meets the needs of your wine business, you can find in-depth product details in our Equipment section (page 48).

▍▏Assay Kit Formats

Our standard kits are designed for use with a manual spectrophotometer as described in the Beginner’s Guide, but most kits in the range can also be used with microplate readers and/or auto-analysers. For some of our most popular products, we offer specialised kits that are optimised for use with auto-analyser formats.

▍▏Method Validation and Accreditation

Many of the analytical methods developed by Megazyme have been validated as official standard methods, recommended by the relevant regulatory bodies and scientific associations following rigorous inter-laboratory evaluations.

These stamps of approval demonstrate that these methods deliver accurate, reliable, quantitative and easy-to-use test methods that meet our customers’ exacting specifications.

The following official bodies have approved one or more Megazyme test procedures for wine. Also shown are their abbreviations which are used in the individual assay kit descriptions, where applicable.

▍▏Precision of Megazyme Kits

‘Precision’ refers to the capability of an assay to produce the same result from the same sample measured under the same conditions, that is, the repeatability or reproducibility of its results.

This characteristic is of vital importance for winemakers comparing outcomes within batches or across vintages.

▍▏Quantifying Precision

The precision of the individual values generated by a specific assay kit can be measured in terms of the Coefficient of Variation (%CV).

The tables below show repeatability performance data for assays performed at Megazyme laboratories. The results demonstrate that Megazyme assay kits provide for extremely precise biological assays, generating %CVs well within the target %CV of < 3 for both red and white wine samples.

▍▏White Wine Samples

▍▏Red Wine Samples

Accuracy refers to the agreement between results from one form of analysis with the results of other analysesof the same sample.

▍▏Quantifying Accuracy

The tables below show accuracy performance data for assays performed by a number of laboratories around the world. Accuracy is measured in terms of the Bias (b) of the assay or method. Here, a mean is calculated from the analyses performed by all laboratories. The Bias (b) for the Megazyme kit is the relative percentage recovery of the analyte in comparison with the mean. The data below demonstrate the excellent performance of Megazyme kits relative to other enzymatic assay kits and to alternative methods of analysis.