In eukaryotes, mRNA is modified after transcription to form a special structure at the 5' end, i.e., the cap structure, which plays a more important role in the stabilization, transport and translation process of mRNA. Cowpox virus capsid enzyme is an effective enzyme that catalyzes the formation of the cap structure, which consists of two subunits, D1 and D12, and combines RNA triphosphate esterase activity, guanylate acyltransferase activity and guanine methyltransferase activity, which can connect the 7-methylguanine cap structure (m7Gppp) to the 5' end of RNA (m7Gppp5'N). Cowpox Virus Capping Enzyme, in the presence of appropriate concentrations of capping buffer, guanosine triphosphate (GTP), and S-adenosine methionine (SAM), is capable of capping RNAs in less than an hour and ensures proper orientation.

This kit uses the principle of double-antibody sandwich enzyme-linked immunosorbent assay (sandwich ELISA) to detect the residual Vaccinia Capping Enzyme in samples. First, add the Vaccinia Capping Enzyme standard and test sample to the Anti-Vaccinia Capping Enzyme coated microtiter strips (36709-A), then add the diluted biotin labeled Anti-Vaccinia Capping Enzyme antibody (36709-C), and finally add Streptavidin-HRP (36709-D) to form an antibody + antigen + antibody-Biotin + SA-HRP complex. Subsequently, add TMB substrate (36709-H) into the complex to observe color reaction after washing the complex. TMB is converted into blue under the catalysis of HRP enzyme and finally converted into yellow in the presence of acid, and the shade of color is positively correlated with the amount of Vaccinia Capping Enzyme in the sample.

The detection range of this kit is 0.125~8 ng/mL; the lower detection limit is 0.065 ng/mL.

Features

• Highly sensitive: Detecting as little as 0.065 ng/mL of Vaccinia Capping Enzyme residuals in in-process and final biological samples.
• Ensures accuracy: The Vaccinia Capping Enzyme residuals can be accurately detected with a recovery of 75%-125%.
• Specificity: Cooperate use with YEASEN Vaccinia Capping Enzyme and can accurately detect the residuals.
• Stable: The difference between lot-to-lot is low, the kit property is not impacted under 37℃ for 7 days.
• Easy signal collection: Using Biotin-Streptavidin system, the signal can be stably enlarged.

Application

• Residual Vaccinia Capping Enzyme test in biological products

Specification

Product Performance

Components

^*The Standard (36709-B) is Lyophilized powder.

Storage

This product should be stored at 2°C ~8°C. Unopened product is valid for one year.

^*Upon receipt of the kit, please check whether all components are complete and immediately store them in corresponding condition.

Figures

Specificity Demonstration

Figure 1. The specificity of Vaccinia Capping Enzyme ELISA kit was shown in the figure above

7 samples were estimated for the non-specific reaction with the antibodies in the kit. The concentration of the 7 samples were all below the Limit of Quantitation (LOQ) (0.065ng/mL).

Precision Display

A) Repeatability

B) Intermediate precision

Table 1. The precision of Vaccinia Capping Enzyme ELISA was assayed as the above scheme

10 duplicated were tested at 3 concentrations on the standard curve, and all the CV% were lower than 10% (Table 1A). 3 lot of Vaccinia Capping Enzyme ELISA kits were tested and the CV% were lower than 15% (Table 1B).

Accuracy Validation

Three concentrations of Vaccinia Capping Enzyme standards, high, medium and low, were spiked to the matrix samples in medium proportions, while the high value spiked samples were made multiplicative dilutions.

The Vaccinia Capping Enzyme was assayed in doubly diluted samples, as well as in samples with different spiked concentrations, and in unspiked samples, with linear and spiked recoveries ranging from 80% to 120%.

Spiked recovery = measured value / (spiked standard x 50% + spiked specimen value x 50%)

Table 2. Accuracy Validation

The linear and spiked recoveries of Vaccinia Capping Enzyme in the doubly diluted samples, as well as the samples with different spiked concentrations, and in the unspiked samples, ranged from 80% to 125% (Table 2).

Document: