Recombinant Human Intrinsic Factor (IF) from Scripps Laboratories is offered as a high-quality replacement for native porcine IF. The quality of the native porcine IF available to the clinical diagnostic industry has declined in recent years, so Scripps Laboratories developed a highly purified recombinant IF that is a reliable and economical alternative to native IF. Data are presented here to demonstrate the suitability of Recombinant Human Intrinsic Factor for research use or Vitamin B12 assay development.
Figure 1 presents an SDS-PAGE image of highly purified Recombinant Human IF, Mat. No. I0821-90658. It displays an approximate molecular weight greater than 40kDa and less than 50 kDa. This is consistent with the reported molecular weight of 44-48 kDA for Native Human IF.1 In addition, the purified product shows no visible contaminating proteins, both in the reduced/heated (lanes 1,2) and non-reduced/not heated (lanes 6,7) samples.
Recombinant Human IF is a glycosylated protein, so we employed Isoelectric Focusing to visualize the heterogeneity of the glycosylation patterns. Figure 2 shows a variety of distinct isoelectric points, ranging from 4.75 to 6.3, demonstrating the microheterogeneity of this purified, glycosylated protein. The Recombinant Human IF sample was run in duplicate.
BIOTIN CONJUGATION &
VITAMIN B12 ELISA
Recombinant Human IF and Native Porcine IF were conjugated to biotin and each conjugate was used side-by-side as a component of a Vitamin B12 ELISA.2
Figure 3 shows the generation of standard curves, using the Vitamin B12 standards included with the ELISA kit. The top graph was prepared using the Recombinant Human IF-Biotin conjugate, while the bottom graph was prepared with the Native Porcine IF-Biotin conjugate. (Note: The Native Porcine IF-Biotin used is the reagent included with the ELISA kit.) The charts demonstrate good correlation, indicating Recombinant Human IF-Biotin is suitable for use in generating a standard curve in ELISA.
Figure 4 presents a scatter plot showing good correlation when Recombinant Human IF-Biotin is substituted for Native Porcine IF-Biotin in the Vitamin B12 ELISA. The data represent the recoveries of a series of 36 Vitamin B12 samples with both biotin conjugates. With an R-squared (R2) value of 0.9979, the linear regression shows a strong positive correlation between Recombinant Human IF and Native Porcine IF.
Of note, Recombinant Human IF-Biotin demonstrated good thermal stability after storage at 37°C for 2 weeks.
Analyzed by HPLC, the elution profile of Recombinant Human IF in Figure 5 displays a single peak with an elution time of 17.092. The absence of any minor peaks confirms what is corroborated by SDS-PAGE: Recombinant Human IF is a highly purified product with no discernible contaminants.
The data presented here for Recombinant Human IF demonstrate it possesses the physical characteristics and Vitamin B12-binding capabilities to make it a suitable replacement for Native Porcine IF in a Vitamin B12 assay. It is a highly purified, glycosylated protein and, as a renewable recombinant product, it is ideal for research work as well as long-term use in the development and production of a commercial assay.
Recombinant Human Intrinsic Factor is in stock and available now:
(1) Allen and Mehlman. J. Biol. Chem., 1973, (248): 3660.
(2) Vitamin B12 ELISA data generated by Calbiotech - El Cajon, CA USA.