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Technical Briefs |
1 Department of Clinical Biochemistry, AKH; Aarhus University Hospital, DK-8000 Aarhus C, Denmark;2 The Protein Laboratory, Aarhus University, DK-8000 Aarhus C, Denmark;3 Cobento Biotech A/S, DK-8000 Aarhus C, Denmark
aaddress correspondence to this author at: Department of Clinical Biochemistry, AKH, University Hospital of Aarhus, Nørrebrogade 44, DK-8000 Aarhus C, Denmark; fax 45-89493060, e-mail vakurbor{at}hotmail.com
Competitive binding approaches with use of specific binding proteins are the most commonly used methods to measure vitamin B12 in laboratory medicine. Various binding proteins have been used in these methods, including intrinsic factor (IF), pooled human or chicken serum, transcobalamin (TC), and saliva (1)(2). The most widely used of these are non-human IF preparations, usually obtained from hog gastric mucosa. Numerous problems have been reported with their application, however, including difficulty of purification, instability on storage, and variation in the binding capacity connected with changes in serum protein and vitamin B12 concentrations (1)(2). If IF is not highly purified, it may contain haptocorrins (also called R proteins), which bind not only vitamin B12 but also related metabolically inactive compounds that may be present in the sample, thereby causing artificially increased vitamin B12 results (1)(3).
To circumvent the problems associated with nonhuman IF, we recently expressed human IF in plants and obtained a product free of endogenous vitamin B12 and contaminating vitamin B12-binding proteins (4). In the current study, we examined the feasibility of using this recombinant human IF for measurement of vitamin B12 bound to TC.
Human IF was expressed in the recombinant plant Arabidopsis thaliana and purified as described previously (4). As a first step, recombinant human IF was coupled to magnetic beads (Dynabeads, M-280 Tosylactivated; DYNAL) according to the protocol for ligand-binding applications as recommended by the manufacturer. The beads from 1 mL of the standard suspension were washed three times in 0.2 mol/L phosphate-buffered saline, pH 7.4. The washed beads were mixed with 1 mL of recombinant human IF (0.5 g/L) and incubated for 24 h at 37 °C with gentle and continuous agitation. The beads were precipitated, 1 mol/L Tris (pH 7.5) was added to the pellet, and the incubation was then continued for 4 h. The treated beads were then washed five times with 2 mL of 0.05 mol/L Tris (pH 7.5)–0.5 mol/L NaCl and suspended in this buffer.
Recombinant human IF coupled to magnetic beads was used for measurement of vitamin B12 in the last step of the Axis-Shield Holo-TC assay in place of the binding protein supplied with the assay. This step measures the vitamin B12 bound to TC trapped by insolubilized antibodies (5). The Axis-Shield Holo-TC assay was used to demonstrate the feasibility of using recombinant human IF for measurement of vitamin B12 because it was the only commercial assay that allowed us to use our binder instead of the one supplied by with the assay. The vitamin B12 content of the sequestered holo-TC is released under reducing and alkaline conditions, converted to the stable cyano form with potassium cyanide, and quantified in a competitive binding assay with [57Co]-cobalamin as tracer and immobilized IF as the vitamin B12-binding protein. In this last step, we modified the assay and used recombinant human IF coupled to Dynabeads in place of the hog IF provided. The recombinant human IF was dissolved in the buffer supplied with the assay (borate buffer, pH 9.6) in a stock solution of 
0.1 µmol/L. This stock solution was stored at -4 °C until application. The stock solution was further diluted (50 µL of stock solution in 9 mL of borate buffer) to obtain "zero" binding of 75% of the total count, which was comparable to the binding recommended in the assay insert. Fig. 1A
shows a calibration curve generated using the manufacturer-supplied calibrators at 0, 10, 20, 40, 80, and 160 pmol/L vitamin B12.
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The performance of the vitamin B12 assay using human recombinant IF was evaluated by analyzing three serum samples with low, intermediate, and high holo-TC concentrations in five consecutive analytical runs (interassay variation) with each sample analyzed in four replicates (intraassay variation). This enabled us to determine intraassay and total variation for the assay. The imprecision (intraassay and total variation) and mean values (in parentheses) were 8% and 8% (41 pmol/L), 3% and 6% (63 pmol/L), and 3% and 6% (123 pmol/L). These results indicate that the imprecision of the assay using human recombinant IF was comparable to that for the original assay, which was reported to be <10% for the three different sera (5).
In a buffer matrix stored at -4 °C, recombinant human IF coupled to Dynabeads was stable for 2 months as determined by the analysis of the controls for the holo-TC assay in three concentrations (data not shown).
We compared the vitamin B12 assay for measurement of holo-TC using recombinant human IF as binding protein with the original Axis-Shield assay on a total of 96 samples ranging from 10 to 151 pmol/L [71 human serum samples and 25 controls (3 in-house controls and 2 control materials supplied by the manufacturer)]. For all samples and controls (Fig. 1B
), the regression equation relating the new assay (y) with the original method (x) was: y = 1.04x + 2.2 pmol/L (Sy|x = 6.91 pmol/L; r = 0.98). The 95% confidence intervals for the slope and intercept were 1.002–1.008 and -0.88 to 5.2 pmol/L, respectively.
The recent availability of recombinant human IF expressed in plants allowed us to use this product for diagnostic purposes for the first time. Recombinant human IF was first coupled to magnetic beads and then used as a binder in the last step of the Axis-Shield Holo-TC assay for the measurement of the vitamin B12 bound to TC. Previously, our human recombinant IF was shown to be stable during storage and to have properties comparable to those obtained for human gastric IF in terms of the IF-aquocobalamin spectrum, the relative affinity for cobalamin or the analog cobinamide, and binding to the IF receptor cubilin (4). In the current study, use of the Axis-Shield assay allowed us to search for weaknesses in use of the recombinant IF because this assay was designed to measure low concentrations of vitamin B12.
We conclude that recombinant human IF produced in plants may well be an optimal binder for use in assays of vitamin B12 in plasma and/or serum.
Acknowledgments
This work is part of an EU BIOMED Project (QLK3-CT-2002-01775) and a EUREKA Project (CT-T2006). We warmly acknowledge the excellent technical assistance of Anna-Lisa Christensen and Jette Fisker.
References
The following articles in journals at HighWire Press have cited this article:
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  R. Carmel The Disappearance of Cobalamin Absorption Testing: A Critical Diagnostic Loss J. Nutr., November 1, 2007; 137(11): 2481 - 2484. [Full Text] [PDF]
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 M. V. Bor, M. Cetin, S. Aytac, C. Altay, and E. Nexo Nonradioactive Vitamin B12 Absorption Test Evaluated in Controls and in Patients with Inherited Malabsorption of Vitamin B12 Clin. Chem., November 1, 2005; 51(11): 2151 - 2155. [Abstract] [Full Text] [PDF]
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) and 25 controls (
)] ranging from 10 to 151 pmol/L holo-TC were analyzed by the holo-TC assay using recombinant human IF as binding protein and the original Axis-Shield assay. The regression lines obtained were as follows: for human serum samples (n = 71; solid line), y = 1.05x + 2.8 pmol/L (r = 0.98); for controls (n = 25; dashed line), y = 0.99x + 1.7 pmol/L (r = 0.99).