Vitamin B12（VB12）

Nanjing OkayBio launches its innovative Vitamin B12 (VB12) sandwich assay series, featuring a groundbreaking 3-component chemiluminescent immunoassay（CLIA） bulk reagent. This advancement streamlines traditional 5-component systems, reducing detection time by 30% through optimized reaction protocols. Validated on CLIA platforms, the reagent achieves a sensitivity of?5 pg/mL?and demonstrates exceptional correlation (R2 >0.95) with Roche-assigned samples.

• Chemiluminescent Immunoassay (AE)

1.Calibration Curve

VB12 antibody (H835e1) and intrinsic factor (C1770) were used to detect VB12 samples (0–2000 pg/mL).

Figure 1. VB12 Calibration Curve

Sample	Concentration （pg/mL）	RLU
1	0.0	8308
2	22.2	64128
3	52.3	109666
4	499.5	957793
5	1004.1	2084186
6	2015.4	4067850

Table 1. VB12 Calibration Curve Data

2.Sample Concordance

Roche-assigned samples (35.1–1934 pg/mL, n=48 ) tested with H835e1 and C1770 showed?R2 = 0.9926.

Figure 2. VB12 Sample Concordance
(Roche-assigned)

Sample	Assigned Conc.（pg/mL）	Detected Conc.（pg/mL）
1	35.1	43.8
2	36.2	32.4
3	54.3	59.8
?	?	?
47	1872	1931
48	1934	1889

Table 2. VB12 Sample Concordance Data
(Roche-assigned)

3.Limit of Detection (LoD)

Calibrator	Concentration (pg/mL)	RLU Mean	Linear Equation
S1	0	8308	y=2514.4x+8308
S2	22.2	64128
Mean RLU values of zero - conc. diluent measured 20 times		8701
SD		427.7
Mean +2SD		9556
LoD (pg/mL)		0.496

Table 3. Limit of Detection of VB12

4.Specificity

Antigens (VB12, Adenosylcobalamin [AdoCbl], Hydroxocobalamin [HOCbl], and Methylcobalamin [MeCbl]) were diluted to 500 and 2000 pg/mL in 10mM PBS for detection.

Figure 3. Specificity of VB12 Antibody Recognition

Theoretical Conc.（pg/mL）	Detected Conc.（pg/mL）
	VB12	AdoCbl	HOCbl	MeCbl
0	0	0	0	0
500	499	487	705	656
2000	2015	1615	1943	2224

Table 4. Specificity of VB12 Antibody Recognition Data

5.Repeatability

The same batch of VB12 antibody (H835e1) and intrinsic factor (C1770) were used to detect Roche-assigned samples at concentrations of?50.26 pg/mL?and?523.7 pg/mL 10 times each. The measured concentrations exhibited?coefficients of variation (CV)?of?< 3%?for both low- and high-value samples.

Low-Concentration Samples
Sample	RLU	Concentration (pg/mL)
1	107100	50.26
2	107866	50.87
3	107502	50.58
4	107213	50.35
5	107112	50.27
6	108017	50.99
7	106987	50.17
8	106547	49.82
9	104710	48.36
10	108306	51.22
Mean	107136	50.29
SD	/	0.80
CV	1.6%

Table 5. VB12 Detection Repeatability
(Low-Concentration)

High-Concentration Samples
Sample	RLU	Concentration (pg/mL)
1	991892	514.2
2	1010307	522.4
3	1003793	519.5
4	1037952	534.7
5	1009633	522.1
6	1074624	551.0
7	1041325	536.2
8	986505	511.8
9	1026037	529.4
10	1033005	532.5
Mean	1021507	527.4
SD	/	11.79
CV	2.2%

Table 6. VB12 Detection Repeatability
(High-Concentration)

Vitamin B12 is the collective term for a number of substances with qualitatively equivalent biological effects. These compounds consist of a corrin ring system that is similar to porphyrin with four reduced pyrrole rings and a central cobalt ion and are therefore referred to as cobalamins. Characteristically, all cobalamins have an α-axial ligand at the cobalt ion consisting of a phosphoribosyl-5,6-dimethylbenzimidazole side chain. The β-axial position can be occupied by various substituents. Depending on the group (R), a distinction is made between cyano- (R = CN), aquo-(R = H₂O), hydroxo- (R = OH), methyl- (R = CH₃), and adenosyl-(R = 5’-deoxyadenosyl) cobalamin. Cobalamins are synthesized only by microorganisms, and humans and animals receive them solely through the food chain. In natural foods, vitamin B12 is present mainly as hydroxocobalamin and adenosylcobalamin and is found in milk as methylcobalamin. Due to its stability, cyanocobalamin is used in fortified foods as well as in pharmaceuticals and dietary supplements. Some pharmaceuticals and dietary supplements also contain hydroxocobalamin and methylcobalamin.

Vitamin B12 is necessary for the transformation of methyltetrahydrofolate to tetrahydrofolate for DNA synthesis. B12 deficiency can therefore lead to functional folate deficiency and impaired DNA synthesis, resulting in pernicious (megaloblastic) anaemia.