CDSImple™ Folate & Vitamin B12 ELISA Kit

Sample

Serum and Plasma

Species Reactivity

Human

Detection Method

cELISA

Intended Use

The Quantitative Determination of Vitamin B12 and Folate Concentration in Human Serum and Plasma by a Microplate Enzyme Immunoassay, Colorimetric.

Contents of Kit

A. Folate/Vit B12 Calibrators -1 ml/vial
Six (6) vials containing references for markers at levels indicated below. A preservative has been added. The calibrators, human serum based, were calibrated using a reference preparations indicated in the chart.

B. Folate Enzyme Reagent -7.0 ml/vial
One (1) vial containing Folate (Analog)-horseradish peroxides (HRP) conjugate in a protein-stabilizing matrix with dye. Store at 2-8°C.
C. Folate Biotin Reagent -7.0 ml/vial
One (1) vial containing biotinylated purified folate binding protein conjugate in buffer, dye and preservative. Store at 2-8°C.
D. Vitamin B12 Enzyme Reagent -7.0 ml/vial
One (1) vial containing Vitamin B12 (Analog)-horseradish peroxides (HRP) conjugate in a protein_stabilizing matrix. Store at 2-8°C.
E. Vitamin B12 Biotin Reagent -7.0 ml/vial
One (1) vial containing anti-Vitamin B12 biotinylated purified rabbit IgG conjugate in buffer, dye and preservative. Store at 2-8°C.
F. Wash Solution Concentrate -20.0 ml/vial
One (1) vial containing surfactant in buffered saline. A preservative has been added. Store at 2-8°C for up to 60 days.
G. Streptavidin Coated Microwells -96 wells
One 96-well microplate coated with streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C.
H. Substrate Reagent -12.0 ml/vial
One (1) vial containing tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂) in buffer. Store at 2-8°C.
I. Stop Solution -8.0 ml/vial
One (2) vial containing a strong acid (0.5M H₂SO₄). Store at 2-8°C.
J. Releasing Agent - 14.0 ml/vial
One (1) vial containing a strong base (sodium hydroxide) and potassium cyanide. Store 2-8°C.
K. Stabilizing Agent -0.7 ml/ vial
One (1) vial containing tris (2-carboxyethyl) phosphine (TCEP) solution. Store at 2-8°C.
L. Neutralizing Buffer -7.0 ml/ vial
One (1) vial containing buffer with dye that reduces the pH of sample extraction. Store at 2-8°C.
M. Product Insert
Note 1: Do not use reagents beyond the kit expiration date.
Note 2: Avoid extended exposure to heat and light. Opened reagents are stable for sixty (60) days when stored at 2-8°C. Kit and component stability are identified on label.
Note 3: Above reagents are for a single 96-well microplate

Storage

2-8°C

Performance Characteristics

Accuracy
The Folate CLIA Test System was compared with a reference method. Biological specimens were used with values that ranged from 3.2ng/ml – 13.7ng/ml. The total number of such specimens was 30. The least square regression equation and the correlation coefficient were computed for this Folate CLIA in comparison with the reference method. The data obtained is displayed in Table.

The Vitamin B12 CLIA Test System was compared with a reference method. Biological specimens from low, normal and relatively high Vitamin B12 level populations were used (values ranged from 156 pg/ml – 1830 pg/ml). The total number of such specimens was 56. The least square regression equation and the correlation coefficient were computed for this Vitamin B12 test in comparison with the reference method. The data obtained is displayed in Table.

Only slight amounts of bias between the Folate/Vit B12 CLIA Test System and the reference methods are indicated by the closeness of the mean values. The least square regression equation and correlation coefficient indicates excellent method agreement.

Precision

The within and between assay precision of the Folate ELISA Test System were determined by analyses on three different levels of pool control sera. The number, mean values, standard deviation and coefficient of variation for each of these control sera are presented in Tables.


*As measured in ten experiments in duplicate over a ten day period.
The within and between assay precision of the Vitamin B12 ELISA Test System were determined by analyses on three different levels of pool control sera. The number, mean values, standard deviation and coefficient of variation for each of these control sera are presented in Tables.


*As measured in ten experiments in duplicate over a ten day period.

Sensitivity

The Folate/Vit B12 ELISA Test System has sensitivity for different
analytes as listed in the following Table. The sensitivity was ascertained by determining the variability of the 0ng/ml serum calibrator and using the 2б (95% certainty) statistic to calculate the minimum dose.
Folate: 0.52ng/ml
Vitamin B12: 70.13 pg/ml

General Description

Folate supplementation has escalated over recent years with the knowledge of its many benefits. As one of the B vitamins, folate, or Vitamin B9, is involved in many bodily functions and deficiency can cause disease in not only the elderly but infants too. Folate deficiency is associated with megaloblastic anemia, neural tube defects, and cardiovascular dieases.
Folate plays an important role in brain development and therefore is vital during growth. The most common defects resultant from folate deficiencies are neural tube defects. With a vital role in nucleic acid synthesis, folate has been found to be beneficial as supplementation during pregnancy and other times of rapid tissue growth. Folate also plays a vital role in maintaining proper balance of homocysteine, a contributing factor in occurrences of occlusive vascular diseases and stroke. Individuals with susceptibility to heart disease and several forms of cancer may also benefit from supplementation.
Major sources of folate include green leafy vegetables, legumes, beans and fortified cereals. Foods fortified with folate are actually fortified with folic acid because of the higher bioavailability for absorption by the body. In circulation, folate is present in several different forms, some of which are more stable than others. Folic aicd and methyltetrahydrofolate are two common forms, the latter being more stable and found in higher concentrations in serum. Due to the stability of the molecule, methytetrahydrofolate is very often used as the form focused on during methods of analysis.
Folate binding proteins are responsible for folate metabolism. Two types exist in circulation; one type aids in binding to the cell surface and the other soluble form exists in circulation. These folate binding proteins also have the capability of binding several different folate derivatives including folic acid and methytetrahydrofolate. The interaction between folic acid and folate binding protein is greater than methyltetrahydrofolate. Current assays on the market require an extraction step to release the folate derivatives from the folate binding protein.
Vitamin B12 is one of the nine water soluble vitamins important for healthy body functioning. The most important roles Vitamin B12 plays in the human body are in the formation of red blood cells and the formation of the myelin sheath around the nerves. Since the effects are seen in body systems with a large range of function, the symptoms of Vitamin B12 deficiency can sometimes be very ambiguous. A deficiency may also take from months to years to manifest depending on the cause and severity.
Two of the most common causes of Vitamin B12 deficiency are diet and age. Because most sources of dietary Vitamin B12 come from animals, vegans who do not efficiently supplement their diet are at risk. The elderly community is also at high risk because of their diet, as well as the less efficient functioning of their digestive system.
Two very useful tests to distinguish between Vitamin B12 deficiency and folate deficiency are methylmalonyl CoA (MMA) and homocysteine (hcy). Both deficiencies are represented by similar symptoms; however, even though both show increased levels of homocysteine, only Vitamin B12 deficiency causes an increase in methylmalonyl CoA. The increase in levels of methylmalonyl CoA and homocysteine is thought to be the root cause of any symptoms that accompany a Vitamin B12 deficiency. High levels of these two analytes in the blood stream causes increased oxidative stress to cells therefore causing increased apoptosis. In turn, vascular disease results in the form of atherosclerosis, coronary heart disease and/or neurodegeneration (ex. Parkinson's Disease).