KIT OF PARTS AND MICROBIOLOGICAL METHOD FOR ASSESSMENT OF THE FOLATE STATUS IN SERUM AND RED BLOOD CELLS
Kit and method for the microbiological determination of folate and folic acid in a whole blood sample and for the assessment of the folate status of an individual, comprising steps for complete lysis of erythrocytes (red blood cells) and release of folate species, as well as steps for release of lysosomal γ-glutamyl hydrolase from cells contained in the whole blood sample and/or addition of γ-glutamyl hydrolase and complete enzymatic hydrolysis of the γ-glutamyl chains of folypolyglutamate species, folytetraglutamates, folypentaglutamates and folyhexaglutamate, followed by a microbiological assay for comparative study of growth and metabolism in the absence and presence of various amounts of treated whole blood sample and/or folate calibrator to assess the folate status of an individual in comparison to the folate references.
1 . A method for microbiological determination of folate and folic acid in a whole blood sample and assessment of the folate status of an individual, comprising the steps of:
preparing one or more culture vessels containing a predetermined number of viable cells of Lactobacillus rhamnosus for microbiological growth and metabolism determination;
obtaining a defined sample amount of whole blood from an individual whose folate status is to be determined;
adding to the sample of whole blood a predetermined amount of red blood cell lysis buffer to obtain lysis of red blood cells for a release of folate species;
adding an amount of γ-glutamyl hydrolase and/or a surfactant capable of lysosomal permeabilization to release lysosomal γ-glutamyl hydrolase from cells contained in the whole blood sample;
treating and incubating the lysed blood sample at pH 5.5 to 7 for a sufficient time to allow enzymatic hydrolysis of the γ-glutamyl chains from folypolyglutamate species, folytetraglutamates, folypentaglutamates, and folyhexaglutamates;
performing a microbiological assay to compare growth and metabolism in the absence or presence of varying amounts of the treated whole blood sample and/or folate calibrator to assess the individual's folate status against folate references.
2 . The method of claim 1 , wherein the red blood cell lysis buffer contains ascorbate/ascorbic acid, at pH 4.2 to 5.0, and a detergent for permeabilization of lysosomes.
3 . The method of claim 1 , wherein the surfactant for lysosomal permeabilization is selected from the group consisting of sapogenins, steroidal sapogenins, saponins, triterpene glycosides, terpenoids, alkylphenol ethoxylates, (Triton® X-100), nonylphenol ethoxylates, and octylphenol ethyxylates.
4 . The method of claim 1 , wherein the enzymatic hydrolysis of γ-glutamyl chains from folypolyglutamate species is carried out in a phosphate buffer of pH 5.5 to 6.5 at ambient temperature to 37 degrees Celsius for 10 to 30 minutes.
5 . The method of claim 1 , wherein Lactobacillus rhamnosus is grown in an assay medium buffered at pH 6 to counteract the inhibitory effects of lactic acid production.
6 . The method of claim 1 , wherein the Lactobacillus is Chloramphenicol-resistant Lactobacillus rhamnosus ATCC 7469.
7 . The method of claim 1 , wherein a microtiter plate is used as the culturing vessel.
8 . The method of claim 1 , wherein a test kit is used for assessing the folate status of an individual by microbiological assay of folate and/or folic acid in a whole blood sample, wherein the test kit comprises:
a microtiter plate which cavities each contain a predetermined number of viable cells of Lactobacillus rhamnosus , that have been rendered durable for dry storage at ambient temperature by shock-freezing and freeze-drying;
an ascorbic acid/ascorbate buffer system, pH 4.0 to 4.5.