Methods for determination of an analyte
View Patent ↗The invention relates to analytical methods in which the partition of a labeled substance between a liquid and a solid phase is determined. The assays include solid-phase reagents which can be particulate or monolithic such as, for example, a coated tube. Assays of this type are known per se to the person skilled in the art and include immunoassays and immunometric assays.
1. A heterogeneous immunochemical method for the determination of an analyte in a sample in a liquid medium, comprising:
a) incubating the sample in a reaction vessel with a particulate solid-phase on which a first binding partner which specifically binds to the analyte is immobilized; wherein the particles of the particulate solid phase have a size of 0.1-5.0 μm; and with a second binding partner which specifically binds to the analyte, wherein a fluorescent or chemiluminescent label is conjugated to the second binding partner, and wherein after addition of the solid-phase and the second binding partner the solid-phase is suspended in the liquid medium, whereby the second binding partner is partitioned between the liquid medium and the particulate solid-phase and the solid-phase is subjected to magnetic or gravitational attraction to attract the solid-phase in the liquid medium to an interior surface of the reaction vessel;
b) removing the first liquid medium from the solid-phase prior to adding a second liquid medium to the solid-phase;
c) resuspending the solid-phase in the second liquid medium;
d) adding a reagent A, which causes a separation of the solid-phase and the label by elimination of at least one intermolecular linkage which is essentially composed of hydrogen bonds and ionic, hydrophobic and van der Waals interactions;
e) removing the solid-phase by magnetic or gravitational attraction; and
f) determining the amount of analyte in the sample, wherein the reagent A contains in aqueous solution one substance selected from the group of reagents consisting of:
a chaotropic agent, and
a buffer for adjusting the pH to a range of 1.5 to 3;
wherein a) through f) are performed in sequential order.
2. The method of claim 1 , wherein the particulate solid-phase is magnetically attractable.
3. The method of claim 1 , wherein the label is chemiluminescent.
4. The method of claim 1 , wherein the reagent A contains said buffer for adjusting the pH to a range of pH 1.5 to 3 in a concentration of 0.1 to 1 mol/l; and an ionic detergent.
5. The method of claim 4 , wherein the ionic detergent has a concentration of 0.01 to 10% (w/v).
6. The method of claim 4 , wherein the ionic detergent is sodium dodecylsulfate.
7. The method of claim 4 , wherein the buffer is glycine.
8. The method of claim 5 , wherein said detergent concentration is 1% (w/v).
9. The method of claim 1 , wherein the particles of the particulate solid phase have a size of 0.2-2.5 μm.
10. The method of claim 1 , wherein the particles of the particulate solid phase have a size of 0.2-1.0 μm.
11. A heterogeneous immunochemical method for the determination of an analyte in a sample in a liquid medium, comprising:
a) incubating the sample in a reaction vessel with a particulate solid-phase on which a first binding partner which specifically binds to the analyte is immobilized; wherein the particles of the particulate solid phase have a size of 0.1-5.0 μm; and with a second binding partner which specifically binds to the analyte, wherein a fluorescent or chemiluminescent label is conjugated to the second binding partner, and wherein after addition of the solid-phase and the second binding partner the solid-phase is suspended in the liquid medium, whereby the second binding partner is partitioned between the liquid medium and the particulate solid-phase and the solid-phase is subjected to magnetic or gravitational attraction to attract the solid-phase in the liquid medium to an interior surface of the reaction sample;
b) removing the first liquid medium from the solid-phase prior to adding a second liquid medium to the solid-phase;
c) resuspending the solid-phase in the second liquid medium;
d) adding a reagent A, which causes a separation of the solid-phase and the label by elimination of at least one intermolecular linkage which is essentially composed of hydrogen bonds and ionic, hydrophobic and van der Waals interactions;
e) removing the solid-phase by magnetic or gravitational attraction; and
f) determining the amount of analyte in the sample, wherein the reagent A contains in an aqueous solution one substance selected from the group of reagents consisting of:
KSCN,
guanidinium hydrochloride,
NaClO 4 ,
urea,
propionic acid,
dimethylformamide, and
glycine for adjusting the pH to a range of 1.5 to 3;
wherein a) through f) are performed in sequential order.