Automated immunoanalyzer system for performing diagnostic assays for autoimmune and infectious diseases
A quantitative method for diagnosing an autoimmune disease or an infectious disease comprising performing an automated diagnostic assay, comprising: incubating a capture reagent with a streptavidin-coated medium to form a solid phase complex, wherein the capture reagent is a biotinylated autoantigen or infectious disease antigen; washing the solid phase complex to remove excess capture reagent; incubating the solid phase complex with a serum sample to form an immune complex; washing the immune complex to remove any unbound sample; incubating the immune complex with a conjugate to create an immune-conjugate complex; washing the immune-conjugate complex to remove any unbound conjugate; introducing a substrate capable of generating a quantifiable response; and calibrating the response generated from introducing the substrate.
1 . A method of performing a diagnostic assay, comprising:
a) combining a capture reagent-coated separation medium, wherein the separation medium comprises fluorescently-labelled particles, with an analyte-containing sample in a reaction cuvette, wherein the capture reagent has been biotinylated with PEG 12 -Biotin and the fluorescently-labelled particles have been coated with streptavidin;
b) binding luminescent labels to the separation media in proportion to a number of bound analyte molecules, to form a complex;
c) measuring an initial fluorescent signal associated with an initial quantity of the fluorescently-labelled particles;
d) washing the complex;
e) adding a luminescing reagent or substrate to the cuvette to generate a luminescent signal in proportion to the number of bound analyte molecules,
f) measuring the luminescent signal and a final fluorescent signal associated with a final quantity of the fluorescently-labelled particles in the cuvette; and
g) normalizing the luminescent signal by a ratio of initial fluorescent signal to final fluorescent signal to calculate a reported value.
2 . The method of claim 1 , wherein the fluorescently-labelled particles are magnetic.
3 . The method of claim 1 , wherein the analyte-containing sample is a serum sample.
4 . The method of claim 1 , wherein the capture reagent will be bound by analyte molecules in the analyte-containing sample.
5 . The method of claim 1 , wherein the analyte molecules are capture reagent-specific immunoglobulins.
6 . The method of claim 1 , wherein the binding of luminescent labels is mediated by a labeled anti-immunoglobulin conjugate.
7 . The method of claim 6 , wherein the labeled anti-immunoglobulin conjugate comprises an antibody, which is specific for immunoglobulin, and a luminescent label.
8 . The method of claim 7 , wherein the luminescent label is horseradish peroxidase (HRP), alkaline phosphatase (ALP), or beta galactosidase (BGAL).
9 . The method of claim 1 , wherein the capture reagent has been bound to the fluorescently-labelled particles through a biotin-streptavidin interaction.
10 . The method of claim 1 , wherein binding luminescent labels to the separation media comprises incubating a labeled anti-immunoglobulin conjugate with the capture reagent-coated separation medium.
11 . The method of claim 10 , wherein the conjugate is in a diluent including polyethylene glycol.
12 . The method of claim 1 , wherein measuring the fluorescent signals and the luminescent signal takes place within an optics box.
13 . The method of claim 1 , wherein the reported value is a bead retention adjusted relative light unit signal.
14 . The method of claim 13 , wherein the bead retention adjusted relative light unit signal is compared to a calibration curve relative light unit signal.
15 . The method of claim 1 , wherein a ratio of the final fluorescent signal to the initial fluorescent signal is used to quantify a fraction of particles that were lost during the wash step.