Differential diagnostic method and kit for infectious and parasitic diseases, using flow cytometry

Abstract

The present invention relates to a differential diagnostic method using flow cytometry, performed by means of differential fluorescent marking of biological agents, such as cells and pathogens of interest, with fluorescent substances. The diagnostic method generally consists in performing fluorescent marking of biological agents with gradual concentrations of fluorescent substances, and in analyzing the reactivity profile of IgG1 to the biological agents. The present invention further relates to a diagnostic kit.

What is claimed is: 
     
         1 . A method for differential diagnosis of infectious and parasitic diseases, said method comprising the following steps:
 (a) differential labeling of biological agents with a first fluorescent substance to provide differentially labeled biological agents, wherein a first type of biological agent is labeled with the first fluorescent substance at a first concentration, a second type of biological agent is labeled with the first fluorescent substance at a second concentration different from the first concentration, and a third type of biological agent is labeled with the first fluorescent substance at a third concentration different from the first and second concentrations;   (b) combining and suspending the differentially labeled biological agents in a buffer solution to provide a suspension;   (c) incubation of aliquots of the suspension with serial dilutions of a heat-inactivated human serum sample;   (d) incubation of the aliquots from step (c) with human antibody anti-IgG1 conjugated with biotin, in a presence of streptavidin conjugated with a second fluorescent substance to provide a fluorescent labeled anti-IgG1 compound;   (e) incubation of the aliquots from step (d) with fixative solution for cytometry to provide flow cytometry samples;   (f) obtaining size parameters, granularity and fluorescence during analysis of the flow cytometry samples on flow cytometry equipment;   (g) analysis of an IgG1 reactivity profile of the flow cytometry samples, by determination of a percentage of positive fluorescent biological agents relative to a fluorescence of the fluorescent labeled anti-IgG1 compound; and   (h) applying a desynchronized algorithm to analytical results obtained in step (g) to determine whether the human serum sample contains antibodies indicative of the infectious and parasitic diseases.   
     
     
         2 . The method in accordance with  claim 1 , wherein each of the first fluorescent substance and the second fluorescent substance is a member independently selected from the group consisting of Alexa-fluor, Fluorescein Isothiocyanate, Chicago Sky Blue, Rhodamine, Phycoerythrin, and Allophycocyanine. 
     
     
         3 . The method in accordance with  claim 1 , wherein the second fluorescent substance conjugated with anti-IgG1 is detected in a second fluorescence channel of a flow cytometer different from a first fluorescence channel for detecting the first fluorescent substance of the biological agents. 
     
     
         4 . The method in accordance with  claim 1 , wherein the algorithm of the step (h) further comprises steps to detect and analyze IgG1 present in the same human serum sample. 
     
     
         5 . The method according to  claim 1 , wherein steps of the algorithm comprise detecting a percentage of positive fluorescent biological agents in a series of pre-established dilutions of human serum sample. 
     
     
         6 . The method according to  claim 1  wherein the diseases subject to the differential diagnosis are Leishmaniasis and Chagas' disease. 
     
     
         7 . A diagnostic kit comprising:
 preparations of biological agents stained with incremental concentrations of fluorescent substance;   a preparation of human biotinylated antibody anti-IgG1;   a fluorescent reagent for detecting human anti-IgG1;   serum samples from negative control subjects;   serum samples from a positive control subject;   a solution to wash plates and dilute the samples;   a fixative solution for preparation for flow cytometry reading;   96-well plates; and   sealing adhesives.   
     
     
         8 . The kit in accordance with  claim 7 , wherein the biological agents are parasites stained with incremental concentrations of the fluorescent substance.