Method for detecting chemical substances in samples of material that can be taken from a subject, in particular for detecting embryotoxic factors

Abstract

A method for detecting chemical substances in samples of material that can be taken from a subject, comprising a step of preparing a sample of a substance coming from a donor organism; a step of associating with said sample of a substance a control element suitable for verifying a presence of at least one active agent in the sample of the substance and a step of verifying the presence or absence of the active agent based on a change in state in said control element; the control element comprising at least one living organism having the ability to nourish itself autonomously through temporary organs associated with the living organism itself and able to supply nutritive substances to the latter without taking said nutritive substances from an outside environment.

1 . A method for detecting chemical substances in samples of material that can be taken from a subject, said subject being human or animal, in particular for detecting embryotoxic factors, comprising the following steps:
 preparing a sample of a substance, said sample of a substance preferably comprising organic fluids and/or portions of tissues coming from a donor organism; and   associating with said sample of a substance a control element suitable for verifying a presence of at least one active agent in said sample of a substance; and   verifying a presence or absence of said active agent based on a change in state in said control element,   
       characterised in that said control element comprises at least one living organism having the ability to nourish itself autonomously through temporary organs associated with the living organism itself and able to supply nutritive substances to the latter without taking said nutritive substances from an outside environment. 
     
     
         2 . The method according to  claim 1 , wherein said living organism comprises at least one fish belonging to the class Actinoptetygii and the genus  Xenopus  of the class Amphibia. 
     
     
         3 . The method according to  claim 2 , wherein said fish is of the species  Danio rerio  or “zebrafish” and is preferably a  Danio rerio  belonging to a strain of genotype “AB”, said fish even more preferably having a lifetime less than or equal to 120 hours of development and/or cell multiplication after fertilization. 
     
     
         4 . The method according to  claim 2 , wherein said fish is of the species  Oryzias Latipes  or “medaka”, said fish preferably having a lifetime less than or equal to 288 hours of development and/or cell multiplication after fertilization. 
     
     
         5 . The method according to  claim 1 , wherein said living organism is able to feed itself autonomously via a temporary organ of the “yolk sac” type or the like, said temporary organ being developed integrally with the living organism. 
     
     
         6 . The method according to  claim 1 , wherein said step of associating a control element with said sample of a substance comprises the following sub-steps:
 exposing a predetermined number of living organisms to said sample, said predetermined number of living organisms preferably being at least 20 embryos of  Danio rerio  having a development and/or cell multiplication time no greater than 72 hours after fertilization;   placing equal groups of said embryos of  Danio rerio , preferably in a number equal to 10, in respective verification containers;   adding the sample of a substance to said verification containers, said sample of a substance comprising blood serum and/or plasma taken from a donor subject.   
     
     
         7 . The method according to  claim 6 , wherein said verification containers comprise at least two adjacent wells of a culture plate, said culture plate preferably comprising a total of 24 wells. 
     
     
         8 . The method according to  claim 1 , wherein said step of verifying a presence or absence of said active agent is designed to verify an absence or presence of embryotoxic factors (ETFs) and comprises the follow sub-steps:
 maintaining an environmental interaction, preferably in said verification containers of said culture plate, between the embryos and the sample of a substance for a defined period of interaction ranging from twelve hours or, preferably, multiples of twelve hours, up to a maximum of 48 hours;   counting a number of deceased embryos after said twelve hours and/or after said multiples of twelve hours have elapsed; and   attributing to the sample of a substance a toxicity level that is proportional and quantitatively correlated to a quantitative presence or a level of chemical activity of said embryotoxic factors (ETFs).   
     
     
         9 . The method according to  claim 8 , wherein said toxicity level is defined as “toxic” when the number of dead embryos exceeds 50% of the total number of embryos placed in said verification containers of the culture plate, said toxicity level being alternatively defined as “moderately toxic” when the number of dead embryos ranges between 50% and 30% of the total number of embryos placed in said verification containers of the culture plate, and said toxicity level being alternatively defined as “non-toxic” when the number of dead embryos is less than or equal to 20% of the total number of embryos placed in said verification containers of the culture plate. 
     
     
         10 . The method according to  claim 1 , wherein there is also a step of operating on the donor subject based on a result of said step of verifying the presence or absence of embryotoxic factors (ETFs), said step of operating on the donor subject comprising exposure to immune system-suppressing agents and preferably comprising:
 infusions of immunoglobulin and/or suppressors of cytokine production, still more preferably administered intravenously; and/or   topical applications of progesterone and/or progesterone oil on the donor subject or culture medium suitable as a host for artificial insemination processes associated with the donor subject; and/or   administering vitamin or non-vitamin or nutraceutic compounds or natural and/or phytotherapeutic compounds as well as other food supplements.   
     
     
         11 . The method according to  claim 2 , wherein said amphibian is of the genus  Xenopus , said amphibian preferably having a lifetime less than or equal to 96 hours of development and/or cell multiplication after fertilization.