Chemistry: analytical and immunological testing – Involving diffusion or migration of antigen or antibody
Reexamination Certificate
2001-08-23
2002-12-31
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving diffusion or migration of antigen or antibody
C436S517000, C436S518000, C422S051000, C422S068100, C422S105000, C435S285200, C435S287200, C435S204000, C435S164000, C435S091500, C435S091500
Reexamination Certificate
active
06500673
ABSTRACT:
The devices and methods of the prior art for performing precise immunological diagnoses generally use devices having most often already been the object of an immunological preparation (by immobilization of a first antibody). Thus the wells of the plates used in the ELISA type processes comprise in general a coating comprising first antibodies to capture an antigen, which will then be recognized by a second antibody, the whole being then revealed by a third anti-immunoglobulin antibody. The good practice of this process requires at least six washings and three hours of incubation.
Rapid tests have appeared during the 1980s, that use microballs previously clad with a first antibody and which migrate on a nylon surface to come to rest ultimately on a first line (presence of antigens) of second antibodies, for stopping in all cases on a second line of third antibodies. The whole takes place in several minutes, using a minimum of 7 nanograms of antigen per milliliter of specimen. This process does not permit detecting serum antibodies.
The object of the present invention is no longer an electrophoresis which is characterized by a more or less prolonged migration between two electrical poles (one at the beginning and the other at the end), as a function of molecular weight of different proteins. There exist more or less sophisticated gel techniques conducted between two electrodes and which permit separating a group of proteins in different bands according to their molecular weight.
The present invention has for its object to overcome the insufficiency of sensitivity of the known methods, in particular to provide a process permitting obtaining a qualitative or quantitative signal of the presence, at infinitesimal concentrations, of a substance in fluids of a specimen, even if it is not constituted by a nucleic acid. The so-called PCR (polymerase chain reaction) technique detects very small quantities of fragments of chromosomic material (RNA or DNA). It is not capable of measuring proteins such as new generations of tumoral markers or cytokenes circulating at quantities that can be less than a picogram per milliliter (a billionth of a gram). The immunological techniques, even the most sensitive such as radioimmunoanalysis, do not permit it.
The process according to the invention is applicable to the detection of any analyte that may be contained or adapted to be included in a fluid or a liquid which can react with a specific ligand to form a complex, the analyte or the ligand or both being carriers of electronic charges which are neutralized, or even reverse in the complex when the latter is formed. It is characterized in that:
the fluid or liquid is caused to migrate, after placing it in contact with the specific ligand, in an electrically inert porous material interposed between opposite surfaces of two electrically charged electrodes of the same polarity, and
there are detected in the liquid that has migrated into the porous material between the two electrodes, those components, analyte, ligand or complex, which were not captured by the electrodes.
Needless to say, the total volume of fluid or liquid used, which is that contained in the dissolved condition of one of said components or both, particularly as a result of their possible mixing, must be sufficient to permit its diffusion in the porous material to the place where the detection is carried out.
According to the nature of the constituent detected, or not detected, therefore depends the conclusion that can be drawn as to concentration. In a preferred embodiment of the method according to the invention, one of the constituents, for example the ligand, is marked. It is for example an antibody which carries free NH
2
groups, therefore positive charges (the analyte being thus an antigen), and if the electrodes are negatively charged, the detection of the marker in the fluid or liquid having migrated into the porous material between the electrodes will show that the antibody is engaged in a complex, hence that the antigen was present in the original liquid or fluid. Thus following the antigen-antibody reaction, the NH
2
groups of the antibodies are masked in the formed complex, the positive charge of the antibody being thus neutralized, the antigen-antibody complex being again, as the case may be, able to have a negative polarity. On the contrary, the absence of detection will translate into the absence of the antigen in the fluid or liquid studied. Thus the marked antibodies which have not encountered “partners” are thus captured and blocked by the negatively charged electrodes.
Other examples show in what follows this description, important possibilities offered by the invention. But for good understanding of the invention, it may be useful to define the expressions “analyte” and “ligand”, as used in the present description. Thus, the application of the invention is not limited to the detection of analytes constituted only by antigens or antibodies, even if it is in this connection that the invention will be most often used.
By analyte, should be understood any molecule or entity which it is desired to detect, no matter what its nature: haptene, protein, antigen, antibody, nucleic acid fragments, substance, even a group of molecules having common characteristics, this analyte being adapted to give rise to a specific reaction with a ligand to form a complex adapted to take part in a reaction seeking to detect in a selective manner the presence or absence of the analyte in a fluid or a liquid, for example a fluid of biological origin.
In the case of an analyte constituted by an antigen, it will be appreciated that the antibody, which thus plays the role of the ligand, is the carrier of NH
2
sites which give it a positive polarity, giving rise to the possibility for this antibody to be captured by an electrode having negative charges, whether the latter is of electrostatic nature or the result of placing the electrode under voltage in the suitable direction. Naturally, the converse can also take place. It can happen, for example, that in other types of analyte-ligand couples, the polar group will be formed from a negatively charged group, for example a COOH group, which will be neutralized in the analyte-ligand complex that is formed.
Accordingly, the ligand can as needed be itself modified by a group giving it its properties, for example the required electrical polarity for its use in the method of the invention. This could, by way of example, be the case when the analyte is constituted by a DNA fragment, whose presence can be searched for in a liquid after it has itself been used in a complementary RNA detection test. In such a case, the DNA fragment will be for example rendered the carrier of a biotin group, the ligand thus acceptable to be used-being adapted to be constituted by avidine, carrying itself, as the case may be, a marker, for example colorimetric, fluorescent, chemiluminescent, radioactive or the like and, if there was further need, a supplemental group giving it an electrostatic charge, which could thus be neutralized during the production of the analyte-ligand complex whilst susceptible to being formed, via the biotin-avidine reaction. Or else the analyte could be constituted by a DNA fraction and the ligand by a DNA probe itself marked by an enzyme, particularly peroxidase, by means of spacer arms produced by means of heterobifunctional systems, for example of the (DNA (FMCC)-peroxidase (SPDP) type.
Preferably, the analyte is in solution in a liquid or a fluid. But for example when the analyte consists of an antigen, it can also be contained for example in a biopsy or mucous fragment. But it then should be extracted by, or carried in, the liquid which will then migrate in the porous material between the electrodes.
As to the “porous” material, it must necessarily be meant by this expression any material permitting to propagation or diffusion of a liquid through its mass, when it has been moistened with this liquid. It could be constituted of any open pore material, fibrous material or which can behave as a
Counts Gary W.
Le Long V.
Young & Thompson
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