Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
1999-09-10
2001-08-14
Snay, Jeffrey (Department: 1743)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C422S082080
Reexamination Certificate
active
06274085
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a method of ascertaining the mutual binding of a first reactant and a second reactant, which method comprises the following steps:
providing a substance comprising the first reactant in a holder so that the substance is adsorbed at the inner side of the holder,
adding a solution containing the second reactant to the holder, and
irradiating the holder with electromagnetic radiation and detecting whether a parameter of radiation from the holder has changed with respect to said parameter of the radiation incident on the holder.
The invention also relates to a device for performing this method. In known methods, the radiation parameter which may change is, for example, the color or the state of polarization.
2. Description of the Related Art
The method is often used as an antibody virus detection in medical diagnostics for detecting infectious diseases at an early stage, with the virus being the first reactant and the antibody being the second reactant. An antibody, or possibly several antibodies in succession, are added to a substance containing an unknown virus so as to detect this virus. Moreover, for example, for developing medicines, a material which may be a possible antibody for the virus may be added to a known virus so as to ascertain whether this antibody reacts with the virus. The biological substance which is examined by means of the method may be human or animal tissue, blood or secretion.
Due to the considerable risks which may be involved in viral infections, which may occur, for example during pregnancy or birth, virus-antibody tests are nowadays performed in large numbers.
Therefore, there is a need for inexpensive and rapid virus detection methods, all the more because after a test with a first antibody and after it has been found that the virus does not react to this antibody, a second test and possibly subsequent tests, each time with a different antibody, must be performed.
For detecting the presence of a virus, the method described in the opening paragraph is generally used in which, after addition to the substance of the solution with antibody to which Rhodamine 6G molecules are bound, an incubation time is observed in which the virus can react with the antibody.
Subsequently, the holder with the substance and the antibody is washed several times, for example four times, so that all antibodies which have not reacted with the substance are removed. Subsequently, it is ascertained by means of irradiation with light, whether there are still fluorescent Rhodamine 6G molecules present, hence whether there is still antibody. This antibody is then bound to the substance, in other words, it has reacted with a virus. If no fluorescence is observed, the substance does not contain a virus matching the antibody.
A great drawback of this method is that the washing operation must be performed frequently, which is time consuming and involves a waste of antibodies so that the method cannot be performed in situ. Moreover, the detection method is not very reliable.
To be able to detect in situ and to avoid the washing steps, alternative methods have been proposed which are based on optical techniques such as the detection of the quantity of light reflected by the substance, or of the change of the state of polarization of the light coming from the substance (ellipsometry) or of a technique known as the Fourier-transformed infrared (FTIR) technique.
PCT Patent Application No. 94/03774 describes a number of these techniques for detecting, inter alia, viruses which to this end are contacted with a thin layer in which antibodies are present and, if they react with an antibody, cause a change of an optical property such as the reflection coefficient, the polarization selectivity or the color selectivity.
For a reliable virus detection, only radiation from the surface of the antibody which is in contact with the substance to be examined should be detected. However, in the above-mentioned in situ detection methods, radiation from the holder may reach the detector, which radiation does not originate from this surface, hence of Rhodamine 6G molecules which are bound to antibody which has not reacted with the virus. Since no distinction can be made between Rhodamine 6G molecules which are bound to an antibody which has reacted with a virus and molecules which are bound to antibody but has not reacted with a virus, said in situ methods are not very reliable either.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method of ascertaining the mutual binding of a first and a second reactant for providing, inter alia, virus detection which is simple, inexpensive and reliable. This method is characterized in that it is detected whether a layer having a non-linear optical behavior is formed at the interface between the first reactant and the second reactant.
This method provides the possibility of direct detection in situ, inter alia, whether a virus has reacted with an antibody. Then, a polar ordered molecular layer is produced at the interface between the substance and the antibody, which layer exhibits a non-linear optical behavior. As a result, a radiation component having a frequency which essentially differs from that of the radiation with which the surface of the holder is irradiated is produced when irradiating the holder surface on which the substance to be examined has deposited. If the substance does not contain any virus which reacts with the antibody, there is no formation of a polar ordered layer and no wavelength change. Due to the selective detection of radiation at a given frequency which has a given relation with that of the radiation with which the holder is irradiated, it is only ascertained that the dipolar layer has been formed or not formed, while possible radiation coming from antibody parts which have not reacted with a virus does not influence the detection. Consequently, the detection method according to the invention is very reliable.
There are different possibilities of ascertaining the formation of said dipolar layer.
A first principal embodiment of the method according to the invention is further characterized in that an incubation period is observed after the solution has been introduced into the holder, in which period the reactants can react with each other, whereafter it is ascertained whether said dipolar layer has been formed.
A second principal embodiment of the method according to the invention is alternatively characterized in that the detection is started immediately after the solution has been introduced into the holder, and in that it is ascertained whether and at which rate said dipolar layer is formed.
In the latter embodiment, use is made of the fact that also the rate at which a reaction between an antibody and a virus takes place, hence the rate at which a dipolar layer is formed, is characteristic of the kind of virus or of the kind of antibody, so that this kind can be determined by measuring the increase as a function of time of the radiation intensity incident on the detector.
The two principal embodiments may be further characterized in that the holder is irradiated with monochromatic radiation having a first frequency, and in that it is detected whether radiation from the substance has a frequency which is twice the first frequency. Use is then made of the second harmonic effect of the non-linear dipolar layer; that is, this layer doubles the frequency of radiation incident thereon and converts, for example, red light into blue light.
Alternatively, the two principal embodiments may be further characterized in that the holder is irradiated with two radiation components having a first and a second frequency, respectively, and in that it is detected whether radiation from the substrate has a frequency which is a mixed term of the first and the second frequency.
The mixed term may be the sum of the first and the second frequency, but also the difference between these frequencies.
To promote the formation of the dipolar layer and hence
Barmentlo Maarten
Hollering Robertus W. J.
Snay Jeffrey
U.S. Philips Corporation
Vodopia John F.
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