METHOD FOR APPLYING, ON A SOLID SUPPORT, AT LEAST ONE BINDING PARTNER TO A MOLECULE

Abstract

A process for applying, in a tubular solid support, at least one binding partner P1 to an analyte to be detected or quantified in a test sample, including steps: (i) connect the support to a suction-discharge device, (ii) draw up into the support, by one of its ends, a solution including the binding partner P1, called sensitization solution S1, contained in a container, called container C1, (iii) continue contact between solution S1 and the inner surface of the support for a time between 0 s and 11 min, (iv) discharge the solution S1 into a container, which is optionally the container C1, steps (ii) to (iv) forming a cycle that can be repeated at least once, over a total duration of at least 1 min and at most 2.5 h. Also, the use of the coated support for the detection or quantification of an analyte in a test sample.

Claims

1. A process for applying, in a tubular solid support, optionally flared, having a circular or ellipsoidal opening at each end, at least one binding partner P1 to an analyte to be detected or quantified in a test sample, comprising the following steps: (i) connect the solid support to a suction-discharge device, (ii) draw up into the support, by one of its ends, a solution comprising the at least one binding partner P1, called sensitization solution S1, contained in a container, called container C1, (iii) continue contact between the sensitization solution S1 and the inner surface of the solid support for a time between 0 s and 11 min, (iv) discharge the sensitization solution S1 into a container, which is optionally the container C1, steps (ii) to (iv) forming a cycle that can be repeated at least once, over a total duration of at least 1 min and at most 2.5 h.

2. The application process as claimed in claim 1, wherein the contact time of step (iii) between the sensitization solution S1 and the inner surface of the solid support is between 2 s and 1 min.

3. The application process as claimed in claim 1, wherein steps (ii) to (iv) are repeated from 10 to 100 times.

4. The application process as claimed in claim 1, wherein the total of the repeated cycles is between 10 and 20 min.

5. The application process as claimed in claim 1, wherein it also includes the following steps, carried out when the suction (ii)/contact (iii)/discharge (iv) cycles are completed: (v) draw up into the solid support in which the at least one binding partner P1 is applied, a wash solution W1 contained in a container called container CW1, (vi) continue contact between the wash solution W1 and the inner surface of the solid support for a time between 0 s and 11 min, (vii) discharge the wash solution W1 into a container, which is optionally the container CW1, steps (v) to (vii) forming a cycle that can be repeated at least once, over a total duration of at least 1 min and at most 2.5 h.

6. The application process as claimed in claim 1, wherein the sensitization solution S1 also contains at least one binding partner P2 to the binding partner P1 to the analyte.

7. A process for applying, in a tubular solid support, optionally flared, having a circular or ellipsoidal opening at each end, at least one binding partner P2 to a binding partner P1 to an analyte to be detected or quantified in a test sample, comprising the following steps: (a) connect the solid support to a suction-discharge device, (b) draw up into the solid support, by one of its ends, a solution comprising the at least one binding partner P2, called sensitization solution S2, contained in a container, called container C2, (c) continue contact between the sensitization solution S2 and the inner surface of the solid support for a time between 0 s and 11 min, (d) discharge the sensitization solution S2 into a container, which is optionally the container C2, steps (b) to (d) forming a cycle that can be repeated at least once, over a total duration of at least 1 min and at most 2.5 h.

8. The application process as claimed in claim 7, wherein it also includes the following steps, carried out when the suction (b)/contact (c)/discharge (d) cycles are completed: (e) draw up into the support in which the at least one binding partner P2 is applied, a wash solution W2 contained in a container called container CW2, (f) continue contact between the wash solution W2 and the inner surface of the solid substrate for a time between 0 s and 11 min, (g) discharge the wash solution W2 into a container, which is optionally the container CW2, steps (e) to (g) forming a cycle that can be repeated at least once, over a total duration of at least 1 min and at most 2.5 h.

9. The application process as claimed in claim 7, also comprising the application, in the solid support, after the suction (b)/contact (c)/discharge (d) cycles, of the at least one binding partner P1.

10. (canceled)

11. The application process as claimed in claim 5, wherein the container C1, the container CW1, and optionally the container C2 and/or the container CW2 are contained in the same test strip, which consists of several containers.

12. The application process as claimed in claim 11, wherein the test strip also includes other containers containing other components necessary for the detection or quantification of the analyte.

13. A process for the in vitro detection or quantification of an analyte in a test sample likely to contain the analyte, the process using at least one tubular solid support, optionally flared, having a circular or ellipsoidal opening at each end, in which at least one binding partner P1 to an analyte to be detected or quantified is applied in the test sample according to an application process as defined in claim 1, which detection or quantification process comprises the steps of contacting the test sample with the solid support and detecting the binding, if the analyte is present, of the analyte and the at least one binding partner P1.

14. The detection or quantification process as claimed in claim 13, wherein the binding partner P1 is an immunoassay partner and the detection of the binding of the analyte is carried out by a sandwich test using another binding partner to the analyte, optionally of a different nature, which is labelled.

15. The detection or quantification process as claimed in claim 13, wherein the binding partner P1 to the analyte is an immunoassay partner and the detection or not of the binding of the analyte is carried out by a competitive test using a labelled compound competing with the analyte to be detected or quantified.

16. The detection or quantification process as claimed in claim 13, wherein the detection of the analyte is carried out using an enzyme and an enzymatic substrate catalyzed by the enzyme.

17. (canceled)

18. The detection or quantification process as claimed in claim 13, wherein the steps of contacting the test sample with the solid support on which the at least one binding partner P1 is applied, and detecting the binding of the analyte and the at least one binding partner P1, include the following steps consisting in: (1) drawing up into said solid support, by one of its ends, the sample contained in a container, called container CE, leaving it in contact and discharging the sample into a container, which is optionally the container CE, (2) drawing up into the solid support, at the same end, a solution comprising a compound conjugated to a label, called conjugate solution CS, the conjugate solution C being contained in a container called container CC, allowing the conjugate solution CS to contact and discharge into a container, which is optionally the container CC, (3) if the label uses a detection substrate, drawing up into the solid support, at the same end, a solution comprising a detection substrate with which the label will react, called substrate solution SS, the substrate solution SS being contained in a container called container CS, leaving in contact and discharging the substrate solution into a container, which is optionally the container CS, and (4) measuring the transmitted signal.

19. The detection or quantification process as claimed in claim 18, wherein the container CE, the container CC and the container CS are contained in the same test strip.

20. The detection or quantification process as claimed in claim 13, wherein the test sample to be analyzed is a sample of biological, food or environmental origin.

Description

[0065] Various other features emerge from the description given below with reference to the appended FIGS. 1 to 8, which show, by way of non-limiting examples, embodiments of the subject matters of the invention.

[0066] FIG. 1 shows a diagram for the application of a binding partner, in a solid pipette tip-type support, with a loop coating system.

[0067] FIG. 2 shows a diagram for the application of a binding partner, in a solid pipette tip-type support, with an in-line system.

[0068] FIG. 3 shows a diagram for the application of a binding partner, in a solid pipette tip-type support, with a reciprocating system, as implemented for example in the VIDAS® instrument.

[0069] FIG. 4 shows a strip and a VIDAS® pipette tip when implementing a suction cycle (FIG. 4A)/contact (FIG. 4B)/discharge (FIG. 4C) according to an embodiment of the invention for the application of a binding partner, the strip being made up of several containers including the container S1 or S2.

[0070] FIG. 5 is a histogram-type representation of the relative fluorescence values (RFVs) for both sample types 1 and 2 when detecting TSH with the VIDAS® instrument, as a function of the different application conditions of the binding partner (dynamically with a concentration of binding partner and a contact time that varies—2.5 μg/mL for 5, 10 or 15 min or 5 μg/mL for 5, 10 or 15 min, or static with a concentration at 6 μg/mL for 20 h).

[0071] FIG. 6 is a histogram-type representation of the relative fluorescence values (RFVs) for the three sample types (blank, low and high—FIG. 6A) or only for the blank sample (representation of the histograms in FIG. 6A with enlarged scale—FIG. 6B) when detecting anti-Tg antibodies with the VIDAS® instrument, as a function of the different application conditions of the binding partner (dynamic for 5, 15 or 30 min, or static for 20 h).

[0072] FIG. 7 is a graph showing the RFV signal as a function of TnI concentration when detecting TnI with the VIDAS instrument (FIG. 7A: low TnI concentration and FIG. 7B: high TnI concentration) using a pipette tip coated with 3 binding partners according to the process of the invention (invention) or according to the prior art (reference), namely biotinylated BSA, then streptavidin, then a mixture of biotinylated anti-TnI antibodies.

[0073] FIG. 8 is a histogram giving the RFV signal for five samples with increasing TSH concentration using either a pipette tip coated with an anti-TSH antibody according to the invention (dynamic coating at 4 μg/mL) or a pipette tip coated with an anti-TSH antibody according to the prior art (static coating at 6 μg/mL).

[0074] FIGS. 1 to 3 are particular embodiments of implementation of suction/contact/discharge cycles according to the process of the invention.

[0075] According to FIG. 1, the device for applying the binding partner P1 in an open hollow support 1, of pipette tip type, comprises a container C1 4 containing the sensitization solution S1 containing said binding partner P1, a pump 2 which allows the sensitization solution to circulate, via a tubing system 3, in the indicated arrow direction. The first step consists of drawing up, in the direction of the arrow, using pump 2, the sensitization solution from the container C1 4 into the support 1, then optionally leaving the sensitization solution and the support 1 in contact, then discharging, still in the direction of the arrows, using the pump 2, the sensitization solution from the support 1 into the container 4, these steps being repeated. Thus, the sensitization solution is taken up at each step into the container 4, enters through one of the two ends of the support 1 and is discharged into the container 4 through the other end of the support 1.

[0076] According to a variant of this embodiment and as indicated above, the sensitization solution S1, after being drawn up into the support 1 by one of its ends, is discharged into the container C1 4 by the same end.

[0077] According to FIG. 2, the device for applying the binding partner P1 in an open hollow support 1, of pipette tip type, comprises a container C1 4 containing the sensitization solution S1 containing said binding partner P1, a pump 2 which allows the sensitization solution to circulate, via a tubing system 3, in the indicated arrow direction. The first step consists of drawing up, in the direction of the arrow, using the pump 2, the sensitization solution from the container C1 4 into the support 1, then optionally leaving the sensitization solution and the support 1 in contact, then discharging, still in the direction of the arrows, using the pump 2, the sensitization solution from the support 1 into the container 5 which is a waste container, these steps being repeated and the sensitization solution being taken up at each step into the container C1 4. Thus, the sensitization solution is taken up at each step into the container 4, enters through one of the two ends of the support 1 and is discharged into the container 5 through the other end of the support 1.

[0078] According to a variant of this embodiment, the suction/contact/discharge cycles are carried out using the same end of the support 1 and it is only during the last cycle that the sensitization solution is discharged into a waste container.

[0079] According to FIG. 3, the device for applying the binding partner P1 in an open hollow support 1, of pipette tip type, comprises a container C1 4 containing the sensitization solution S1 containing said binding partner P1, a pump 2 which allows the sensitization solution to circulate, via a tubing system 3, in the indicated arrow direction. The first step consists of drawing up, in the direction of the arrow going to the right, using the pump 2, the sensitization solution from the container C1 4 into the support 1, then optionally leaving the sensitization solution and support 1 in contact, then discharging, still in the direction of the arrow to the left, using the pump 2, the sensitization solution from the support 1 into the container 4, these steps being repeated. Thus, the sensitization solution is taken up at each step into the container 4, enters through one of the two ends of the support 1 and is discharged into the container 4 from the same end.

[0080] An example of a process consisting of drawing up and discharging the sensitization solution using the same end of the hollow support is shown in FIG. 4 which represents a strip 6 and a VIDAS® pipette tip 1. The test strip 6 contains different wells, including a well 10 to receive the test sample, a well 4 containing the sensitization solution S1, a reading well 11 in which the fluorescence will be read after the biological analysis of the sample is carried out, and wells 7, 8, 9 which may contain different elements such as wash buffers or a waste well in which the fraction of sensitization solution that comes into contact with the pipette tip is discharged. FIG. 4A shows the first step of the process of the invention, consisting in drawing up into the pipette tip 1, via a tubing 3 connected to a suction-discharge device (not shown in FIG. 4, but as shown in FIG. 3), a fraction of the sensitization solution contained in the well 4. FIG. 4B shows the second step during which the sensitization solution and the pipette tip 1 are left in contact. Finally, FIG. 4C shows the discharge step consisting of discharging the sensitization solution into the well 4.

[0081] The contact step (iii) between the sensitization solution S1 and the solid support is performed for a time between 0 s and 11 min. When the contact time is 0 s, it means that the suction and discharge steps follow one another, without any pause time between the two. The contact time can also be at most 10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, 55 s, 50 s, 45 s, 40 s, 35 s, 30 s, 25 s, 20 s or 15 s. The contact time can be at least 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s, 10 s, 11 s, 12 s, 13 s, 14 s, 15 s, 20 s, 30 s, 35 s or 40 s.

[0082] The contact time also varies according to the number of cycles implemented, which is at least two. The minimum and maximum number of cycles depends on the total duration of the application process, depending on the contact time chosen. Thus, the number of repetition cycles of steps (i) to (ii) may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20 or 25 and at most 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 120, 139, 150, 185, 200, 232, 250, 278, 300, 325, 371, 400, 417, 464, 500, 510, 556, 600, 603, 649, 700, 800, 900, 1000, 2000, 2500, 3000, 4000, 5000 or 6000 times.

[0083] The total time of the process is at least 1 min and at most about 2.5 h. This total time can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 min and at most 2 h, 1.5 h, 1 h, 55 min, 50 min, 45 min, 40 min, 39 min, 38 min, 37, min, 36 min, 35 min, 34 min, 33 min, 32 min, 31 min, 30 min, 29 min, 28 min, 27, min, 26 min, 25 min, 24 min, 23 min, 22 min, 21 min, 20 min, 19 min, 18 min, 17, min or 16 min.

[0084] The combination of contact time, number of cycles and total process time will be chosen by skilled persons according to the sensitivity of the analyte detection or quantification test they want to implement. This combination will condition the amount of binding partner P1 that will be applied to the inner surface of the solid support.

[0085] The temperature at which the application process of the invention is carried out is any temperature compatible with the binding partner P1, i.e. at any temperature that maintains the binding capacities to the analyte of said binding partner P1. In general, the temperature is between 10° C. and 45° C. Room temperature, from about 17° C. to about 25° C., is therefore appropriate. The temperature can also be a function of the instrument used. Thus, with regard to the VIDAS® instrument, the temperature used is 37° C.

[0086] Once the binding partner P1 has been applied to the inner surface of the solid support, it can be washed with wash solution W1 to remove unbound partners. The wash solutions useful for this purpose are classic and known to the skilled person. They include buffers, such as phosphate, Tris, HEPES buffers, salts such as NaCl, detergents such as 0.2% Triton X-100, Tween 20, etc. They may also contain saturation agents, also called passivation agents, such as BSA or milk proteins, to saturate the inner surface of the solid support, thus avoiding non-specific bonds that could occur when the test sample and the solid support are contacted. Again, the only condition for this wash solution W1 is that it must not destroy the binding capacity to the analyte of said binding partner P1.

[0087] The washing of the inner surface of the solid support may be carried out by any method known to the skilled person, such as soaking the solid support in a container containing said wash solution W1. It can also be implemented by reproducing the suction (ii)/contact (iii)/discharge (iv) cycles as described above. Thus, according to an embodiment of the invention, the application process also includes the following steps, implemented when the suction (ii)/contact (iii)/discharge (iv) cycles are completed:

[0088] (v) draw up into the support in which the at least one binding partner P1 is applied, a wash solution W1 contained in a container called container CW1, (vi) continue contact between the wash solution W1 and the inner surface of the solid support for a time between 0 s and 11 min,

[0089] (vii) discharge the wash solution W1 into a container, which is optionally said container CW1,

[0090] steps (v) to (vii) forming a cycle that can be repeated at least once, over a total duration of at least 1 min and at most 2.5 h.

[0091] All the features described above for steps (ii), (iii) and (iv) also apply to steps (v), (vi) and (vii).

[0092] As indicated above, at the end of the discharge step, the solution used can be discharged into the wash container CW1 or into another container which can then be the same container as the one used after step (iii). This would then be a waste container which will be identical both for the application of the binding partner P1 and for the wash.

[0093] The binding partner P1 can be applied directly to the inner surface of the solid support. Or it can be applied through another binding partner, called binding partner P2 to binding partner P1 to the analyte. Here again, the concepts of analyte and binding partner P2 are quite distinct from each other. The analyte is the compound present in the test sample, while the binding partner P2 is the compound that will bind to the binding partner P1 that will itself bind to the analyte. These binding partners P2 are optionally of immunological origin, as described above. When the binding partner P1 is an antibody, the binding partner P2 can be an anti-species antibody, for example an anti-mouse antibody when the partner P1 is a mouse antibody. The binding partner P2 can also be an anti-IgM antibody when the binding partner P1 is an IgM. When the binding partner P1 is an antigen, for example a peptide, the binding partner P2 can be an anti-peptide antibody. The binding partner P1 may have been previously modified with a ligand, for example a biotin, the binding partner P2 being an antiligand, for example streptavidin. Other examples of binding partners P2 are widely known to the skilled person.

[0094] The binding partner P2 can be contained in the sensitization solution S1, which constitutes a particular embodiment of the invention. In this case, the binding partner P2/binding partner P1 formation on the solid support will be implemented during the suction (ii)/contact (iii)/discharge (iv) cycles of the application process of the invention. This is applicable when the partner P2 binds more easily to the support than the partner P1, for example when the partner P2 is streptavidin and the partner P1 is a biotinylated antibody.

[0095] The binding partner P2 may also have previously been applied to the inner surface of the solid support according to any process known to the skilled person, for example in static or dynamic mode, reproducing the same cycles as those described above for the application of the binding partner P1.

[0096] Thus, at least one binding partner P2 to a binding partner P1 to an analyte to be detected or quantified in a test sample the binding partner P2 can be applied in the tubular solid support, optionally flared, having a circular or ellipsoidal opening at each end, such as a pipette tip or a pipette, by a method comprising the following steps: (a) connect the solid support to a suction-discharge device,

[0097] (b) draw up into the solid support, by one of its ends, a solution comprising said at least one binding partner P2, called sensitization solution S2, contained in a container, called container C2,

[0098] (c) continue contact between the sensitization solution S2 and the inner surface of the solid support for a time between 0 s and 11 min,

[0099] (d) discharge the sensitization solution S2 into a container, which is optionally said container C2,

[0100] steps (b) to (d) forming a cycle that can be repeated at least once, over a total duration of at least 1 min and at most 2.5 h.

[0101] All the features described above for steps (ii), (iii) and (iv) also apply to steps (b), (c) and (d).

[0102] As indicated above, at the end of the discharge step, the solution used can be discharged into the sensitization container C2 or into another container which can then be the same container as the one used with the other discharge steps previously described (waste container).

[0103] After application of the binding partner P2 to the inner surface of the solid substrate, it can be washed with a wash solution W2 to remove unbound partners.

[0104] Washing may also include the following steps, implemented when the suction (b)/contact (c)/discharge (d) cycles are completed:

[0105] (e) draw up into the support in which the at least one binding partner P2 is applied, a wash solution W2 contained in a container called container CW2,

[0106] (f) continue contact between the wash solution W2 and the inner surface of the solid substrate for a time of between 0 s and 11 min,

[0107] (g) discharge the wash solution W2 into a container, which is optionally said container CW2,

[0108] steps (e) to (g) forming a cycle that can be repeated at least once, over a total duration of at least 1 min and at most 2.5 h.

[0109] The wash solution W2 has the same features as the wash solution W1 described above. It may be identical (in which case it will be referred to indifferently as wash solution W for solution W1 and solution W2) or it may be different from wash solution W1, both in terms of nature and in terms of its container. It is preferably in a different container CW2 from CW1, but washing can be carried out with the same solution contained in the same container (this is called container CW).

[0110] As before, the discharge of the wash solution can be carried out in the wash container itself (CW2 or CW) or in a waste container, for example common for all discharge steps.

[0111] Whether or not there are washing steps, in order to use the solid support to detect or quantify an analyte likely to be contained in a test sample, it will then be coated with the binding partner P1. According to an embodiment, the application process of the binding partner P2 also includes the application, in said solid support, after the suction (b)/contact (c)/discharge (d) cycles, of said at least one binding partner P1.

[0112] The binding partner P1 can be applied to the inner surface of the solid support according to any process known to the skilled person, for example in static or dynamic mode, such that the same cycles of steps (ii) to (iv) described above can be reproduced for the application of said binding partner P1, which is another embodiment.

[0113] The application process of the binding partner P1 can be delayed relative to the application process of the binding partner P2, i.e. there is a waiting time of at least 5 min between the two processes, or it can be implemented immediately after the application process of the binding partner P2. Preferably, they should be used with the same instrument or with two instruments from the same range.

[0114] The different containers used in the application process of the binding partners can be physically separated, handled independently from one another, such as vials that are placed in a carousel of an instrument. Or the containers may be integral, for example in the form of a well in a strip, such as the VIDAS® strip (bioMérieux) shown in FIG. 4 (strip 6). According to an embodiment, the container C1, the container CW1, and if necessary the container C2 and/or the container CW2 are contained in the same test strip, which consists of several containers.

[0115] Since the test strip or instrument incorporating the different containers is intended for the in vitro detection or quantification of an analyte, it may also include other containers containing other components necessary for the detection or quantification of said analyte. The components necessary for the detection or quantification of said analyte are known to the skilled person and will be described below in the context of the detection or quantification of the analyte. Examples of such components include labelled binding partners, also known as conjugates, wash solutions and detection substrates. Examples of containers include a VIDAS® strip with all reagents (for coating and detection or quantification), or two VIDAS® strips, one for coating and the other for detection or quantification.

[0116] The in vitro detection or quantification of an analyte likely to be contained in a test sample may be carried out by any in vitro process known to the skilled person using at least one binding partner P1 to the analyte. It comprises the steps of contacting the test sample with the solid support coated with the binding partner P1 and detecting the binding, if the analyte is present, of said analyte and said at least one binding partner P1.

[0117] The application process of the binding partner P1 has been described above. The support can also include a binding partner P2, also applied as described above.

[0118] The analyte and the test sample that may contain it are also as described above. According to one embodiment, the test sample is a sample of biological, chemical, food or environmental origin.

[0119] The detection or quantification of analyte may be carried out by any analytical method known to the skilled person, such as immunoassay methods. These so-called enzyme-linked immunoassay (EIA) methods are coupled to an enzyme-catalyzed reaction using an enzyme substrate. Depending on the enzyme substrate chosen, a colorimetric signal (enzyme-linked immunosorbent assay, ELISA) (Rassasie, M. J., et al., 1992), a fluorescence signal (enzyme-linked fluorescent assay, ELFA) or a chemiluminescent signal (chemiluminescence immunoassay, CLIA) (Stabler T. V., et al., 1991) can be produced.

[0120] These methods are based on measurements to quantify the signals emitted during the analysis of the test sample. The quantity of signals detected is generally proportional to the amount of analyte to be measured (for example in a sandwich test) or inversely proportional to the amount of analyte to be measured (for example in a competition test).

[0121] Conventional steps in the in vitro detection and/or quantification process of an analyte by sandwich immunoassay, in a test sample likely to contain said analyte, include or consist of: [0122] the presence of the solid support inside which is applied said at least one binding partner P1 and said sample for fixing the analyte on the partner P1, [0123] the addition of a detection partner, which is directly or indirectly coupled to a label, such as an enzyme capable of lysing an enzymatic substrate, for example fluorogen for ELFA detection, for its binding to the binding partner P1-analyte complex, [0124] when the label is an enzyme, contacting an enzyme substrate and the binding partner P1-analyte-detection partner complex coupled to an enzyme to form a reaction medium, and [0125] detection, for example by immunofluorescence in ELFA detection, of the presence and/or amount of analyte by measuring the signal (for example fluorescence) emitted in the reaction medium.

[0126] Detection partner means any partner capable of binding to the analyte to be detected or quantified, which will be coupled directly or indirectly to a label, for example an enzyme. It may be of the same nature as the binding partner P1 or of a different nature. Examples are given above with the binding partner P1.

[0127] Direct or indirect coupling of the label to the detection partner means that the label is attached directly to the detection partner recognizing the analyte (direct coupling) or the enzyme is coupled to a binding partner that recognizes the detection partner which recognizes the analyte itself (indirect coupling).

[0128] Thus, in the context of direct coupling, the complex formed at the end of the assay, called a conjugate, will consist of: “Capture partner/analyte/detection partner coupled to the label”.

[0129] As part of the indirect coupling, the complex formed at the end of the assay will consist of: “Capture partner/analyte/detection partner/binding partner coupled to the label”.

[0130] In the latter case, the binding partner is well known to the skilled person and may be, for example, an anti-IgG (immunoglobulin) antibody when the detection partner is an IgG recognizing the analyte of interest.

[0131] Label means in particular any molecule containing a reactive group with a group of the detection partner, directly without chemical modification, or after chemical modification to include such a group, which molecule is capable of generating a detectable signal directly or indirectly. A non-exhaustive list of these direct detection markers consists of:

[0132] enzymes that produce a detectable signal for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase,

[0133] chromophores such as fluorescent compounds, luminescent compounds, dyes,

[0134] radioactive molecules such as 32P, 35S or 125I,

[0135] fluorescent molecules such as Alexa or phycocyanins, and

[0136] electrochemiluminescent salts such as organometallic derivatives based on acridinium or ruthenium.

[0137] According to a particular embodiment, the binding partner P1 used in the detection or quantification process is an immunoassay partner and the detection of the binding of said analyte is implemented by a sandwich test using another binding partner to the analyte, called the detection partner, optionally of a different nature, which is labelled by a label.

[0138] Conventional steps in the process of in vitro detection and/or quantification of an analyte by competitive immunoassay in a test sample likely to contain said analyte include: [0139] the presence of the solid support inside which is applied said at least one binding partner P1, an analyte analogue coupled to a label, for example an enzyme capable of lysing an enzymatic substrate, for example fluorogen, and said sample, which compete for binding to the binding partner P1, [0140] when the label is an enzyme, contacting an enzyme substrate, binding partner P1-analyte and binding partner P1-analogue to the analyte complexes to form a reaction medium, and [0141] the detection, for example by immunofluorescence, of the presence and/or amount of analyte by measuring the signal, for example fluorescence, emitted in the reaction medium.

[0142] Analogue to the analyte means any molecule that has the same binding capabilities to the binding partner P1 as the analyte.

[0143] The label coupled to the analogue to the analyte is equivalent to the label used in a sandwich test.

[0144] According to another particular embodiment of the invention, the binding partner P1 used in the detection or quantification process is an immunoassay partner and the detection of the binding or not of said analyte is implemented by a competitive test using a labelled compound competing with the analyte to be detected or quantified, otherwise called the labelled analogue to the analyte.

[0145] Regardless of the type of method used, whether in sandwiches or in competition, the enzyme is a widely appropriate label and examples include sulfatase, alkaline phosphatase (ALP), acid phosphatase, glucose oxidase (GOx), glucose-6-phosphate dehydrogenase (G6PD) and β-galactosidase (β-gal). The corresponding enzyme substrates are widely known to the skilled person and include, for example, 4-methylumbelliferyl phosphate or 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside.

[0146] According to a particular embodiment, in the detection or quantification process according to the invention, the detection of the analyte is carried out using an enzyme and an enzymatic substrate catalyzed by said enzyme, preferably alkaline phosphatase and 4-methylumbelliferyl phosphate.

[0147] The analyte detection or quantification process may also include one or more additional washing steps after each step, such as: [0148] before the addition of the detection partner, a washing step to remove the analyte not bound to the binding partner P1-analyte complex; and [0149] after the addition of the detection partner, a washing step to eliminate the unbound detection partner.

[0150] The washing steps are steps known to the skilled person. They are implemented with buffers compatible with the reaction medium and signal reading.

[0151] The method of detection or quantification of an analyte can be implemented at any time after the application process of the binding partner P1 to the inner surface of the solid support, and if applicable the binding partner P2. It can be implemented one or more days after the application of the binding partner P1 and, if necessary, the binding partner P2, or even one or more weeks later. In this case, the solid support must be dried and then stored in a desiccant bag to avoid any stability problems. Advantageously, the steps of contacting the test sample with the solid support and detecting the binding, if the analyte is present, of said analyte and said at least one binding partner P1 of the detection or quantification process of the invention are carried out immediately after the process of applying at least one binding partner P1 in said solid support as defined above. Immediately after means that the detection or quantification process is implemented within minutes or even seconds of the application process of the binding partner P1. There are no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 min between the last step of the binding partner P1 application process and the first step of the analyte detection or quantification process. This short time may be useful to introduce the test sample and/or containers that will be used for detection or quantification. In other words, the application process of at least one binding partner P1, or even at least one partner P2 beforehand, and the detection or quantification process are implemented successively.

[0152] In order to facilitate the user's organization, to control the costs and space of the laboratory, the analyte detection or quantification process is implemented with the same instrument or type of instrument as that used for the application of the binding partner P1, or even the binding partner P2, or even the different washing processes. Also, the steps of contacting the test sample with the solid support on which said at least one binding partner P1 is applied, and detecting the binding of said analyte and said at least one binding partner P1, include or consist of the following steps consisting in:

(1) drawing up into said solid support, by one of its ends, said sample contained in a container, called container CE, leaving in contact and discharging said sample into a container, which is optionally said container CE,
(2) drawing up into said solid support, at the same end, a solution comprising a compound conjugated to a label, such as another binding partner to the analyte or a compound competing with the analyte, called conjugate solution CS, said conjugate solution C being contained in a container called container CC, leaving in contact and discharging said conjugate solution CS into a container, which is optionally said container CC,
(3) if the label uses a detection substrate, drawing up into said solid support, at the same end, a solution comprising a detection substrate with which the label will react, called substrate solution SS, said substrate solution SS being contained in a container called container CS, leaving in contact and discharging said substrate solution into a container, which is optionally said container CS, and
(4) measuring the transmitted signal.

[0153] If necessary, these steps are preceded by a step of connecting the solid support to a suction-discharge device such as a pump unit.

[0154] Steps (1) and (2) of suction and discharge can be implemented as described above.

[0155] The different components used, for example the binding partners P1, the detection partners, markers, etc. are as described above.

[0156] The different containers CE, CC and CS used are as described above and can be separated or contained in the same test strip, for example, which is a particular embodiment.

[0157] The last step (4) consists in measuring the transmitted signal. This step is well known to the skilled person. This measurement can be transformed into a representative amount by the user such as the concentration of analyte using a standard curve, also called a calibration curve, the obtaining of which is widely known to the skilled person and can be obtained by i) measuring the signal generated by standards, also called standards or calibrators, then ii) plotting the curve giving the signal as a function of amount or concentration. Very often, it is common practice to find a mathematical model that represents, as accurately as possible, this relationship between signal and amount or concentration, in order to be able to easily calculate the results of a quantitative immunoassay.

[0158] In addition to steps (1) to (4), the process may also include washing steps, as described above.

[0159] The invention will be better understood using the following examples, which are given by way of non-limiting illustration.

EXAMPLES

Example 1: Application of an Anti-TSH Antibody in a Pipette Tip and Detection of TSH

[0160] Thyrotropin-releasing hormone or thyroid stimulating hormone (TSH) is a hormone secreted by the thyroid cells of the anterior pituitary gland. This hormone is the main stimulating factor of the thyroid gland that determines the production of the thyroid hormones T3 and T4. In return, these thyroid hormones exert a feedback control on the anterior pituitary gland, which slows down the secretion of TSH. The secretion of TSH is also controlled by the central nervous system through a hypothalamic neuropeptide, TRH, and neurotransmitters such as somatostatin or dopamine. The blood test for TSH is an aid in the diagnosis of thyroid or pituitary disorders.

[0161] 1.1 Application of an Anti-TSH Antibody in a VIDAS® Pipette Tip According to the Invention

[0162] VIDAS® pipette tips are sensitized with 300 μL of a solution of mouse monoclonal anti-TSH antibody (bioMérieux product number 30400) at 2.5 or 5 μg/mL in a Tris HCl buffer pH 7.3 (sensitization solution) contained in one of the wells of a VIDAS® strip, using the VIDAS® instrument, as follows:

Five-Minute Protocol:

[0163] 100 μL/s aspiration of the sensitization solution [0164] incubation 10 seconds [0165] solution discharge.

[0166] This cycle is repeated 20 times.

Ten-Minute Protocol:

[0167] 100 μL/s aspiration of the sensitization solution [0168] incubation 18 seconds [0169] solution discharge.

[0170] This cycle is repeated 25 times.

Fifteen-Minute Protocol:

[0171] 100 μL/s aspiration of the sensitization solution [0172] incubation 20 seconds [0173] solution discharge.

[0174] This cycle is repeated 35 times.

The emptied pipette tips are ready to be used with no further preparation for the determination of TSH in a biological sample.

[0175] 1.2 Application of an Anti-TSH Antibody in a VIDAS® Pipette Tip According to the Prior Art

[0176] For comparison purposes, the VIDAS® pipette tips were sensitized statically as follows: The pipette tips are sensitized with 300 μL of the same mouse anti-TSH monoclonal antibody solution except that the antibody concentration is 6 μg/mL. After about 20 hours at room temperature (18-25° C.) with the sensitization solution, the pipette tips are emptied. Then, 330 μL of a saturation solution containing in particular animal proteins are added for passivation of the pipette tips for about 6 hours. The pipette tips are then emptied, dried and stored at 4° C. until use, away from moisture.

[0177] 1.3 Determination of TSH in a Sample

[0178] The pipette tips coated with anti-TSH antibodies according to points 1.1. and 1.2. above are used with strips from the VIDAS® TSH Kit (bioMérieux product number 30400), which include the other reagents of the immunological reaction.

[0179] All assay steps are performed automatically by the VIDAS® instrument according to a standard procedure for this instrument.

[0180] The sample contained in the sample well (200 μL) of the strip is collected and transferred to the well containing the alkaline phosphatase (conjugate) labelled anti-TSH antibody. The sample/conjugate mixture is drawn up into and then successively discharged by the pipette tip for about 16 minutes. This operation allows the antigen to bind to the immunoglobulins attached to the pipette tip and to the conjugate forming a “sandwich”.

[0181] Three successive washing steps of 3 cycles of 5 seconds each, carried out by the instrument using the wash solution contained in the VIDAS TSH Kit strip, remove the unbound compounds.

[0182] In the final detection step, the substrate (4-methylumbelliferyl phosphate) contained in a well of the strip is drawn up into the pipette tip and then discharged into the detection well; the enzyme of the conjugate catalyzes the hydrolysis reaction of this substrate into a product (4-methylumbelliferone) whose emitted fluorescence is measured at 450 nm in the detection well. The value of the fluorescence signal (RFV=relative fluorescence value) is proportional to the concentration of the antigen present in the sample.

[0183] Two types of human serum samples containing TSH were used for each concentration condition and protocol duration, namely a sample 1 containing a normal TSH concentration (3 μIU/mL TSH) and a sample 2 containing a high TSH concentration, corresponding to hypothyroidism (40 μIU/mL TSH).

[0184] The results are given in FIG. 5 which is a histogram-type representation of the relative fluorescence values (RFVs) for both types of sample 1 and 2 when detecting TSH with the VIDAS® instrument, as a function of the different application conditions of the binding partner (dynamically with a binding partner concentration and contact time that vary—2.5 μg/mL for 5, 10 or 15 min or 5 μg/mL for 5, 10 or 15 min, or static with a concentration at 6 μg/mL for 20 h).

[0185] FIG. 5 shows that the RFV signal obtained for sensitization concentrations of 2.5 μg/mL and 5 μg/mL in dynamic mode is quite comparable, regardless of the duration of sensitization. From 5 min of dynamic coating, the RFV signal for sample 1 is about 500 RFV. Sample 2 yields RFVs greater than 3500. All the dynamic application conditions of the anti-TSH antibody used according to the invention have excellent signal dynamics, equivalent to the reference condition obtained in several days by a static incubation process.

Example 2: Application of a Tg Antigen in a Pipette Tip and Detection of Anti-Tg Antibodies

[0186] Thyroglobulin (Tg) is a glycoprotein produced in the thyroid gland and is the main component of follicular colloid. Its main role is the storage and synthesis of thyroid hormones. Antithyroglobulin autoantibodies are often present in patients with autoimmune thyroid disease. For example, they are detected in 30% of patients with Graves' disease (Basedow) and in 85% of patients with Hashimoto's disease (2). Anti-Tg antibodies are associated with hypothyroidism or mild hyperthyroidism and are frequently present in patients with other autoimmune diseases such as rheumatoid arthritis, pernicious anemia and type I diabetes (3, 4).

[0187] 2.1. Application of a Tg Antigen in a VIDAS® Pipette Tip According to the Invention

[0188] VIDAS® pipette tips are sensitized with 300 μL of a native Tg antigen solution (bioMérieux product number 30462) at 7 μg/mL in a phosphate buffer (sensitization solution) contained in one of the wells of a VIDAS® strip, using the VIDAS® instrument, as follows:

Five-Minute Protocol:

[0189] 100 μL/s aspiration of the sensitization solution [0190] incubation 10 seconds [0191] solution discharge.

[0192] This cycle is repeated 20 times.

Fifteen-Minute Protocol:

[0193] 100 μL/s aspiration of the sensitization solution [0194] incubation 20 seconds [0195] solution discharge.

[0196] This cycle is repeated 35 times.

Thirty-Minute Protocol:

[0197] 100 μL/s aspiration of the sensitization solution [0198] incubation 30 seconds [0199] solution discharge.

[0200] This cycle is repeated 50 times.

The emptied pipette tips are ready to be used with no further preparation for the determination of anti-Tg antibodies in a biological sample.

[0201] 2.2 Application of a Tg Antigen in a VIDAS® Pipette Tip According to the Prior Art

[0202] For comparative purposes, the VIDAS® pipette tips were sensitized statically as follows:

The pipette tips are sensitized with 300 μL of the same native Tg antigen solution. After about 6 hours at room temperature (18-25° C.) with the sensitization solution, the pipette tips were emptied. Then, 330 μL of a saturation solution containing in particular animal proteins are added for passivation of the pipette tips for about 6 hours. The pipette tips are then emptied, dried and stored at 4° C. until use, away from moisture.

[0203] 2.3 Determination of Anti-Tg Antibodies in a Sample

[0204] The pipette tips thus coated with Tg antigen according to points 2.1. and 2.2. above are used with strips from the VIDAS anti-TG kit (product number 30462).

[0205] The sample (100 μL) is taken by the instrument from the sample well of the strip and then transferred to the well containing a sample diluent. The diluted sample is aspirated and then discharged for about 3 minutes. This step allows the anti-Tg antibodies present in the sample to bind to the antigen attached to the pipette tip. The unbound components of the serum are removed by 3 washes in wells of the VIDAS anti-Tg strip for about 3 minutes. An incubation step with the detection conjugate is performed for about 6 minutes, respecting suction/discharge cycles of 30×8 seconds. The conjugate binds specifically to the anti-Tg antibodies of the previously bound sample. A washing cycle identical to the previous one removes the excess of unbound conjugate before the detection.

[0206] In the final detection step, the substrate (4-methylumbelliferyl phosphate) contained in a well of the strip is drawn up into the pipette tip and then discharged into the detection well; the enzyme of the conjugate catalyzes the hydrolysis reaction of this substrate into a product (4-methylumbelliferone) whose emitted fluorescence is measured at 450 nm in the detection well. The value of the fluorescence signal (RFV=relative fluorescence value) is proportional to the concentration of the antigen present in the sample.

[0207] The assayed samples are samples of natural human serum with a concentration corresponding to 60 IU/mL (low sample) and 1000 IU/mL (high sample). The blank sample is a mixture of negative samples.

[0208] The results are given in FIG. 6 which is a histogram-type representation of the relative fluorescence values (RFVs) for the three sample types (blank, low and high—FIG. 6A) or only for the blank sample (representation of histograms in FIG. 6A with enlarged scale—FIG. 6B) when detecting anti-Tg antibodies with the VIDAS® instrument, as a function of the different application conditions of the binding partner (dynamically for 5, 15 or 30 minutes, or statically for about 6 hours).

[0209] FIG. 6A shows that the dynamic application conditions for binding the Tg antigen to the support according to the process of the invention give signals equivalent to or better than those obtained with the static reference condition. The signal obtained on low and high human serum samples is satisfactory after 5 minutes of dynamic adsorption time. It is at its maximum for the 30-minute protocol, allowing a higher signal than the reference with the high sample.

[0210] The signal of the blank sample is used to evaluate the non-specific signal. The signal for this sample must be at the lowest level. As shown in FIG. 6B, with dynamic application conditions, this non-specific signal is weaker (less than 20 RFV) than for the static reference for which the signal is 25 RFV.

[0211] The conditions of application of the Tg antigen on the support according to the invention lead to an immunoassay equivalent to or better than the reference condition.

Example 3: Application of Several Binding Partners to Form a Biotinylated BSA/Streptavidin/Biotinylated Anti-cTni Antibody Complex and Detection of TNI

[0212] Troponin is a protein complex that sensitizes muscle cells to the calcium responsible for inhibiting the binding between myosin and actin (by masking the actin site that is used for binding to myosin). It therefore has an inhibitory function that has the effect of initiating muscle relaxation. protein used. Troponin I (TnI) is a subunit. Its determination is widely used as a tool to assist in the diagnosis of myocardial infarction (MI) and 30-day risk stratification for all-cause mortality and major adverse cardiac events (MACE) including myocardial infarction and revascularization in patients with acute coronary syndrome (ACS)-like symptoms.

[0213] 3.1 Dynamic Sensitization of Pipette Tips with Biotinylated BSA and Streptavidin for the Binding of Specific Biotinylated Anti-cTni Antibodies

[0214] VIDAS® pipette tips are sensitized with 300 μL of a biotinylated BSA solution (bioMérieux product number 30448) at 1 μg/mL in a carbonate buffer contained in one of the wells of a VIDAS® strip. The cycles performed by the VIDAS® and repeated 50 times are: [0215] 100 μL/s aspiration of the biotinylated BSA solution, [0216] incubation for 20 s then [0217] solution discharge.

[0218] Next, these same pipette tips are incubated with 300 μL of a 5 μg/mL streptavidin solution (bioMérieux product number 30448) diluted in PBS buffer for 50 cycles of 20 seconds (aspiration, incubation of 20 s and discharge).

[0219] The pipette tips are then sensitized with a mixture of biotinylated anti-cTnI antibodies (bioMérieux product number 30448) at 2 μg/mL and 3 μg/mL respectively, on 300 μL. The incubation time is 50 times 20 seconds.

[0220] After each of these 3 incubation steps, the pipette tips are washed twice successively on 300 μL for 8 times 1 second in wells containing a wash solution in the VIDAS cartridge. The emptied pipette tips are ready to be used with no further preparation for the determination of TnI in a biological sample.

[0221] 3.2. Application of Biotinylated BSA and then Streptavidin for the Binding of Specific Biotinylated Anti-cTni Antibodies in a VIDAS® Pipette Tip According to the Prior Art

[0222] For comparative purposes, the VIDAS® pipette tips were sensitized statically as follows:

The pipette tips are sensitized with 300 μL of the same biotinylated BSA solution for about 20 hours at room temperature (18-25° C.). The pipette tips are then emptied and filled with 300 μL of streptavidin solution for about 20 hours at room temperature (18-25° C.). After emptying the pipette tips, the sensitization step on approximately 300 with the mixture of biotinylated anti-cTni antibodies at the same concentration as in point 3.1. continues for approximately 20 hours. The pipette tips are then emptied, dried and stored at 4° C. until use, away from moisture.

[0223] 3.2. Determination of cTnI

[0224] All test steps are performed automatically by the instrument according to a standard instrument operating mode. They consist of a succession of suction/discharge cycles of the reaction medium. The sample (200 μL) is collected and transferred to the well containing the alkaline phosphatase (conjugate) labelled cardiac anti-troponin antibodies. The sample/conjugate mixture is drawn up into and then successively discharged by the pipette tip for about 10 minutes (50×8 seconds). This operation allows the antigen to bind to the immunoglobulins attached to the pipette tip and to the conjugate forming a “sandwich”.

[0225] Three successive washing steps of 3 cycles of 2 seconds each remove unbound compounds.

[0226] Two detection steps are then carried out successively. At each step, the substrate (4-methylumbelliferyl phosphate) is drawn up into and then discharged into the pipette tip; the enzyme of the conjugate catalyzes the hydrolysis reaction of this substrate into a product (4-methylumbelliferone) whose emitted fluorescence is measured at 450 nm. The value of the fluorescence signal is proportional to the concentration of the antigen present in the sample.

[0227] At the end of the test, the results are automatically calculated by the instrument against two stored calibration curves corresponding to the two detection steps. A threshold signal controls the choice of the calibration curve to be used for each sample. Then the results are printed.

[0228] The assayed samples are human sera with Tni concentrations ranging from 0.001 μg/L to 14.03 μg/L (samples 1 to 11).

[0229] RFV signal results as a function of TnI concentration are given in FIG. 7 (FIG. 7A: samples 1 to 6 and FIG. 7B: samples 7 to 11) which shows that the signal is equivalent between the two types of coating, regardless of the concentration of the sample tested. The application of the biotinylated BSA/streptavidin/biotinylated antibody complex according to the invention makes it possible to obtain an efficient immunoassay, according to a much faster process than with the prior art.

Example 4: Variation of Binding Partner Concentrations in the Sensitization Solution

[0230] 4.1. Application of an Anti-TSH Antibody in a VIDAS® Pipette Tip According to the Invention

[0231] VIDAS® pipette tips are sensitized with 300 μL of a 4 μg/mL solution of mouse monoclonal anti-TSH antibody in a Tris HCl buffer pH 7.3 (sensitization solution) contained in one of the wells of a VIDAS® strip, using the VIDAS® instrument, as follows:

Sixty-Minute Protocol:

[0232] 100 μL/s aspiration of the sensitization solution [0233] incubation 20 seconds [0234] solution discharge.

[0235] This cycle is repeated 139 times.

[0236] The empty pipette tips are ready for use with no further preparation for the determination of TSH in biological samples.

[0237] 4.2. Application of an Anti-TSH Antibody in a VIDAS® Pipette Tip According to the Prior Art

[0238] For comparative purposes, the VIDAS® pipette tips are sensitized according to the prior art as follows:

[0239] The pipette tips are sensitized with 300 μL of a solution of mouse monoclonal anti-TSH antibody (bioMérieux product number 30400) at 6 μg/mL in a Tris HCl pH 7.3 buffer. After about 20 hours at room temperature (18-25° C.) with the sensitization solution, the pipette tips are emptied. Then, 330 μL of the same solution containing animal proteins are added for passivation of the pipette tips for about 6 hours. The pipette tips are then emptied, dried and stored at 4° C. until use, away from moisture.

[0240] 4.3. Determination of TSH

[0241] The TSH assay is implemented as described in Example 1. The assayed samples (SC13, SC14, SC15, SC16 and SC17) are human sera with increasing TSH concentrations.

[0242] The results of these assays are given in FIG. 8 which is a histogram-type representation giving the RFV signal for each sample using either a pipette tip coated with an anti-TSH antibody according to the invention (dynamic coating at 4 μg/mL) or a pipette tip coated with an anti-TSH antibody according to the prior art (static coating at 6 μg/mL).

[0243] FIG. 8 shows that the signals obtained between the two pipette tip fabrications (according to the invention and according to the prior art) are very comparable, whereas the fabrication of the invention uses ⅓ less anti-TSH antibodies in the sensitization solution (4 μg/mL versus 6 μg/mL) and allows a production of these pipette tips for use in immunoassay in 1 hour versus 2 steps of more than 6 hours each according to the prior art. All other experimental conditions being identical, the application of anti-TSH antibodies according to the invention allows substantial savings in raw materials and time.

REFERENCES

[0244] Boersma Y L and Plütckthun A, 2011, Curr. Opin. Biotechnol, 22:849-857 [0245] Ellington A D and Szostak J W., 1990, Nature, 346:818-822 [0246] Rassasie M. J. et al., 1992, Steroids, 57:112 [0247] Stabler T. V., et al., 1991, Clin. Chem., 37(11):1987