Washing solution for solid-phase immunometric methods which...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...

Reexamination Certificate

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C435S014000, C435S021000, C435S025000, C435S026000, C435S028000, C435S962000, C435S967000

Reexamination Certificate

active

06664070

ABSTRACT:

The invention relates to a washing solution, containing stabilizers for the labeling enzyme, for solid-phase immunometric assays, and to the use of this washing solution. Specifically the stabilizers for enzymes are phenol and phenol derivatives.
Solid-phase immunometric assays, for example the enzyme-linked immunosorbent assay (ELISA), require one or more washing steps in the procedure. This entails the solid phase being rinsed with the washing solution in order to remove substances which have undergone nonspecific attachment, for example immunoglobulins, or excess reagents, for example an enzyme conjugate. If this is carried out in a suitable manner, the result of the assay is a measured signal which corresponds to the concentration of the detected analyte. In addition, this result can be reproduced.
Enzyme immunoassays as such are known to the person skilled in the art and described in the literature, see, for example, KYREIN, H. J., Ärztl. Lab. 24, 57-65 (1978).
Solid phases for use in such enzyme immunoassays are likewise known to the person skilled in the art and described in the literature, see, for example, VOLLER, A. et al., Bull. World Health Organ. 53, 55-65 (1976).
Such solid-phase immunometric assays can also be completed using instruments. This entails the washing steps being carried out by the instrument. The known washing solutions, which are composed, for example, of detergent-containing phosphate buffers in the neutral range, have certain disadvantages in these systems. When such instruments, are used to complete the washing step, both the accuracy and the reproducibility of the measured signal reach an acceptable level only after some time, i.e. after some plates have been completed (BURROWS, P. M. et al., J. Virol. Meth. 8, 207-216 (1984)).
The present invention was therefore based on the object of finding a washing solution whose use., in instruments makes possible correct completion of the ELISA even on immediate use of these devices. A measured signal which correlates with the concentration of the detected analyte, and the reproducibility of the results obtained, are regarded as criteria for correct assay procedure.
Instruments within the meaning of this invention are all instruments with whose aid washing steps in enzyme immunoassays can be carried out mechanically, irrespective of whether these instruments are able to carry out further steps in completing ELISA assays.
It has now been found, surprisingly, that the addition of stabilizers achieves this object, irrespective of the buffer basis, the pH or other additives to the washing solution.
Stabilizers within the meaning of this invention are substances which stabilize the labeling enzyme, such as, for example, tobramycin, phenol and phenol derivatives, and phenols and phenol derivatives which carry one or more substituents, which can be C
1
-C
3
-alkyl, chlorine and/or bromine, are preferred.
It is also generally possible to stabilize enzymes by substrates and competitive inhibitors.
The invention therefore relates to a washing solution for heterogeneous enzyme immunoassays which contains a stabilizer for the marker enzyme.
The invention also relates to the use of a washing solution as described above in a heterogeneous enzyme immunoassay.
The invention further relates to heterogeneous enzyme immunoassays entailing the use of a washing solution as described above in at least one washing step.
The invention furthermore relates to the use of stabilizers for the labeling enzyme in washing solutions for heterogeneous enzyme immunoassays.
The stabilizer is added in a concentration of 0.01 to 20 mM. A concentration of 0.1 to 5 mM is preferred, and 1 mM is very particularly preferred. The stabilizer can be added to previously known washing solutions or buffers for solid-phase immunometric assays.
In a preferred embodiment, the washing solution is buffered. Buffer systems which can be used for this are known to the person skilled in the art. The specific pH used depends on the assay system and can be determined where appropriate by experiment.
Preferred stabilizers are phenols and phenol derivatives, in which case phenol can also carry one or more substituents which can be C
1
-C
3
-alkyl groups and chlorine and/or bromine atoms.
The solutions described in the examples are particularly preferred, and phenol is very particularly preferred.
Heterogeneous enzyme immunoassays are known per se to the person skilled in the art. They can be used to detect antigens and antibodies and can be additive, such as, for example, a sandwich immunoassay, or competitive. The various possible embodiments have been adequately described in the literature. The ELISA method is preferred within the scope of the invention.
Marker enzymes for enzyme immunoassays as such are disclosed in the literature, and alkaline phosphatase, &bgr;-galactosidase and horseradish peroxidase are preferably used, and horseradish peroxidase is particularly preferably used.
Solid phases for heterogeneous enzyme immunoassays are known per se to the person skilled in the art, and concave shaped articles such as, for example, tubes or wells, convex shaped articles such as, for example, beads, stars or the like and microparticles (particle size<1,000 nm) such as, for example, latex particles and magnetically attractable particles are preferably used. Particularly preferred in this context are wells in the form of microtiter plates, latex particles and magnetically attractable particles. Microtiter plates are very particularly preferred.
Materials for solid phases are known to the person skilled in the art. Unless already fixed by the nature of the solid phase, such as, for example, in the case of latex particles, polystyrene is preferably used.
Buffer systems for use in enzyme immunoassays are known to the person skilled in the art. The person skilled in the art is also aware that the nature of the buffer system used in each case depends inter alia on the pH to be achieved.
Detergents for use in washing solutions for heterogeneous enzyme immunoassays are likewise known to the person skilled in the art (see, for example, VOLLER, A. et al., Bull. World Health Organ. 53, 55-65 (1976)), and non-ionic and zwitterionic detergents are preferably used; polyoxyethylenes are particularly preferred, and
R
Tween 20 is very particularly preferred.
Neutral proteins for use in enzyme immunoassays are known to the person skilled in the art; examples which are preferably used are serum albumins, gelatin, chemically modified gelatin such as, for example, polygeline, and milk proteins such as, for example, lactoferrin, particularly preferred are human or bovine serum albumin, polygeline and lactoferrin, very particularly preferred are polygeline and lactoferrin, the latter prepared as described in German Patent Application 36 38 767.
The person skilled in the art is aware that neutral salts such as, for example, NaCl are added to solutions used in enzyme immunoassays in order to adjust to a defined osmolarity.
Said substances are employed in aqueous solution for use; until used they can be, for example, in lyophilized or granulated form, as dry mixture or in liquid form as final dilution or concentrate.
A preferred embodiment of the washing solution according to the invention has the following composition:
Buffer
0 . . . 100 mmol/l, preferably
10-20 mmol/l, very preferably
10 mmol/l
Detergent
0 . . . 1%, preferably 0 . . . 0.2%, very
preferably 0.1% (w/v)
Neutral protein
0 . . . 1% (w/v)
Stabilizer
0.1 . . . 20 mmol/l, preferably
1 mmol/l.
The composition and components of conventional washing solutions are known to the person skilled in the art.
One example of them is a 10 mM phosphate buffer composed of Na
2
HPO
4
, KH
2
PO
4
, NaCl 0.45% w/v and 0.1% (w/v) Tween
R
20 with a pH of 6.5. This washing solution was mixed according to the invention as example with 1 mM phenol and was used to demonstrate the improvement in the measured signal and in the reproducibility achieved therewith in the ELISA.
The following example serves only for illustration and in no way represen

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