Purification of substances from a biological sample

Details Purification of substances from a biological sample Purification of substances from a biological sample

2000-10-13

2003-10-21

Le, Long V. (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S004000, C435S007100, C435S007900, C435S007920, C435S007940, C435S174000, C435S176000, C435S177000, C435S803000, C435S810000, C435S814000, C435S815000, C435S971000, C435S975000, C436S063000, C436S501000, C436S518000, C436S513000, C436S523000, C436S524000, C436S527000, C436S528000, C436S532000, C436S534000, C436S536000, C436S823000, C436S824000

Reexamination Certificate

active

06635420

ABSTRACT:

This application claims priority to parent application DE 1039218 filed Sep. 8, 1997, now German patent no. 19739218, granted Mar. 11, 1999, and to PCT application no. PCT/EP98/05521 filed Aug. 31, 1998 and published as WO 9913330 on Mar. 18, 1999.
BACKGROUND
The invention concerns a method for the preparative purification of a target substance from a biological sample by immobilizing the target substance on a solid phase by means of a high affinity binding pair and subsequently eluting it by adding a partner of the binding pair in a free form. In addition reagent kits for carrying out the method are disclosed.
The immunosorptive purification of biological substances, e.g. macromolecules such as proteins, is a method that has been known for a long time which allows the efficient isolation of such substances from complex biological materials like serum, urine or cell lysates (Eveleigh and Levy, J. Solid-phase Biochem. 2 (1977), 45-78; Cooper, “Biochemische Arbeitsmethoden”, pp. 222-241 (1981), Walter de Gruyter, Berlin; Pharmacia Fine Chemicals, Affinity Chromatography: Principles and Methods (1983), pp 92-95; Wawrazynczak and Cumber, in: Immunochemical Protocols, M. M. Manson ed. Methods in Molecular Biology 10, chapter 32 (1992), Humana Press, Totowa, N.J. USA). The desorption of the substances bound to the immobilized antibodies is usually achieved with unspecific methods e.g. by adding buffers with a low (<2.5) or high (>10) pH value, chaotropic reagents such as urea or guanidine hydrochloride, organic solvents or detergents to the elution buffer.
In some cases specific desorption methods have also been described e.g. to isolate haptens by displacement with a hapten analogue or to isolate proteins by displacement with a low molecular peptide. However, such specific desorption processes are an exception in immunosorptive processes since they can only be carried out at all with a very limited number of substances. Hence the unspecific desorption methods described above are mainly used and especially elution at a low pH such as e.g. with 1 M propionic acid.
However, in immunosorptive purification methods from biological materials it has been shown that the biomolecules that are present e.g. proteins in some cases also bind unspecifically to the immunoadsorber to a considerable extent i.e. not via an antibody-antigen interaction. If the desorption is unspecific, e.g. using an acidic buffer, these substances are then also eluted from the immunoadsorber and are present in addition to the desired substance as an impurity. This is a particular disadvantage especially when the substance to be purified is only present at a very low concentration since it then only represents a small proportion of the overall material that is eluted from the immunoadsorber so that additional laborious purification steps are necessary before the desired substance is available in sufficient purity, if this is possible with the very small amounts of substance that may be present.
Thus for example the prostate-specific antigen (PSA) is present in human serum at a concentration of ca. 1 to 1500 ng/ml, whereas other proteins such as human serum albumin (HSA) are present at a 10
5
to 10
7
-fold higher concentration of ca. 70 mg/ml. Since these proteins bind unspecifically to the immunoadsorber, when a substance such as PSA that is present at very low concentration is purified by immunosorption, the desired substance may only represent a very small proportion of the eluted total protein as a result of unspecific desorption and for example may not be identifiable at all in a gel electrophoretic separation of the eluate (cf. e.g. FIG.
2
). In this case it may not be possible to characterize the protein or only with great difficulty e.g. after a protease digestion due to the presence of the contaminating proteins. Similar problems also occur with other analytical methods such as e.g. mass spectrometry since also in this case the analysis is very difficult or impossible due to the many contaminating proteins especially when the exact mass of the protein to be examined is unknown. Thus although a combination of MALDI-TOF mass spectroscopy and immunosorption has been described in which the immunosorptively-bound material is eluted through the acidic MALDI matrix (cinnamic acid derivatives dissolved in trifluoroacetic acid/acetonitrile) and is measured directly (Nelson et al., Anal. Chem. 67 (1995), 1153-1158). However, it is clear that when the substance to be purified is only present at very low concentrations, numerous other proteins occur some of which result in considerably higher signals than the desired target substance (cf.
FIG. 1
taken from Krone et al., Mass Spectrometric Immunoassay, Poster Session ABRF'96: Biomolecular Techniques (1996), San Francisco, USA).
DESCRIPTION
Hence the object of the present invention was to find a generally applicable method for purifying substances from biological samples in which the disadvantages of the prior art are at least partially eliminated. In particular the method should utilize the advantages of specific immunosorption while avoiding the disadvantages of unspecific desorption. In particular the method according to the invention should enable an elution in an essentially neutral pH range to avoid damage to the target substance associated with a loss of immune reactivity or/and biological activity by extreme pH values.
This object is achieved by a method for purifying a target substance from a sample comprising the steps:
a) adsorbing the target substance onto a solid phase in which the binding of the target substance to the solid phase comprises the interaction between a first and a second partner of a high affinity binding pair, and the first partner of the binding pair is bound directly or indirectly to the target substance and the second partner of the binding pair is bound to the solid phase,
b) separating non-adsorbed sample components from the solid phase,
c) contacting the solid phase with the first partner of the binding pair or with an analogue thereof in a free form resulting in a desorption of the target substance from the solid phase and
d) separating the target substance from the solid phase.
A first preferred embodiment of the invention is a method for purifying a target substance from a sample comprising the steps:
a) Providing a solid phase with an immobilized reactant R
1
where R
1
contains at least one group that can bind specifically to the target substance and at least one group comprising a first partner of a high affinity binding pair, and the binding of R
1
to the solid phase occurs by means of an immobilized reactant R
2
which contains a group comprising the second partner of the high affinity binding pair,
b) contacting the sample with the solid phase resulting in an adsorption of the target substance onto the solid phase via a binding to R
1
,
c) separating non-adsorbed sample components from the solid phase
d) contacting the solid phase with the first partner of the binding pair or with an analogue thereof in a free form resulting in a desorption of the target substance as a complex with R
1
from the solid phase and
e) separating the complex from the solid phase.
In this embodiment of the method according to the invention a reactant R
1
is used that can bind specifically to the target substance to be purified and which is coupled to a first partner of a high affinity binding pair e.g. a hapten. In addition a second reactant R
2
is used which can bind specifically to the first binding partner and contains the second partner of the binding pair e.g. an anti-hapten antibody and has such an affinity for the first binding partner that the reactant R
1
can be efficiently displaced by the first binding partner in a free form or by a derivative thereof. Elution with the free first binding partner enables the complex composed of the reactant R
1
and the substance to be detected to be very specifically eluted from the solid phase and free from unspecifically adsorbed impurities. The principle and result of this purification

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