Methods and reagents for determining protein S

Details Methods and reagents for determining protein S Methods and reagents for determining protein S

1997-11-25

2002-04-30

Saunders, David (Department: 1644)

Chemistry: analytical and immunological testing

Biospecific ligand binding assay

C435S007940, C436S518000, C436S821000

Reexamination Certificate

active

06379975

ABSTRACT:

The present invention is related to detection and determination of protein S in biological fluids and to reagents for use therein. More specifically, free protein S is determined as a receptor/ligand complex formed between free protein S and a molecule comprising a ligand that binds specifically to protein S.
Protein S is a member of the naturally occurring anticoagulant protein C system (a part of the blood coagulation system) and acts as a cofactor to the activated state of protein C, APC (Activated Protein C), the other cofactor being intact Factor V. This system expresses anticoagulant action since APC acts so as to degrade the coagulation promoting Factors V
a
and VIII
a
.
In human plasma, protein S circulates both as free protein and in complex with another plasma protein, the C4b-binding protein (C4BP) (Dahlbäck, Thromb. Haemostas. 1991, 66:49-61). Approximately 60% of the total protein S in plasma is bound to C4BP and it is noteworthy that this form of protein S is not functionally active as APC-cofactor. Thus, the binding of C4BP to protein S leads to a loss of the APC-cofactor function of protein S. The importance of protein S as an anticoagulant protein is illustrated by the association between protein S deficiency and thromboembolic disorders. Homozygous deficiency, which is extremely rare, gives a neonatal fatal disease, whereas heterozygous deficiency is a risk factor for venous thrombosis in adult life. Indeed, protein C deficiency or protein S deficiency is found in approximately 5 to 10% of all individuals exhibiting venous thrombosis.
An individual having protein S deficiency, thus, runs an increased risk of experiencing venous thromboembolic events. Accordingly, methods for determining blood or plasma levels of protein S have a potential clinical use. Particularly, methods for measurement of the levels of free protein S would be appreciated, since several investigators have shown that for the diagnosis of protein S deficiency, the level of free protein S should be measured rather than the level of total protein S or the bound form of protein S (Zöller et al, Blood, 1995, 85:3524-3531). The reason for this is that higher sensitivity and specificity as regards the genetic defect causing protein S deficiency are achieved with the free protein S assays than with assays for measuring total protein S or bound protein S.
Previously known methods for determining free protein S are based on two different test principles, viz. differential polyethylene glycol precipitation properties and use of monoclonal antibodies, resp.
Methods using polyethylene glycol (PEG) to selectively remove bound protein S from a fluid comprising bound protein S and free protein S prior to measurement of protein S are based on the discovery that the complex bound form of protein S (PS:C4BP) precipitates already at a PEG concentration of approximately 3.75-5%, whereas most of the free protein S remains in solution. This principle has been used extensively in different commercially available protein S assays. Thus, in assays for free protein S, plasma samples are usually subjected to precipitation with PEG (3.75-5%) whereafter the protein S remaining in the supernatant after centrifugation is measured with immunological methods, such as ELISA, RIA or Laurell rockets. Such methods are disclosed in Am. J. Clin. Path. 94:176-186 (1990), Anal. Biochem., 10:358-361 (1985) and Blood, 67:504-508 (1986). However, these methods suffer from some disadvantages, mainly due to the PEG precipitation procedure. Thus, even when the PEG precipitation is highly standardized, this procedure is plagued by poor reproducibility and by its laborious and time consuming nature.
The second test principle mentioned above is based on use of monoclonal antibodies. Such monoclonal antibodies are specific for the free form of protein S, i.e. the epitopes for these antibodies are located at or close to the binding site for C4BP on the protein S molecule (Amiral et al, Blood Coag. Fibrinol. 1994, 5:179-186 and Wolf et al., Blood Coag. Fibrinol. 1994, 5:187-192).
C4BP, which as stated above binds to protein S and thereby reduces the amount of free protein S circulating in blood, is composed of approximately seven identical &agr;-chains, each of which contains a binding site for the complement protein C4b, and one single &bgr;-chain. The &agr;-chains are linked in their C-terminal regions to each other and in addition to the single &bgr;-chain. These seven &agr;-chains and the single &bgr;-chain of the C4BP molecule are arranged like wheel-spokes to form a spider-like molecular structure (Dahlbäck and Stenflo in The molecular basis of blood disease, eds Stammatoyannopolous et al. WB Saunders 1994, p 599-627). The protein S binding site is known to be located on the single &bgr;-chain and very recently (Härdig and Dahlbäck, J. Biol. Chem. 1996, Volume 172, p. 20861-20867) the entire protein S binding site has been localized to the extreme N-terminal SCR-module (SCR stands for Short Consensus Repeat, which is a protein module containing approximately 60 amino acid residues) of the &bgr;-chain. Although, this module has previously been proposed to contain the protein S binding site (Fernandez and Griffin, J. Biol. Chem. 1994, 269:2535-2540), it was not known before, that the entire protein S binding site is located in this first (extreme) SCR-module of the &bgr;-chain.
The knowledge of the complex formation between protein S and C4BP, has been used to develop antibodies, which are specific for the free form of protein S. Thus, attempts have been made to raise antibodies that bind specifically to the region of protein S that is involved in the binding of C4BP, which antibodies obviously would only bind to free protein S, since in the C4BP-bound form of protein S, such binding sites in protein S, which are specific for these antibodies, are already occupied by C4BP. A prerequisite for development of antibodies with the said specificity is, however, specific knowledge of the C4BP binding site on the protein S molecule. Whereas this binding site has not been elucidated in detail in prior art, two areas in a large C-terminal module of protein S designated SHBG have been claimed to be involved. The first report suggested residues number 605-614 of mature protein S to be involved (Walker, J. Biol. Chem. 1989, 264:17645-17648) whereas another region comprising residues 413-433 (Fernandez et al., J. Biol. Chem. 1993, 268:16788-16794) has more recently been suggested to be important for the binding of C4BP to protein S.
In WO 93/01209 monoclonal antibodies directed to specific regions of mature protein S, contemplated to be involved in C4BP binding, are disclosed, which antibodies are useful in diagnostic methods and systems for purifying or detecting free protein S. Protein S polypeptides comprising these specific regions are also disclosed. These regions differ, however, from the C4BP binding regions disclosed below.
Moreover, assays for free protein S based on immobilized monoclonal antibodies directed to free protein S, which are used as immobilized antibody in standard ELISA (Enzyme Linked Immuno Sorbent Assay) to capture free protein S in plasma, have been described in the literature and are also commercially available from Stago (Amiral et al., Blood Coag: Fibrinol. 1994, 5:179-186, and Wolf et al., Blood Coag. Fibrinol. 1994, 5:187-192). In such tests, plasma dilutions in buffer containing calcium are incubated in microtitre plates containing monoclonal antibodies specific for free protein S, and, subsequent to washing steps, protein S bound to the monoclonal antibodies can be detected with the use of a second mono- or polyclonal antibody directed to protein S. However, such assays are extremely expensive. Furthermore, the antibodies used in these tests are not well characterized and they have not been raised specifically against any region of protein S suggested to be involved in the binding of C4BP to protein S. Rather, these antibodies have been raised against the entire protein S molecule, whereafter antibodies having specific

Affiliated with

Also associated with

Rating

Methods and reagents for determining protein S does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Methods and reagents for determining protein S, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods and reagents for determining protein S will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2912108