Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-08-04
2004-08-17
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007200, C435S007940, C435S007920, C435S174000, C435S176000, C435S177000, C435S287100, C435S287200, C435S287900, C435S975000, C436S506000, C436S507000, C436S518000, C436S519000, C436S513000, C436S524000, C436S528000, C436S533000, C436S534000, C436S536000, C436S538000, C436S547000, C436S548000, C436S811000, C436S815000, C436S829000, C422S068100
Reexamination Certificate
active
06777193
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to obtaining antibodies recognizing lipids and more particularly, is related to methods for obtaining antibodies against lipidic structures different from the lipidic bilayer, and to the use of these antibodies in diagnostic and/or treatment of diseases associated with the antiphospholipid syndrome; as well as for the determination of physiological states of the cell.
BACKGROUND OF THE INVENTION
Considering the state of the art there are different studies in which evidence of the existence of antibodies that recognize lipids can be found. For example, they have been detected in the serum of patients with antiphospholipid syndrome, as was described by Asherson et al. in their book “The antiphospholipid syndrome” in 1996 (
CRC Press,
Boca Raton). In the same way, antiphospholipid antibodies have been obtained from animals that were experimentally treated with lipids by active immunization, in accordance with Alving in 1992 (
Biochim. Biophys. Acta
1113:307-322) or, in animals that received antiphospholipid antibodies by passive immunization, as Tincani and Shoenfeld described in 1996 in the above mentioned book.
The anti-lipid antibodies have been classified into two major subgroups according with the method used for their determination. These groups are anti-cardiolipin antibodies and anticoagulant antibodies (Guglielmone y Fernandez, 1998,
J. Rheumatol.
26:86-90).
The anti-cardiolipin antibodies are determined by methods in which cardiolipin immobilized in a solid phase is used. This was described by Harris et al. in 1985 (
Clin. Rheum. Dis.
11:591-609), such as the enzyme-linked immunosorbent assays and the radioimmunoassays better known by their respective initial abbreviations as ELISA and RIA which have been broadly used in the above mentioned technique.
The anticoagulant antibodies are detected by methods in which the prolongation in the coagulation time of plasma samples is measured in vitro, according with Bevers et al. 1991(
Thromb. Haemost.
66:629-632). Some of these methods are: activated partial thromboplastin time (APTT), dilute Russell's viper venom time (dRVVT), protein C, and protein S among others. In these methods, the anticoagulant antibodies are bound to phosphatidylethanolamine or to phosphatidylserine which are intermediary factors in the blood coagulation cascade, and when the concentration of these lipids decrease due to the immune reaction, the coagulation time is prolongated.
The anti-cardiolipin antibodies have the disadvantage of producing crossed reaction with other anionic lipids such as phosphatidylserine and phosphatidylglycerol. Due to the lack of specificity for a certain type of lipid, the above mentioned antibodies are generally known as antiphospholipid antibodies.
In addition, antibodies against phosphatidylethanolamine have been detected in the sera from patients with antiphospholipid syndrome. Also, antibodies against phosphatidylcholine are detected in patients with hemolytic anemia, as was described by Sugi and McIntyre (
Blood
86:3083-3089) and Arvieux et al. (
Thromb. Haemost.
74:1120-1125), respectively, in 1995.
On the other hand, some studies have demonstrated that the binding of antiphospholipid antibodies to the lipidic antigen increases in the presence of a plasmatic protein. For example, in 1990, McNeil et al., determined that the binding of antibodies to the cardiolipin was markedly enhanced by the plasma protein &bgr;
2
-glycoprotein I or apoprotein H (
Proc. Nat. Acad. Sci. USA
87:4120-4124). Additionally, some anti-cardiolipin antibodies are bound directly to &bgr;
2
-glycoprotein I, as was described by Roubey et al. in 1995 (
J. Immunol.
154:954-960). These findings suggest that the anti-cardiolipin antibodies may recognize either a cryptic epitope on &bgr;
2
-glycoprotein I exposed on the complex of &bgr;
2
-glycoprotein I-cardiolipin, or &bgr;
2
-glycoprotein I alone but with a very low affinity towards he glycoprotein, as was described by Pengo et. al. (1995
, Thromb. Haemost.
73:29-34).
In accordance with these studies, it may be concluded that the binding of antiphospholipid antibodies to lipidic antigens is also associated with proteins. Sugi and McIntyre (op. cit., 1995) found that the proteins called kininogens are involved in the binding of antibodies to phosphatidylethanolamine, whereas the proteins that are bound to phosphatidylserine, such as prothrombin, protein C, protein S and annexin V, have been implicated in the binding of anticoagulant antibodies to phosphatidylserine, according with the studies in 1994 by Nakamura et al. (
Biochim. Biophys. Res. Commun.
205:1488-1493) and by Roubey (
Blood
84:2854-2867).
These studies indicate that the antigen of some antiphospholipid antibodies is really a complex formed by phospholipids and specific plasma proteins, but these proteins differ from those required for reactivity of antiphospholipid antibodies with cardiolipin. Nevertheless, in other studies, antiphospholipid antibodies that bound directly to the phospholipid have been identificated such as the anti-cardiolipin antibodies that do not require the &bgr;
2
-glycoprotein I. Such studies were carried out by McNeil et al. in 1989 (
Br. J. Haematol.
73:506-513) and by Pengo and Basiolo in 1993 (
Thromb. Res.
72:423-430).
On the other hand, some anti-cardiolipin antibodies, purified by affinity chromatography, do not show anticoagulant activity (McNeil et al., op. cit., 1989; Shi et al., 1993,
Blood
81:1255-1262). However, other studies demonstrated that the anti-cardiolipin and the anticoagulant antibodies were removed by adsorption with cardiolipin (Pengo and Biasiolo, op. cit., 1993; Pierangeli et al. 1993,
Br. J. Haematol.
85:124-132).
Additionally, during studies in experimental animals, treated by passive or active immunization, the employed methods for the detection of antiphospholipid antibodies are the same as those described for the detection of human antiphospholipid antibodies. Furthermore, in these animal models, the different organs and tissues were analized by anatomical and histopathological studies, by immunofluorescent studies, and even by fetal resorption analysis and consequently the produced lessions in fetuses and placentas of the female animal models were also analyzed. These works were performed by Tincani y Shoenfeld (op. cit. 1996) and by Shoenfeld and Ziporen (Lupus 7:S158-S161, 1998).
The previously mentioned studies, show that the antiphospholipid antibodies described in human patients and in animal models have a broad specificity towards the lipidic antigens. This broad specificity of the antibodies may be attributed, among other causes, to the lack of specificity of the methods used for the detection of the above described antibodies.
In such methods, it has not been considered the chemical structure and the molecular association of lipidic antigens, as well as the chemical properties that the lipidic antigens have in the nature. As a consequence, in the lipidic antigens that have been used in those methods, the phospholipids are bound to artificial solid supports, such as in the ELISA and RIA methods, or they are in a molecular association that is not completely characterized, like in tests where the prolongation in the coagulation time is detected.
There are only a few studies in which the molecular structure of the phospholipid employed as antigen has been considered. For example, the reports of Rauch et al. in 1989 and in 1998 (
Thromb. Haemost.
62:892-896 and
Thromb. Haemost.
80:936-941, respectively) and that of Berard et al. (
J. Lab. Clin. Med.,
1993, 122:601-605). In these reports, the authors demonstrated that the sera from some patients with systemic lupus erythematosus is inhibited in its anticoagulant activity by phosphatidylethanolamine associated in the hexagonal tubular II phase. This inhibition was not observed when the phospholipid was associated into the bilayer phase. However, the properties of the cellular membrane can not be related with the tubular association of phospholipids be
Aguilar-Faisal José Leopoldo
Baeza-Ramirez María Isabel
Hernández Miguel Angel Ibáñez
Ramirez Carlos Wong
Uc Mónica Lara
Escuela Nacional de Ciencias Biologicas, del Instituto Politecni
Le Long V.
Padmanabhan Kartic
Rothwell, Figg Ernst & Manbeck, PC
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