Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells
  • G01N33/56972—White blood cells

Definitions

• the present invention relates to an assay for the diagnosis of schizophrenia.
• the schizophrenic disorders as defined by DSM-III
• Schizophrenia occurs worldwide. Although it is one of the most severe and prevalent mental disorders of well documented symptomatology and has been extensively investigated over the past decades, the etiology of this disease is still an enigma.
• Alzheimer-type and multi-infarct dementia both treated with neuroleptics and untreated
• normal control subjects was determined by the use of an enzyme-linked immunoassay based on color development after reaction with anti-human immunoglobulin (anti-human IgA, IgE, IgG and IgM antibodies) bound to horseradish peroxidase.
• anti-human immunoglobulin anti-human IgA, IgE, IgG and IgM antibodies
• PAA isolated from schizophrenic patients were contacted with proteins isolated from platelets (hereinafter "platelet antigens") and all cases developed a similar binding pattern. It was surprisingly found that the PAA from schizophrenic patients did not recognize and bind to well- known antigenic proteins from platelets involved in ITP, but recognized and bound to unknown platelet antigens which, on the other hand, were not recognized by the PAA from demented patients.
• the present invention provides an assay for the diagnosis of schizophrenia in an individual, comprising the following steps:
• a sample from said individual being a blood sample, a platelet-containing fraction thereof, or a fraction containing platelet-associated antibodies (PAA) shed from the platelets;
• PPA platelet-associated antibodies
• the sample of the individual to be tested may be a blood sample, e.g. serum, a platelet-containing fraction thereof or a PAA-containing fraction shed from the platelets.
• the sample is platelet-rich plasma (PRP) obtained by known methods, e-g-*, treatment of blood with an anticoagulant, e.g. heparin or sodium citrate solution, and centrifugation.
• PRP platelet-rich plasma
• the sample is a fraction containing PAA, obtained by centrifugation of PRP, incubation of the pelleted platelets with glycine-HCl buffer, pH 2.5-3.0 at room temperature and centrifugation, thus obtaining a PAA- containing supernatant.
• the platelet antigens are obtained e.g. by disintegration of platelets derived from PRP and separated according to their molecular weights by polyacrylamide gel electrophoresis (PAGE) and transfer to a solid support, e.g., nitrocellulose.
• PAGE polyacrylamide gel electrophoresis
• the antibody detection systems to detect PAA levels comprises anti-human immunoglobulin (anti-hlg) antibody, or a fragment thereof, linked to a marker.
• a marker may, for example, be a radioactive group, a fluorescent group, an enzyme that can catalyze a reaction yielding a detectable product such as, for example, horseradish peroxidase (HRP) and alkaline phosphatase, a biotin group that can be detected by avidin, etc.
• HRP horseradish peroxidase
• alkaline phosphatase a biotin group that can be detected by avidin
• the antibody is rabbit anti-human IgG (anti-hlgG) covalently linked to HRP and ortho-phenyl-enediamine is the reagent used to detect bound HRP.
• a fragment of IgG is used which is Fc-depleted, such as an Fab or an F(ab ) 2 fragment or a part thereof which contains the fragment's binding domain, or a single-chain antibody, or the like.
• the degree of binding of the antibodies to PAA can be determined by the use of a second antibody directed against said single domain antibody, which second antibody is bound to a marker as above.
• a solid support having immobilized thereon a PAA target antigen(s) specific for schizophrenia, i.e., the antigen(s) to which the PAA of schizophrenic patients are directed, is used.
• the PAA target antigen( ⁇ ) specific for schizophrenia will be isolated from the antigenic proteins recognized by the PAA from schizophrenic patients, purified and characterized.
• the platelets are contacted with the support and following an incubation period, the immobilized platelets are reacted with the anti-hlg antibodies, which are preferably labelled and the number of bound PAA can thus be determined.
• the platelets are first treated to shed their PAA, the PAA-containing fraction is then reacted with the support and following incubation and washing, the supports are reacted with the anti-hlg antibodies which are preferably labelled, and the number of bound PAA is thus determined.
• the present invention also provides a kit useful in the above assay.
• the kit of the invention comprises an anti-hlg antibody or a fragment thereof and a support comprising a sole antigen or a plurality of platelet antigens reactive with the PAA immobilized onto the support.
• the antibodies in the kit are conjugated to a detectable marker.
• the kit comprises also a second type of antibodies directed against said single domain antibodies, which second type of antibodies are in turn conjugated to a detectable marker.
• the antibodies are immobilized onto a support and the kit comprises such a support.
• the kit according to all above embodiments may also comprise the various reagents required for carrying out the assay.
• Figs. 1A-B show the pattern of binding by Western blot of PAA obtained from 10 normal control subjects (A) and 8 schizophrenic patients (B) to platelet antigens in a nitrocellulose support, separated by 10% acrylamide gel, as determined by HRP-labelled rabbit anti-human IgG.
• Figs. 2A-C show the pattern of binding by Western blot of PAA from 4 schizophrenic patients (A) , 4 normal control subjects (B) and 4 demented patients (C) to platelet antigens in a nitrocellulose support, separated by 10% acrylamide gel, as determined by HRP-labelled rabbit anti-human IgG.
• Fig. 3 shows the pattern of binding by Western blot of PAA from 10 schizophrenic patients (lanes 1-10) to platelet antigens on a nitrocellulose support, separated by a 7,5-15% acrylamide gradient gel, as determined by HRP-labelled rabbit anti-human IgG.
• PAA levels were determined either with rabbit anti-human IgG antibodies linked to HRP (as in Shinitzky et al.., 1991) or with a Fab fragment of this antibody. Enzyme-linked immunoassay (ELI) based on a color development after binding of the antibody or Fab fragment thereof, was used.
• ELI Enzyme-linked immunoassay
• activated plastic beads (Immunotip, U.S.A. Scientific Plastics) were coupled with papain as follows: 1 mg papain (Worthington, U.S.A.) was mixed in 1 ml of 0.2M sodium cyanoborohydride (Fluka, U.S.A.) and was incubated with a single plastic bead for 5-10 minutes, and then washed extensively with phosphate-buffered saline (PBS) .
• PBS phosphate-buffered saline
• Horseradish peroxidase-conjugated rabbit anti- human IgG (BioMakor, Israel; 740 ⁇ g in 40 ⁇ l) was incubated with gentle shaking with the papain-conjugated bead for 5 hours at 37°C and then passed through a protein A column (Pierce) .
• the platelet suspension was incubated with freshly prepared substrate reagent (19.8 ml PBS - 0.2 ml methanol containing 2 mg ortho- phenylenediamine ⁇ 3 ⁇ l H 2 0 2 30%) for 1 hr at 37°C.
• substrate reagent (19.8 ml PBS - 0.2 ml methanol containing 2 mg ortho- phenylenediamine ⁇ 3 ⁇ l H 2 0 2 30%
• the reaction was terminated by adding 0.1 ml of 6N sulfuric acid. After centrifugation, the O.D. was read at 480 nm. After background substraction, the O.D. was calculated for 10 8 platelets per ml (Shinitzky et al., 1991).
• Platelets were derived from PRP by 3 washings with PBS and separated by PAGE according to their molecular weights as described (Laemmli U.K., Nature, 227. 680 (1970)). Depending on the molecular weight of interest, 5%, 10% or 12.5% acrylamide concentrations were used. Alternatively, a 7,5-15% acrylamide gradient gel was used, leading to a better resolution than homogeneous acrylamide concentration.
• Platelet proteins from control subjects were separated by 10% PAGE and blotted onto nitrocellulose as described in (v) above. All incubation steps were performed at room temperature and under constant shaking (Bellco Rocking Table, speed setting 4) in BioRad Incubation Trays.
• the sample strips and positive control strip (dotted with 0.002 ml human serum) were blocked with 1 ml blocking buffer (5% milk powder dissolved in incubation buffer [60 mM citric acid, 90 mM Na 2 HP0 4 , 200 mM NaCl, pH 7.7] filtered before use) for 30 min. The blocking buffer was then carefully removed (with a Pasteur pipette) and the strips were supplemented with 1 ml incubation buffer.
• PAA 0.2 ml or serum (0.1 ml) was then added to the strips as a sample and incubated for 14 hours. After the incubation, the strips were washed 3 times with 1 ml incubation buffer for 15 min. and one time with 1 ml peroxidase buffer (200 mM Tris, 150 mM KC1, 0.3% Triton X-100, 10 mM phenol, 2 mM CaCl 2 , filtered before use) for 15 min. The strips were incubated with rabbit anti-human IgG covalently linked to peroxidase (1:1000 diluted) in 1 ml peroxidase buffer for 2 hours.
• 1 ml peroxidase buffer 200 mM Tris, 150 mM KC1, 0.3% Triton X-100, 10 mM phenol, 2 mM CaCl 2 , filtered before use
• Fig. IA PAA from 10 blood donors (Fig. IA) and from 8 chronic schizophrenic patients (Fig. IB) were incubated with normal human platelet proteins, separated by gel electrophoresis and blotted onto nitrocellulose as described above. Visual detection of bound PAA was carried out as described above. The results are shown in Fig. 1 , in which the following molecular weight markers (BioRad) were used: lysozyme - 14.4 kD; trypsin inhibitor - 21.5 kD;; carbonic anhydrase - 31 kD; ovalbumin - 45 kD; serum albumin - 66.2 kD and phosphorylase B - 97.4 kD. No bands were observed in any of the 10 examples of PAA from normal donors (Fig. IA) , while in all 8 examples of PAA from chronic schizophrenic patients a series of bands was detected (Fig. IB) .
• BioRad molecular weight markers
• Idiopathic Thrombocytopenia Purpura are Proteins I b ( -unit, 128-141 kD; 3-unit, 22 kD) , II b ( ⁇ -unit, 132 kD; 3-unit, 23 kD) and III a (95 kD) (Williams W.J. et al., eds., Hematology, 4th ed. , 1990, McGraw-Hill Inc., pp. 1189 and 1382) .

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• 1995
  • 1995-02-28 BR BR9507125A patent/BR9507125A/pt unknown
  • 1995-02-28 CA CA 2184602 patent/CA2184602A1/en not_active Abandoned
  • 1995-02-28 EP EP95912625A patent/EP0748447A4/en not_active Withdrawn
  • 1995-02-28 WO PCT/US1995/002426 patent/WO1995023970A1/en not_active Application Discontinuation
  • 1995-02-28 AU AU19716/95A patent/AU695043B2/en not_active Ceased
  • 1995-02-28 JP JP7522960A patent/JPH09510012A/ja active Pending

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