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
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9

Definitions

• the present invention pertains to a systemic lupus erythematosis (SLE) antibody assay method utilizing novel liposome compositions.
• SLE systemic lupus erythematosis
• this assay can be used as a diagnostic tool in the detection of SLE.
• SLE is a serious autoimmune condition in which abnormal humoral and cellular immune responses occur.
• This immune complex disorder manifests symptoms such as erosive inflammation of skin (producing a characteristic rash) , blood vessel lesions, pleuresy, psychiatric disturbances, convulsions, and inflammation of the kidney glomeruli (leading to renal disfunction or failure) .
• erosive inflammation of skin producing a characteristic rash
• blood vessel lesions pleuresy
• psychiatric disturbances convulsions
• convulsions convulsions
• inflammation of the kidney glomeruli leading to renal disfunction or failure
• a wide range of auto-antibodies have been detected in SLE patients including circulating antibodies to lymphocytes, red blood cells, platelets and neutrophils and recognition occurs by all four subclasses of immunoglobulin to cellular components such as nuclei, ribosomes, mitochondria and lysosomes.
• the sites of lupus antibody interaction with subcellular components include nucleoproteins, histones and nucleic acids. In the case of nucleic acids, one antigenic determinent appears to be the carbohydrate-phosphate unit.
• a further SLE screening test involves the agglutination reaction between circulating antibodies and polystyrene particles modified on their surface with dinitrophenyl groups. The interaction of these groups with Fab sites on immunoglobulins produces agglutination and precipitation of the particles.
• This test is essentially qualitative and lacks specificity for SLE antibodies alone, the modified particles interacting with any other circulating immunoglobulin so as to produce false positive results.
• the most commonly used tests for SLE are immunofluorescent assays for the presence of cellular antinuclear antibodies. Human epithelial cells in vitro are exposed to serum of SLE patients and antibodies to cellular components which bind to these cells then are recognized by incubation with fluorescein isothicyanate linked anti-Ig.
• peripheral anti-DNA
• diffuse anti-nucleoprotein
• speckled antiribonucleoprotein
• nucleolar anti-RNA.
• Liposomes are completely closed bilayer membranes containing an entrapped aqueous phase. Liposomes may be any variety of unila ellar vesicles (possessing a single membrane bilayer) or roultilamellar vesicles (onion-like structures characterized by concentric membrane bilayers each separated from the next by a layer of water) .
• aqueous phase was added, the mixture was allowed to "swell", and the resulting liposomes which consisted of multilamellar vesicles (MLVs) were dispersed by mechanical means.
• MLVs multilamellar vesicles
• Lipid vesicles can also be prepared by injection of the lipids in an organic phase into an aqueous solution as described by Batzri and Korn (Biochim. Biophys. Acta, 298:1015 [1973]) using ethanol and by Dea er and Bangham (Biochim. Biophys. Acta, 443:629-634 [1976]) using ether.
• the present invention involves liposomes and diagnostic assays using them; the assays are based on the selective destabilization of liposomes and the inhibition thereof by SLE antibodies.
• the description of the invention will be divided into two major areas: the liposomes and the diagnostic assays.
• Liposomes of the invention can be formed from certain a phipathic lipids. In contrast to lipid aggregates such as micelles and surface monolayers, liposomes involve self-sealing bilayers which form closed vesicles. Such bilayers can be produced from a number of flexible double tailed phosphatidylcholines of the formula
• each of R 1 and R2 is the acyl residue of the same or different fatty acid.
• R 1 and R2 will generally have from 14 to 20 carbon atoms and may be saturated or unsaturated, e.g. , tetradecanoyl, tetradec-9-enoyl, hexadecanoyl, hexadec-9-anoyl, octadecanoyl and octadec-9-enoyl.
• R 2 R can be and often are different. Moreover, when unsaturation is present, the chain generally, but not necessarily, will define the cis configuration and will be present in the R 2 group. Most desirable R1 and R2 groups will be chosen to provide one double bond per phospholipid molecule. Particularly preferred are R
• lipid which also serves as an antigen for antibodies specifically found in patients with SLE.
• a phosphatidic acid or a cardiolipin diphosphatidylglycerol
• the molar ratio of phosphatidylcholine to such lipid antigen will be from about 1:7 to about 7:1.
• One particularly satisfactory ratio is about 3:4 of phosphatidylcholine:lipid antigen.
• head groups generally determine the nature of the bilayer's interfacial region. The nature of either group can also affect the nature of the other.
• the dynamic packing properties of the lipids which comprise a multicomponent liposome are determined by the complex interrelation of the component molecules.
• a rigid molecule such as a steroid (cholesterol or hydrocortisone)
• a rigid molecule such as a steroid (cholesterol or hydrocortisone)
• steroid cholesterol or hydrocortisone
• this amount of steroid will be from about 5 to about 30 molar percent, based on the total amount of phosphatidylcholine and lipid antigen which is present.
• the influence of such stability altering components on the liposome involves a number of_ physicochemical characteristics which are easily determined but which are not characteristic of any given class of chemical compounds.
• a liposome compatible negatively charged compound e.g. , a difatty acid phosphatidyl- glycerol or difatty acid alcohol phosphate ester
• a liposome compatible negatively charged compound e.g. , a difatty acid phosphatidyl- glycerol or difatty acid alcohol phosphate ester
• the function of this component is to increase the net negative surface charge of the bilayer.
• the addition-of DPPG also confers stability. It has also been found desirable to include a small amount, e.g. , from 1 to 5 molar percent based on the total amount of phosphatidylcholine and lipid antigen, of an antioxidant which is lipid-compatible, e.g. , alpha-tocopherol.
• potassium or cesium salt forms of the anionic lipids i.e., cardiolipin and dipalmitoylphosphatidylglycerol
• these forms substantially reduce false positive reactions in the final assay and increase solubility in solvent during the formation step.
• liposomes formulated from the sodium, ammonium or lithium salts of anionic lipids while producing sporadic results for SLE in some instances also produce false positives in the presence of rheumatoid factor.
• the salt form is prepared by reacting a hydroxide of the metal selected with the free acid form of an anionic lipid.
• the free acid form of the anionic lipid is prepared by mixing a lipid with a solvent such as chloroform and adding polyanionic ion exchange beads. The suspension is then centrifuged and the supernatant containing the free acid of the lipid is removed. The free acid of the lipid is neutralized to pH 7 by the addition of the metal hydroxide to form the salt.
• the cardiolipin is associated with cesium cation and dipalmitoylphosphatidylglycerol with the potassium .cation.
• a preferred bilayer composition includes phosphatidylcholine, cardiolipin, cholesterol, alpha-tocopherol and a phosphatidylglycerol in a molar ratio of about 3:4:1.9:0.1:1.
• the liposome bilayers prepared as hereafter defined, also have entrapped therein a metallochromic (colorimetric) indicator which is sensitive to divalent cations.
• a metallochromic (colorimetric) indicator which is sensitive to divalent cations.
• arsenazo III 2,7-bis- (2-arsenophenylazo)-l,8-dihydroxynaphthalene-3, 6-disulfonic acid
• the liposomes of the present invention can be prepared by a modification of a number of known methods [which are reviewed by Szoka and Papahadjopoulos (Ann. Rev. Biosyps. Bioeng. 2:467-508 [1980]).
• the preferred method involves injecting an ethereal solution of the bilayer components into a large volume of aqueous buffer solution containing the dye.
• lipids are prepared and dried, as by rotary evaporation from a suitable nonaqueous liquid such as chloroform, and combined with a solution of the indicator in buffer solution [0.145M NaCl-KCl, about 5mM 4-2(2-hydroxyethyl)piperazine-2-ethane-sulfonic acid] to effect swelling and concommitant entrapment.
• buffer solution 0.145M NaCl-KCl, about 5mM 4-2(2-hydroxyethyl)piperazine-2-ethane-sulfonic acid
• the liposome mixture is chromatographed to separate excess, nonentrapped indicator from liposome-entrapped indicator, elution being readily monitored visually or by spectroscopy. Often it is advantageous or sometimes necessary to hold the liposome mixture under vacuum for a period of time, as for example, one or two hours, prior to chromatography in order to remove substantially all of the remaining solvent.
• This invention involves three major embodiments (I, II, and III) for lupus diagnostic assays as described below.
• the present invention is based on the discovery that the increase in the permeability of the liposome bilayer which is observed in certain environments will be impeded or blocked by SLE antibodies.
• the antibodies are said to stabilize the liposome.
• Stabilization refers to the preservation of the supramolecular structure or membrane architecture of the liposome. The precise mechanism by which this stabilization occurs is not fully understood. It is known that certain liposomes increase their permeability in the presence of divalent cations such as magnesium. Other liposomes are destabilized simply in the presence of normal serum.
• This destabilization will result in lysis of the liposome bilayer with release or "leakage” of any material which is entrapped therein.
• "leakage” refers to the movement (influx) of ions from the external environment into the aqueous spaces of the liposome.
• this destabilization can be selectively blocked, i.e. , the liposome structure is stabilized, by SLE antibodies. Consequently, by entrapping a metallochro ic indicator within the liposome bilayer, the presence of SLE antibodies can be detected by observing the degree of blockage of the destabilization which is reflected by absence of a color change in the indicator.
• the binding of antibodies to the liposomes is restricted to those specific for diagnosis of SLE.
• Antibodies not specific for SLE will not bind.
• the test is exceedingly rapid; e.g. , a response will be observed in a matter of minutes.
• the reagents used in the assay are non-toxic, non-radioactive, present no danger to the investigator, and do not require special handling.
• a liposome composition is prepared which is susceptible to serum (normal) - induced leakage.
• a phosphatidic acid as the lipid antigen in the liposomes, e.g. , a liposome comprising a flexible double tailed phosphatidylcholine 5 and a phosphatidic acid in a molar ratio of from about 1:7 to about 7:1, respectively, preferably from about 2:5 to about 4:3.
• a particularly desirable liposome comprises 3 molar parts of a phosphatidylcholine and 4 molar parts of phosphatidic acid (optionally together with the other 0 components described above) with the indicator entrapped therein (as hereinafter described).
• Futhermore, l-palimitoyl-2-oleoyl-phosphatidic acid is the preferred lipid antigen.
• Such liposomes will be stable in buffer but will demonstrate leakage in normal serum, thereby 5 releasing the indicator. However, if such liposomes are first brought into contact with SLE serum, the leakage which would otherwise be observed is diminished or precluded.
• a liposome composition is prepared similar to that described in Embodiment I but utilizing, cardiolipin in place of the phosphatidic acid. Such a composition will not develop leakage in the presence of serum (or for that matter in the presence of buffer) . Leakage however can be induced by the addition of divalent ions such as magnesium. Again, such leakage is inhibited or reduced if the liposome is first brought in contact with SLE antibodies.
• the addition of magnesium ions not only induces leakage in the liposome composition, it also causes a color change in indicator which has "leaked" out of the liposome.
• SLE serum will show no leakage (or leakage at a greatly reduced rate) in the presence of magnesium ions.
• the concentration of magnesium ion needed according to this embodiment is relatively small; generally a 5 to 80 millimolar solution is adequate. Excessively high concentrations should be avoided as these can produce leakage even in SLE serum treated liposomes. There also is an interplay between the liposome concentration and the amount of magnesium ion added and thus it is convenient to define a . minimum lysis concentration (MLC) for each liposome composition; i.e. , that concentration of magnesium ion which is just sufficient to effect release of the indicator from the_ liposome bilayer in the present of normal serum. This concentration can be easily determined by titration with increasing concentrations of magnesium ion.
• MLC minimum lysis concentration
• liposomes containing a phosphatidic acid and a colorimetric indicator are used.
• these liposomes are treated with Ca , a phase separation or clustering of the phosphatidic acid occurs resulting in the transport of
• the preferred liposome composition for this embodiment comprises a flexible double tailed phosphatidylcholine and a phosphatidic acid in molar ratios of from about 9:1 to about 3:7, respectively.
• the preferable phosphatidic acid is dipalmitoylphosphati ⁇ ic acid and the preferable liposomes comprise egg phosphatidylcholine, dipalmitoylphosphatidic acid, cholesterol and dicetylphosphate in molar ratios of about 7:1:1:1, respectively.
• Aqueous media (other than magnesium solution) should be free of divalent metals such as calcium ion and preferably prepared from deionized water. Detergents and other chaotropic materials should be absent as should any impurities with which the antibodies may react.
• Liposomes are prepared according to a modification of the method of Deamer and Bangham (Biochim. Biophys. Acta, 443: 629-634 [1976]). All materials and equipment should be free of divalent metal cations.
• l-Palimitoyl-2-oleoyl-phosphatidylcholine, cardiolipin, cholesterol, alpha-tocopherol and dipalmitoyIphosphatidy1- glycerol in a 3:4:1.9:0.1:1 molar ratio are solubilized in petroleum ether.
• Ten milliliters of ether solution (40 micromoles of lipid) are placed in a 20ml glass syringe with a Teflon plunger which is attached to a vertical infusion pump.
• the petroleum ether phase is then injected into the aqueous phase at a rate of 0.5 ml/ in. As the ether evaporates and escapes, the liposome suspension remains. This suspension is passed through a column of Sepharose 4B, eluting with buffer, to remove excess and unsequestered A III, the liposome-entrapped A III (red) being eluted from the column in the void volume.
• the suspension to be_ chromatographed can be held under vacuum (water aspirator) for from about one to five hours to remove excess petroleum ether prior to passage through the column. This material is then diluted
• OM?I with Hepes buffer to a concentration at which one can best distinguish between lupus positive sera and normal sera. This is typically an optical density of approximately 0.12 at 750 nanometers.
• cardiolipin cesium salt
• dipalmitoylphosphatidylglycerol when utilized in the procedure of Example 1 produce a liposome composition of increased selectivity to SLE antibodies.
• liposomes are prepared according to the procedure of Example 1 utilizing, however, phosphatidic acid in place of cardiolipin.
• leakage will occur upon addition* of the serum. The leakage will be detected upon addition of magnesium ion.
• Embodiment II aliquots of the liposomes (red) are placed in two glass tubes. To one tube is added an SLE test serum and to the other normal serum (control) . Based upon the volume in each tube, a serum dilution equal to 1:16 may be prepared. (In practice, several different dilutions, and therefore several tubes, could be used) . The same dilution is made for both the SLE test serum and normal serum. After serum addition, the liposomes are incubated for 1 to 5 minutes at .about 25°C. Following the incubation, magnesium chloride solution (e.g. , 50mM final concentration) is added to each of the tubes. The normal serum control will turn blue within 5 minutes while the lupus serum test sample will remain red for at least 3 hours longer than the normal serum.
• SLE test serum SLE test serum
• control normal serum
• test sample and normal serum 5 control are serially diluted. If at any dilution there is a substantial delay in the color change induced by the test sample as compared to the serum control, the test is ' ' considered positive.
• Embodiment III one ( hundred raicroliters of liposomes (red) are placed in three glass tubes and diluted with Hepes buffer up to 0.2ml.
• One tube 5 constitutes a buffer blank
• the second is an SLE test serum
• the third serves as a normal serum control.
• a serum dilution equal to 1:16 is calculated.
• the same dilution is made for both the SLE test serum and normal serum.
• the liposomes are incubated for 5 minutes at about 25°C. Following the incubation, 80mM calcium chloride (final concentration) is added to each of the tubes.
• the buffer control will turn blue within 10 minutes.
• the normal serum control will turn blue before 30 minutes, and the lupus serum test will remain red for at least 30 minutes longer than the normal serum.
• the test sample and normal serum control are serially diluted. If at any dilution there is a substantial delay in the color change induced by the test sample as compared to the serum control, the test is considered positive.
• a blind study included serum samples from the following sources: known lupus patients, patients exhibiting clinical symptoms of lupus but a negative clathridia test, patients showing antinuclear antibodies but no clinical symptoms, patients with scleroderma, patients with a positive VDR test (syphilis), patients with Sjogren's Syndrome and normal patients.
• the assay gave a proper response in 19 out of 20 unknowns. The only false negative was a patient suffering from both syphilis and lupus. Significantly, and in contrast to other tests, a patient with syphilis but not lupus " did not produce a false positive.

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• 1983
  • 1983-03-24 WO PCT/US1983/000420 patent/WO1983003473A1/en unknown
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