WO1986006279A1 - Proteine de phase aigue modulant l'activite endotoxique de lipopolysaccharides, compositions et procedes - Google Patents

Proteine de phase aigue modulant l'activite endotoxique de lipopolysaccharides, compositions et procedes Download PDF

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Publication number
WO1986006279A1
WO1986006279A1 PCT/US1986/000936 US8600936W WO8606279A1 WO 1986006279 A1 WO1986006279 A1 WO 1986006279A1 US 8600936 W US8600936 W US 8600936W WO 8606279 A1 WO8606279 A1 WO 8606279A1
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serum
glycoprotein
lps
lipopolysaccharide
animal
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PCT/US1986/000936
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English (en)
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Richard J. Ulevitch
Peter S. Tobias
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Scripps Clinic And Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/50—Lipopolysaccharides; LPS

Definitions

  • the present invention relates to mitigation of the effects of gram-negative bacterially secreted lipopolysaccharides on an infected host animal, and particularly the mitigation provided by a particular glycoprotein found in acute phase serum that is substantially absent from normal serum and binds to lipopolysaccharides.
  • microorganisms commonly found in the gastrointestinal tract have become increasingly important in clinical medicine. They are the principal organisms found in infections of the abdominal viscera, peritoneum, and urinary tract, as well as being frequent secondary invaders of the respiratory tract, burned or traumatized skin, and sites of decreased host resistance and instrumentation. Currently, they are the most frequent cause of life-threatening bacteremia.
  • the gastrointestinal flora are exceedingly complex. The large intestine contains about 10 to 10 organisms per gram of contents. Of these, 90 to 95 percent are obligate anaerobes.
  • gram-negative bacilli Bacteroides and Fusobacterium
  • gram-positive bacilli including Bifidobacterium, Eubacterium, and Corynebacterium species
  • streptococci Other anaerobes include the gram-positive spore-forming rods of the clostridia species and gram-negative cocci, Veillonella. Enterococci are also present.
  • the well-known aerobic gram-negative rods which are members of the family Enterobacteriaceae, account for only 5 to 10 percent of the total flora. These include the most common, E. coli, as well as the
  • Klebsiella-Enterobacter group Proteus, Providencia, Edwardsiella, Serratia, and under pathologic conditions. Salmonella and Shigella.
  • Drugs often used to treat gram-negative bacterial diseases include aminoglycosides such as gentamicin, tobramycin and amikacin as well as carbenicillin and ticarcillin.
  • Penacillinase- resistant drugs such as ethicillin, oxacillin and nafcillin or members of the cephalsporin family such as cepthlothin, cefazolin and cephaprin are also used. Penicillin, clindamycin and chloramphenicol are often suggested for use when anaerobes are implicated.
  • LPS lipopolysaccharide
  • endotoxins from a wide variey of unrelated species behave quite similarly, regardless of the inherent pathogeni ⁇ ity of the microorganism from which they are derived or their antigenic structure.
  • endotoxins exist as complexes of lipid, polysaccharide, glycolipid and non-covalently-bound protein. The biologic activity seems to be a property of a lipid and carbohydrate portion.
  • the lipopolysaccharides of gram-negative bacteria may be roughly divided into three structural regions.
  • the outer-most' region contains the chains of specific sugars that characterize the O-specific antigens and determine individual serotypes within a 25 species.
  • the specific sugars are linked to a core polysaccharide that is of similar structure among related groups of bacteria.
  • the core is in turn linked through 2-keto-3-deoxyoctonate disaccharides to the major lipid component termed lipid A. t , 30 Evidence has now accumulated to indicate that the properties of endotoxins may be accounted for by this complex lipid substance.
  • Lipid A is a glucosamine disaccharide esterified with phosphoric and pyrophosphoric acid 35 and also contains ester- or amide-linked lauric, palmitic, and myristic acids. Perhaps the most important finding in recent years is that the lipid A and core-polysacchride regions are immunogenic and can induce antibodies that cross-react among the gram-negative bacteria.
  • LPS plasma lipoproteins
  • the complex contains components of HDL including apolipoprotein Al (apo Al) [Ulevitch et al.
  • initial complexes formed in the two serum types also differ in density.
  • the density of the initially formed complex is 1.33 g/cc
  • the density of the intial complex is 1.3.
  • An LPS-containing serum complex with a density of 1.3 g/cc was also reported when Balbc/Strong mice were injected with AgN0 3 or LPS. Tobias and Ulevitch (1983) J. Immunol. 131:1913.
  • Precipitated euglobulin fractions formed from mixtures of LPS and NRS or APRS were examined for their solubilities in saline. It was found that substantially all of the LPS in the NRS-formed precipitate dissolved (52 percent of recovered LPS and 52/53 of precipitate) leaving only about 1 percent of the recovered, precipitated LPS undissolved, while most of the LPS that precipitated from APRS (59 percent of recovered LPS and 59/64 of
  • gp60 material is not the. substance that retards binding between HDL and LPS.
  • One aspect of the invention constitutes a therapeutic composition for introduction into the bloodstream of an animal host that is susceptible to infection by lipopolysaccharide-secreting gram-negative bacteria.
  • the composition comprises a purified glycoprotein that is dispersed in a liquid, physiologically tolerable diluent.
  • the purified glycoprotein (a) is a material that is present in the acute phase serum of the animal host, but is substantially absent from the normal serum of the host; i.e., it is present at a concentration of less than about 0.5 micrograms per milliliter of normal serum; (b) binds to a gram-negative bacterially-secreted lipopolysaccharide when the purified glycoprotein and lipopolysaccharide are admixed ⁇ n vitro in normal serum ' of the animal host; (c) retards the i ⁇ i vitro binding of the lipopolysaccharide to high density lipoprotein present in the normal serum of the host animal; and (d) is substantially homogeneous.
  • the purified glycoprotein is often referred to herein as lipopolysaccharide binding protein (LBP) .
  • the effective amount of the purified glycoprotein is an amount that is sufficient, when administered as a unit dose to the animal host, to provide an animal host serum level of the glycoprotein that is in excess of the level present a-t a time immediately prior to treatment of the animal and is at least an amount sufficient to retard binding in an ⁇ n vitro determination of the lipopolysaccharide endotoxin to high density lipoprotein present in the normal serum of an animal of the same species as the animal host when the lipopolysacchride is present in the normal serum of that jln vitro determination at a concentration of 10 micrograms per milliliter.
  • the purified glycoprotein is preferably isolated from human serum and is an alpha 2 ,beta,-glycoprotein having a molecular weight of about 60,000, a sedimentation coefficient of about 4 S, and contains about 25 weight percent carbohydrate. That carbohydrate includes galactose, mannose, glucosamine, sialic acid and fructose moieties and is free from galactosamine moieties.
  • the glycoprotein is preferably a homolog of the above-described glycoprotein.
  • a method for treating an animal to mitigate the effects of an infection caused by lipopolysaccharide-secreting gram-negative bacteria constitutes another aspect of the present invention.
  • a unit dose of the before-described therapeutic composition is introduced into the bloodstream of the animal to be treated.
  • the unit dose preferably contains about 0.3 to about 5 milligrams per kilogram of treated animal body weight.
  • a further aspect of the present invention contemplates a method of assaying an animal body sample for the presence of a lipopolysaccharide endotoxin secreted by gram-negative bacteria.
  • an aliquot of an animal body sample is provided, and is admixed with an unmasking reagent to to unmask any endotoxin present in the sample aliquot.
  • the aliquot containing unmasked endotoxin is admixed in an aqueous medium with a purified glycoprotein as described hereinbefore.
  • the admixture so formed is maintained for a predetermined time period sufficient for the purified glycoprotein to react with lipopolysaccharide that may be present in the aliquot and form a complex.
  • the presence of a complex formed between the glycoprotein and lipopolysaccharide is then determined, the presence of such a complex indicating that the lipopolysaccharide was present in the body sample aliquot.
  • the above assay method can be carried out using techniques analogous to those of receptor-ligand assays such as antibody-antigen assays wherein the purified glycoprotein is treated as the receptor and the lipopolysaccharide is the ligand.
  • the assay method can also be utilized in a centrifugal density gradient assay where the presence of a formed complex can be determined by its density relative to the densitites of the admixed protein or glycoprotein.
  • Still another aspect of the invention consists essentially of a synthetic polypeptide corresponding in sequence to all or a portion of the amino-terminal 39 residues of the rabbit (lapine) glycoprotein whose amino residue sequence, from left to right and in the direction from amino-terminus to carboxy-terminus, is:
  • residue number 30 (XXX) is indeterminate, and believed to be asparagine (Asn) .
  • the polypeptide of this invention can contain a sequence of about 6 to about 39 residues corresponding to the above sequence, and more preferably contains about 10 to about 25 residues.
  • the present invention provides several benefits and advantages. One benefit is that the invention provides a treating composition that helps mitigate the effects of gram-negative bacterially secreted lipopolysaccharides on a treated animal.
  • Another benefit is that the above-mitigation can be effected by a composition that acts upon the . endotoxin rather than on the infecting bacteria so that useful gastrointestinal flora need not be destroyed during therapy.
  • a salient advantage of the present invention is that the therapeutic composition is preferably free from drugs usually used to treat gram-negative infections.
  • drugs usually used to treat gram-negative infections drugs usually used to treat gram-negative infections.
  • Figure 1 contains two graphs that illustrate the observation and quantitation of the 1.3 grams/cubic centimeter (g/cc) complex (C1.3) formed between the lipopolysaccharide endotoxin (LPS) secreted by Salmonella minnesota Re595 and human serum components in which the serum was collected before and after acute phase induction.
  • g/cc grams/cubic centimeter
  • LPS lipopolysaccharide endotoxin
  • the graph of Figure la is a plot of individual cesium chloride (CsCl) density gradients showing the ability of normal (B) and acute (A) human sera, respectively, to form C1.3.
  • the ordinate of that graph is in counts per minute (CPM) times
  • Figure lb shows the time dependence of (i) the ability of human serum to form C1.3 after acute phase was induced by etiocholanolone (A) and (ii) the C-reactive protein (CRP) concentration in human serum after similar acute phase induction ( ⁇ ) .
  • the left-hand ordinate is in units of percent... H-LPS present as C1.3, while the right-hand ordinate shows the concentration of CRP in nanograms per milliliter (ng/ml) .
  • the abscissa is in units of hours after acute phase induction.
  • Figure 2 is a graph showing plots of the kinetics of Re595 LPS-HDL complex formation in normal human serum that was collected immediately prior to injection of etiocholanolone (A) and in acute phase serum that was collected 32 hours after the etiocholanolone injection (D).
  • the ordinate is in units of log(100-percent H-LPS present as C1.3)
  • the abscissa is time in minutes after admixture of the - ⁇ -LPS with either serum.
  • Arrows indicate the first order one-half times (t, , ⁇ ) in minutes (min) for the respective plots.
  • FIG. 3 is a photograph of a polyacrylamide gel ele ⁇ trophoresis analysis carried out in the presence of sodium dodecyl sulfate (SDS-PAGE) as described by Laemmli (1970) Nature (London) 222:680. A 5 percent stacking gel was used in conjunction with a 10 percent separating gel. Protein-containing bands were visualized with Coomassie blue. Apparent relative molecular weight markers are shown in the right-hand lane. Those markers were phosphorylase B (94,000), bovine serum albumin (67,000), ovalbumin (40,000), and soybean trypsin inhibitor (30,000). The position of the gp60 material described herein and in Tobias and Ulevitch (1983) J. Immun. 131:1913 is indicated on the left by the designation "gp60- ⁇ . Protein preparations fractionated by the
  • Figure 4 is a graph showing the .in vivo clearance from rabbits of H-LPS premcubated with delipoproteinated NRS ( ⁇ ) and APRS (A). Each point in the graphs is the averaged value from 4 to 6 normal rabbits. A catheter was placed in a femoral artery of each rabbit from which blood samples could be taken at desired times.
  • Figure 5 illustrates examples of the NRS reconstitution assay for LBP activity in which mixtures of APRS with NRS (O) or reconstitution of NRS with LBP (t) are shown. Details are given hereinafter.
  • Figure 6 illustrates CsCl density gradient analyses for C1.3 formation in delipoproteinated APRS (A, upper panel) pr delipoproteinated NRS (B, lower panel) reconstituted with lipoproteins from NRS (solid squares) or APRS (open squares) .
  • Figure 7 illustrates ion exchange chromatography on Bio-Rex 70 resin of APRS (A, upper panel) or NRS (B, lower panel) . Fractions pooled are denoted by the horizontal lines and letters with the graph. Solid lines, absorbance at 280 nanometers (nm) ; broken lines, conductivity.
  • Figure 8 illustrates CsCl density gradient analyses of LBP activity in pools from Bio-Rex 70 chromatography of APRS (left set of panels) or NRS
  • Figure 9 illustrates ion exchange chromatography on Mono-Q resin of Pools C from • Bio-Rex 70 chromatography of APRS (A, upper panel) or NRS (B, lower panel). Solid lines, absorbance at 280 nm; broken lines, molarity of ammonium sulfate. One milliliter (ml) fractions were taken using a 1 milliliter per minute (ml/min) flow rate.
  • Figure 10 shows CsCl density gradient analyses of LBP activity in fractions eluting at 20 minutes from Mono-Q chromatography as shown in Figure 9.
  • Panel A fractions collected from APRS
  • Panel B fractions collected from NRS.
  • Figure 11 illustrates SDS-PAGE analyses of chromatography fractions.
  • Lanes 1, 2 and 3 are pools A, B and C from Bio-Rex 70 chromatography of APRS, respectively.
  • Lanes 4, 5 and 6 are pools A, B and C from Bio-Rex 70 chromatography of NRS, respectively.
  • Lane 8 is from the 20 minute fraction from Mono-Q chromatography of pool C, APRS.
  • Lanes 7 and 9 are molecular weight markers having the following apparent molecular masses in kilodaltons (kD) : 94 kD, 67 kD, 43 kD, 30 kD.
  • Figure 12 shows further SDS-PAGE analysis of the 58 kD (lane 1) and 60.5 kD (lane 3) proteins separated electrophoretically from the mixture (lane 2) obtained after Mono-Q chromatography.
  • Figure 13 illustrates CsCl density gradient analyses for LBP activity of immunopre ⁇ ipitate supernates obtained from mixtures of APRS or NRS and rat anti-LBP antiserum.
  • APRS no antiserum, 0; APRS, 1.7% (v/v) antiserum, ; APRS, 4.6% (v/v) antiserum, ; APRS, 14% (v/v) antiserum, ; NRS, 14% (v/v) antiserum, •.
  • Figure 14 shows SDS-PAGE analyses of immunoprecipitates obtained from APRS or NRS and anti-LBP antisera.
  • Lane 1 molecular weight (94 kD, 62 kD, 43 kD, 30 kD) markers; lane 2, NRS plus 14% (v/v) pre-immune serum; lane 3, NRS plus 14% (v/v) antiserum; lane 4, APRS plus 14% (v/v) pre-immune serum; lane 5, APRS plus 14% (v/v) antiserum; lane 6, APRS plus 4.6% (v/v) antiserum; lane 7, APRS plus 1.7% (v/v) antiserum; lane 8, APRS plus 0.6% (v/v) antiserum; lane 9, purified LBP.
  • Figure 15 shows SDS-PAGE analyses of and 3 quantitation of H-LPS immunoprecipitates obtained from mixtures of APRS or NRS and anti-LBP antisera.
  • FIG. 16 illustrates SDS-PAGE analysis
  • Lanes 1-3 contained samples of the reaction mixtures in which samples were applied directly to the gels. Lanes 4-7 contained immunoprecipitates of the reaction mixtures precipitated with 14% (v/v) anti-LBP antiserum. Each pair of lanes, a and b, represents the Coomaassie Blue stained gel and the autoradiographic print, respectively.
  • Lane 1 125 I-ASD-LPS; lane 2, ⁇ J I-ASD-LPS pre-photolysed and then admixted with immunopurified anti-LPS; lane 3, 5 I-ASD-LPS photolysed with immunopurfied anti-LPS; lane 4, pre-photolysed 125 I-ASD-LPS with NRS; lane 5, 125 I-ASD-LPS photolysed with NRS; lane 6 pre-photolysed 125 I-ASD-LPS with APRS; lane 7, 125 I-ASD-LPS admixed with APRS; and lane 8, molecular weight markers, 94 kD, 43 kD, 30 kD.
  • the present invention contemplates a therapeutic composition for treating an animal host that is susceptible of gram-negative bacterial infection, and methods related thereto.
  • compositions and associated methods of this invention relate to the finding "that a glycoprotein present in acute phase serum, but substantially absent from normal serum retards the binding of lipopolysaccharide endotoxins to the high density lipoprotein present in blood serum.
  • the liver is particularly affected during the acute phase response, and causes a rise in concentration of a large number of plasma proteins that have been grouped together as acute phase plasma proteins. Proteins whose concentrations rise by as much as 25 percent have been included in the group of acute phase plasma proteins.
  • the best studied acute phase proteins rise in concentration still more.
  • those proteins are ceruloplasmin and complement component C3 whose concentrations increase by about 50 percent; alpha- ⁇ -acid glycoprotein, alpha-_-antitrypsin, alpha,-antichymotrypsin, fibrinogen and haptoglobin whose concentrations increase about two to about four fold; and C-reactive protein (CRP) and serum amyloid A protein (SAA) whose concentrations usually increase several hundred times.
  • Serum obtained from an animal in an acute phase response is referred to as acute phase serum (APS) .
  • LBP The Glycoprotein
  • LBP The Glycoprotein
  • the compositions and methods of the present invention utilize a purified glycoprotein that is often referred to herein as lipopolysaccharide binding protein (LBP) .
  • purified glycoprotein is used herein to mean that the glycoprotein (LBP) moves as a single band in SDS-PAGE analysis.
  • the purified glycoprotein contains no more than about 30 weight percent of other proteinaceous material that is stainable by Coomassie blue, more preferably no more than about 20 weight percent of such other material, and most preferably no more than about 10 weight percent of such other material. The foregoing percentages are based on the total weight of proteinaceous material present in the single band.
  • the glycoprotein is present in acute phase serum (APS) of animals. In humans, the glycoprotein is present- in an amount of about 5 to about 10 micrograms per milliliter (ug/ml) of APS.
  • the glycoprotein is substantially absent from normal serum (NS) of such animals.
  • substantially absent it is meant that less than about 0.5 ug/ml of the glycoprotein is present in such sera.
  • the useful glycoprotein can be classed as an acute phase protein, as discussed hereinbefore.
  • the useful purified glycoprotein can be further identified by its binding to LPS secreted by gram-negative bacteria when the purified glycoprotein and LPS are admixed jji vitro in a normal animal serum (e.g., a non-acute phase serum) such as that of an animal host to be treated, as is discussed hereinafter.
  • a normal animal serum e.g., a non-acute phase serum
  • the binding of the glycoprotein to LPS can be assayed in several ways as is also discussed hereinafter. However, the centrifugation density gradient method described hereinafter and in Tobias and Ulevitch (1983) J. Immunol. 131:1913, whose disclosures are incorporated by reference, is preferred.
  • the purified glycoprotein also retards the in vitro binding of LPS to high density lipoprotein that is present in a normal animal serum.
  • the method of determining the binding rate retardation caused by the glycoprotein can be assessed in several manners.
  • the centrifugation density gradient technique that assesses rates of density shifts from 1.33 or 1.30 to less than 1.2 g/cc described in Tobias and Ulevitch (1983) J. Immunol. 133 :1913 is preferred.
  • the purified glycoprotein while being pure relative to other proteins and glycoproteins generally, is also substantially free from other acute phase proteins such as those mentioned hereinbefore. Particularly absent are CRP, SAA, murine serum glycoprotein gp70 and their homologs obtained from other animal species.
  • the purified glycoprotein has a molecular weight of about 55,000 to about 70,000.
  • Exemplary of such materials are the newly identified lapine glycoprotein having an apparent relative molecular weight of about 60,000 and the human glycoprotein having an apparent relative molecular weight of about 59,500, both of which are discussed further hereinafter.
  • Apparent relative molecular weights (masses) , "M” are hereinafter referred to as "molecular weights” or "molecular masses”.
  • the protein is an alpha ⁇ eta ⁇ -glycoprotein that was isolated from Cohn Fraction VI.
  • the glycoprotein has a sedimentation coefficient of 4 Svedbergs (S) .
  • S Svedbergs
  • the glycoprotein was denominated 4SGP by Schwick and Haupt and that designation is used herein.
  • 4SGP is a single-chain glycoprotein having about 25 weight percent carbohydrate.
  • the carbohydrate includes galactose, mannose, glucosamine, sialic acid and fructose moieties, but is free of galactosamine moieties.
  • 4SGP is further reported to have an Arg (Arginine) residue at its amino-terminus and a Ser (Serine) residue at its carboxy-terminus, and to be electrophoretically homogeneous at pH values of 8.6 and 5.0. Its isoelectric point is reported to be at pH 4.0. 4SGP was reportedly isolated from a pool of normal serum. It was reported by Iwasaki and Schmid to be present at an estimated amount of 20 milligrams per 100 liters of human serum. That amount corresponds to about 0.2 micrograms per milliliter of normal serum, and is thus substantially absent from normal serum using the before-mentioned definition.
  • One aspect of the present invention is a therapeutic composition for introduction into an animal host such as man, cattle, swine, poultry like chickens, sheep, rabbits, goats and the like that are susceptible (respond) to LPS secreted by gram-negative bacteria.
  • the composition comprises an effective amount of the before-described purified glycoprotein dispersed in a liquid, physiologically tolerable diluent.
  • Exemplary of such diluents are normal saline, phosphate-buffered saline, distilled or deionized water. Ringer's injection, lactated Ringer's injection and the like.
  • the purified glycoprotein is dispersed in an effective amount in that composition.
  • an effective amount is an amount that is sufficient, when administered as a unit dose to the animal host, to provide an animal host serum level of the glycoprotein that is in excess of the level present at a time immediately prior to treatment of the animal, and is at least an amount sufficient to retard binding, in an ji vitro determination, of the lipopolysaccharide endotoxin to high density lipoprotein (HDL) present in the normal serum of an animal of the same species as the animal host when the lipopolysaccharide is present in the normal serum of that ⁇ vitro determination at 10 micrograms per milliliter.
  • the serum level of the glycoprotein is less than about 50 micrograms per milliliter.
  • Typical unit doses contain about 0.3 to about 5 milligrams of purified glycoprotein per kilogram of treated animal body weight. More preferably,' the unit dose contains about 1 to about 3 milligrams per kilogram.
  • Another aspect of the present invention constitutes a method of treating an animal such as those already mentioned to mitigate the effects of an infection caused by lipopolysaccharide-secreting gram-negative bacteria.
  • This method comprises introducing a unit dose of the before-described composition into the bloodstream of an animal to be treated.
  • the composition can be introduced into the animal in a number of ways well known in the art. Exemplary introductions include injections given intramuscularly, intraperitoneally or intraparenterally, infusion as by drip bottle via a catheter, and the like.
  • treatments of gram-negative bacterial infections using a composition of this invention be free of the before-mentioned anti-bacterial agents drugs that are usually used in therapies against gram-negative bacteria.
  • the detriments of such therapies as discussed before can be avoided, the effects of the lipopolysaccharide endotoxin can be mitigated and the body's natural cellular and humoral defensive mechanisms can be utilized to fight the infection.
  • drugs that stimulate the body's natural cellular and humoral defenses can be beneficially included in the therapeutic composition or as a part of the treatment method regimen.
  • the purified glycoprotein described before is also useful in assay methods for the presence of lipopolysaccharide endotoxin secreted by gram-negative bacteria in a liquid animal body sample.
  • exemplary liquid animal body .samples include blood, serum, plasma, abdominal exudate, saliva, urine, cerebrospinal fluid, tears and joint fluid. Serum and plasma are preferred liquid animal body samples.
  • an aliquot of a liquid animal body sample is provided, and is admixed with an endotoxin unmasking reagent to unmask any endotoxin present in the sample aliquot and form a liquid aliquot containing unmasked endotoxin.
  • unmasking reagents and techniques for their use are described in U.S. Patent No. 4,276,050 to Firca and Rudbach whose teachings are incorporated by reference.
  • exemplary unmasking reagents include aqueous solutions containing 2 percent Tween-80 [polyoxyethylene (20) sorbitan monooleate] , 2 percent dextransulfate,.3 percent sodium chloride, 2 percent ammonium thiocyanate, and most preferably 0.002 molar benzamidine and its biologically compatible acid addition salts.
  • the unmasking reagent is preferably admixed with about an equal volume of body sample aliquot, and the composition is agitated gently.
  • the unmasked endotoxin- ⁇ ontaining body sample aliquot is thereafter admixed with a before-described glycoprotein to form an admixture.
  • the admixture so formed is maintained for a predetermined time period sufficient for the purified glycoprotein to react and form a complex with lipopolysaccharide endotoxin present in the body sample; an exemplary time period being about 10 minutes.
  • the maintenance or incubation time is a function, inter alia, of the amounts of both materials that are present in the admixture, e.g., LPS and glycoprotein (LBP) , with lower amounts typically requiring longer maintenance times.
  • the presence of the complex formed between the admixed, purified glycoprotein and lipopolysaccharide endotoxin is determined.
  • the admixed, purified glycoprotein preferably includes a covalently-linked label that provides a means for indicating the formation of the complex, and preferably the amount of complex formed.
  • Section III relates primarily to formation of LPS/glycoprotein complexes in which the LPS bears a radiolabel ( 3 H) and in which the complex formation was assessed by measuring radioactivity in various fractions taken following CsCl density gradient centrifugation.
  • Enzyme labels and their substrates can also be used.
  • Exemplary enzymes and substrates include horseradish peroxidase normally used with hydrogen peroxide and an oxidative dye precursor such as o-phenylenediamine and alkaline phosphatase that is typically used with £-nitrophenyl phosphate.
  • Methods for covalently linking enzymes to proteins are also well known in the art.
  • Substantially any assay method similar to those receptor-ligand assays used in immunological tests between antibody and antigen is also useful herein.
  • Particularly preferred receptor-ligand assays are solid phase assays.
  • a known amount of LPS as ligand is affixed to a solid matrix as a solid phase support.
  • the liquid body sample aliquot and a known, excess amount of the purified, labeled glycoprotein over that of any amount of LPS expected in the sample are admixed to form a liquid phase admixture, and the liquid phase admixture is maintained as described before for a time sufficient for the purified, labeled glycoprotein to react and complex with LPS present in the body sample.
  • An unmasking agent is preferably admixed with the body sample prior to admixture of the body sample and purified glycoprotein.
  • the maintained liquid phase admixture is then admixed with the solid phase to form a solid/liquid phase admixture. That admixture is maintained for a further time period sufficient for a further complex to form between the LPS of the solid phase support and the excess glycoprotein that did not bind with LPS present in the body sample. Separation of the phases and determination of the amount of solid phase-bound, labeled glycoprotein provides a measure of the amount of LPS in the aliquot and therefore in the body sample.
  • glycoprotein can also be used as the solid phase-affixed portion and a known amount of labeled LPS can be added to the body sample aliquot.
  • LPS LPS
  • the amount of LPS present in the liquid body sample can also frequently be augmented by concentrating the sample or its aliquot prior to use in the method.
  • Convenient methods for performing such concentrations include air drying and lyophilization followed by redissolution in a smaller amount of solvent than the original volume, and ultrafiltration as described in Section V F. Ultrafiltration removes many unwanted salts and low molecular weight species.
  • Useful solid matrices are well known in the art. Such materials include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ) , agarose, beads of glass, polyvinyl chloride, polystyrene, cross-linked acrylamide, nitrocellulose or nylon-based webs such as sheets or strips, or the wells of a microtiter plate such as those made from polystyrene or polyvinyl chloride.
  • Latex particles useul in agglutination-type assays are also useful solid matrices.
  • Such materials are supplied by the Japan Synthetic Rubber Company of Tokyo, Japan, and are described as carboxy-functional particles dispersed in an anionic soap. Typical lots of such particles have an average diameter of 0.308 microns, and may have an average carboxy-functional group distribution of about 15 to about 30 square Angstroms per carboxy group.
  • the particles Prior to use, the particles are reacted with a diamine such as l,3-diamino-2-propanol to form a plurality of amide bonds with the particle carboxy groups while maintaining free amine groups.
  • the free amines are thereafter reacted with a dialdehyde such as glutaraldehyde and the glycoprotein to form Schiff base reaction products.
  • the Schiff base reaction products are thereafter reduced with a water-soluble reductant such as sodium borohydride to provide a useful solid support.
  • Section. IV describes work with the lapine (rabbit) homolog of the human glycoprotein (LBP) discussed earlier herein and in the following Section III.
  • the studies described in Section IV particularly describe the amino-terminal thirty-nine residues of lapine LBP.
  • a synthetic polypeptide that consists essentially of about 6 to about 39 amino acid residues, and more preferably about 10 to about 25 amino acid residues, corresponding to all or a. portion of those 39 amino-terminal residues of rabbit (lapine) LBP constitutes another aspect of the present invention.
  • the complete amino-terminal lapine LBP 39-residue sequence, from left to right and in the direction from amino-terminus to carboxy-terminus, is shown below.
  • residue denominated XXX is indeterminate, and believed to be an asparagine (Asn) residue.
  • a synthetic polypeptide of this invention corresponding in sequence to a portion of the amino-terminal 39 residues of lapine LBP that includes position 30 with its indeterminate residue (XXX) includes an Asn residue at that position.
  • Exemplary synthetic polypeptides of the first group include those having the sequences shown below, from left to right and in the direction from amino-terminus to carboxy-terminus,
  • Exemplary more preferred synthetic polypeptides having about 10 to about 25 residues that correspond in sequence to a portion of the amino-terminal 39 residue sequence of the lapine LBP molecule, from left to right and in the direction from amino-terminus to carboxy-terminus, are shown below.
  • parenthesized numerals before the two sets of polypeptide sequences above refer to the numbered positions from the amino-terminus of LBP to which those polypeptides correspond.
  • amino acid residues utilized herein are in the natural, L, form unless otherwise stated.
  • conservative substitution as used above is meant to denote that one amino acid residue has been replaced by another, biologically similar residue.
  • conservative substitutions include the substitution of one hydrophobic residue such as He, Val, Leu or Met for another, or the substitution of one polar residue for another such as between Arg and Lys, between Glu and Asp or between Gin and Asn, and the like.
  • the replacement of an ionic residue by an oppositely charged ionic residue such as Asp by Lys has been termed conservative in the art in that those ionic groups are thought to merely provide solubility assistance.
  • replacements discussed herein are on relatively short synthetic polypeptide antigens, as compared to a whole protein, replacement of an ionic residue by another ionic residue of opposite charge is considered herein to be "radical replacement", as are replacements between nonionic and ionic residues, and bulky residues such as Phe, Tyr or Trp and less bulky residues such as Gly, He and Val.
  • nonionic and ionic residues are used herein in their usual sense to designate those amino acid residues that normally either bear no charge or normally bear a charge, respectively, at physiological pH values.
  • exemplary nonionic residues include Thr and Gin, while exemplary ionic residues include Arg and Asp.
  • a synthetic polypeptide of the present invention can be prepared by several solid and liquid phase techniques as are well known in the art. Preferably, however, the solid phase so-called "Merrified” method is utilized. Exemplary syntheses are discussed in U.S. Patents No. 4,545,931 and No. 4,544,500, whose disclosures are incorporated herein by reference.
  • a polypeptide of the present invention is preferably linked to an immunogenic carrier such as a protein as a conjugate for use in production of antibodies and antibody preparations.
  • Immunogenic carriers are well known in the art and include keyhole limpet hemocyanin (KLH) , edestin, curcubin, human serum albumin, tetanus toxoid, sheep erythrocytes, polyamino acids such as poly(D-lysine D-glutamic acid) and the like.
  • KLH keyhole limpet hemocyanin
  • edestin curcubin
  • human serum albumin tetanus toxoid
  • sheep erythrocytes polyamino acids such as poly(D-lysine D-glutamic acid) and the like.
  • Methods of linking the polypeptide to the immunogenic carrier to form the conjugate are also well known. Exemplary techniques include use of glutaraldehyde, a water-soluble carbodiimide, and those described in United States Patents
  • the polypeptide-carrier conjugate is dissolved or dispersed in an aqueous composition of a physiologically tolerable diluent when used to induce the production of antibodies.
  • a physiologically tolerable diluent include phosphate-bu fered saline (PBS) and 0.9 normal saline, and preferably also include an adjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant.
  • An effective amount of a conjugate- containing composition is introduced into a host animal such as a goat, rabbit, mouse, rat, horse or the like to induce the production (secretion) of antibodies to the polypeptide.
  • a host animal such as a goat, rabbit, mouse, rat, horse or the like to induce the production (secretion) of antibodies to the polypeptide.
  • -Effective amounts of immunogens useful for inducing antibody secretions in host animals are well known in the art.
  • Methods of introduction into the host animal are also well known and are typically carried out by parenteral administration as by injection. A plurality of such introductions is normally utilized so that the host is hyperimmunized to the immunogenic polypeptide-containing conjugate. For example, weekly introductions over a one-to-two-month time period can be utilized until a desired anti-polypeptide antibody titer is achieved.
  • the antibodies so induced are thereafter recovered from the host animal.
  • the recovered antibodies can be utilized as a preparation in the host serum as recovered, or can be in substantially pure form; i.e., substantially free from host serum proteins, polypeptides and cellular debris.
  • the latter antibody preparation can be conveniently prepared by passage of the recovered serum over " an affinity column as prepared from Sepharose 4B
  • the recovered preparation of antibodies immunoreacts with a synthetic polypeptide of the invention such as the polypeptide of the conjugate, as well as with denatured rabbit LBP.
  • a synthetic polypeptide of the invention such as the polypeptide of the conjugate
  • denatured rabbit LBP is that material that has been treated with 2-mercaptoethanol in SDS-PAGE analysis, and typically has a protein structure that is relatively more open or expanded than is that of native protein.
  • the antibodies also immunoreact with native, non-denatured rabbit LBP as is present in APRS.
  • the antibodies immunoreact with human LBP in denatured and/or non-denatured forms are particularly preferred.
  • An antibody preparation of this invention prepared from a polypeptide as described above can be in dry form as obtained by lyophilization.
  • the antibodies are normally used and supplied in an aqueous liquid composition in serum or a suitable buffer such as PBS.
  • the antibodies and polypeptides described herein are useful in assay methods for the determination of the presence and amount of rabbit and human lipopolysaccharide binding protein (LBP) .
  • the polypeptides are particularly useful in these assays for blocking studies as in connection with the Western blot-type assays. Similar blocking can also be carried out in solid phase assays such as the ELISA-type studies that are also described hereinafter.
  • the antibodies are particularly useful in assays because of their unique specificity for im unoreacting with LBP. For example, a liquid body sample as described before can be admixed and contacted with the antibodies affixed to a solid matrix as a solid support to form a solid/liquid phase admixture.
  • the phases are separated to remove any materials that did not immunoreact.
  • the non-immunoreacted antibodies are thereafter visualized or otherwise assayed as with a label linked to a polypeptide of this invention.
  • Solid phase assays such as enzyme-linked im unosorbant assays (ELISA) , radio-labeled immunosorbant assays (RIA) or flurochrome-linked immunosorbant assays (FIA) are particularly contemplated.
  • ELISA enzyme-linked im unosorbant assays
  • RIA radio-labeled immunosorbant assays
  • FFA flurochrome-linked immunosorbant assays
  • the amount of LBP present in a liquid body sample is assayed in an embodiment of this invention.
  • a solid phase matrix such as the sides and bottom of a polystyrene or polyvinylchloride microtiter plate is provided.
  • Antibodies of this invention are affixed to the solid matrix as by physical binding to form a solid phase support, as is known.
  • a predetermined amount of a liquid body sample such as plasma or serum to be assayed for LBP is admixed with the solid phase support to form a solid/liquid admixture.
  • Exemplary predetermined amounts typically are about 25 to about 150 microliters neat, or more preferably present at a known dilution in an aqueous medium such as PBS that contains a total volume of about 25 to about 150 microliters. That solid/liquid admixture is maintained for a period of time sufficient for LBP present in the sample to immunoreact with the solid phase-affixed antibody to form a solid phase-bound immunoreactant, and a liquid phase depleted of LBP.
  • Exemplary maintenance (incubation) times typically range from about 5 minutes to about 6 hours, with the temperature of that maintenance typically being from about 4°C to about 40°C, with room temperature (about 20°C) being exemplary.
  • the solid and liquid phases are then separated as by rinsing to remove any materials from the sample that were not bound to the solid support.
  • the solid phase containing the bound immunoreactant is retained for further use in the assay.
  • the amount of solid phase-bound immunoreactant formed is then determined, and thereby determines the amount of LBP present in the assayed sample. That amount can be determined in a number of well known manners.
  • an aqueous composition containing a polypeptide of this invention operably linked to a label can be immunoreacted with the unreacted affixed antibodies to form a second solid/liquid phase admixture.
  • the second solid/liquid phase admixture is maintained for a second time period sufficient for the labeled polypeptide to immunoreact with the previously unreacted solid phase-bound antibodies and form a second solid phase-bound immunoreactant. Maintenance times and temperatures useful for this maintenance step are similar to those described before.
  • the solid and liquid phases are again separated to remove any labeled polypeptide not present in the second solid phase-bound immunoreactant, and the amount of labeled polypeptides bound is determined. That determination is conveniently accomplished by use of a label such as an enzyme, flurochrome dye or a radiolabel operably linked to the second antibodies.
  • non-specific binding sites on the solid supports be blocked after the solid support is prepared.
  • Such non-specific site blockage can be achieved by known techniques such as by admixture of an aqueous composition of a protein free from immunoreaction in the assay such as bovine serum albumin (BSA) with the solid support prior to admixture of the liquid body sample.
  • BSA bovine serum albumin
  • the admixture so formed is typically maintained for the time period and at the temperature described before.
  • the solid phase having its non-specific binding sites blocked and the liquid phase are then separated as by rinsing, and the liquid body sample is admixed.
  • operably linked is used herein to mean that the label molecules are linked or bonded to the polypeptide molecules so that antibody binding of the polypeptide is not substantially impaired nor is the action of the label substantially impaired.
  • polypeptides containing operably linked label molecules bind to their antibodies and the linked label molecules operate to indicate the presence of the bound polypeptide in the immunoreactant.
  • typically used enzymes operably linked to a polypeptide as a label include horseradish peroxidase, alkaline phosphatase and the like. Each of those enzymes is used with a color-forming reagent or reagents (substrate) such as hydrogen peroxide and -phenylenediamine; and £-nitrophenyl phosphate, respectively.
  • fluorochrome dye operably linked to a polypeptide as a label to signal the presence of polypeptides bound in an immunoreaction product
  • the fluorochrome dye is typically linked by means of an isothiocyanate group to form the conjugate.
  • fluorochrome dyes include fluorescein isothiocyanate (FITC) , rhodamine B isothiocyanate (RITC) and tetramethylrhodamine isothiocyanate (TRITC) .
  • biotin operably linked to an a polypeptide is utilized as a label to signal the presence of the immunoreactant in conjunction with avidin that is itself linked to a signalling means such as horseradish peroxidase.
  • a radioactive element such as H or - *63 i as utilized herein can also be operably linked to the polypeptide to form the label. In this instance, the radioactive decay of the element serves to quantify the assay of bound polypeptide, and thereby bound LBP.
  • gp60 material isolated from acute phase rabbit serum was responsible for the retardation of binding of rabbit HDL by exogenously supplied LPS.
  • APRS acute phase rabbit serum
  • antibodies raised to the isolated gp60 when admixed with both NRS and APRS provided gp60-containing immunorea ⁇ tants from both types of serum. The amounts of gp60 isolated from both serum types using those antibodies were similar.
  • the isolated gp60 when reconstituted with NRS, provided a serum in which no retardation of LPS binding to HDL was observed.”
  • the originally identified and isolated rabbit gp60 was not the material whose presence retards binding of LPS with HDL in rabbit sera.
  • the original results that indicated the presence in APS of a material (i) that is substantially absent from NS (ii) that binds to gram-negative bacterially secreted lipopolysaccharide when both are admixed _i ⁇ vitro in normal serum, and (iii) retards the jj vitro binding of LPS to HDL in serum is still thought to be correct, as is discussed hereinbelow.
  • NRS NRS, and it also retards the .in vitro binding of LPS to HDL in NRS.
  • the human homolog to the newly identified lapine glycoprotein appears to have a slightly higher molecular weight, but is functionally equivalent in its presence and substantial absence in acute phase serum and normal serum, respectively, its binding to LPS .in vitro in normal human serum, and in its retardation of binding of LPS to serum HDL in in vitro determinations.
  • Etiocholanolone is a naturally occurring steroid metabolite experimentally useful for inducing local inflammatory reactions and fever in man. These responses typically begin within 8-20 hours after injection and last 2-6 hours [McAdam et al. (1978) J. Clin. Invest. £1:390; Wolff (1967) Ann. Intern. Med. 6:1268] . Etiocholanolone also induces typical plasma acute phase reactant responses, for example, CRP and serum amyloid A (SAA) , within 24-48 hours after injection [McAdam et al. (1978) J. Clin. Invest. £1:390] .
  • CRP and serum amyloid A SAA
  • Figure la shows a CsCl density gradient using sera collected before and 32 hours after etiocholanolone injection; the appearance of a form of Re595-LPS in the bottom third of the gradient when APHS is used is evident.
  • the density of this form of Re595-LPS was found to be 1.30g/ ⁇ c by measurement of refractive index.
  • Re595-LPS-HDL complexes The formation of Re595-LPS-HDL complexes is plotted as for a first order reaction in Figure 2.
  • Complexation of Re595-LPS with HDL in NHS has a one-half time (t ⁇ ) of about 7 minutes, whereas in APHS the reaction has a one-half time of about 52 minutes. This difference is virtually identical to that seen in rabbit serum where the one-half times are 2-4 minutes and 40-80 minutes for NRS and APRS, respectively [Tobias and Ulevitch (1983) J. Immunol. 13_1:1913] .
  • gp60 is the previously identified and isolated glycoprotein that precipitates from acute phase rabbit serum only in the presence of C1.3.
  • LPS preincubated with delipoproteinated APRS is more rapidly cleared from a rabbit than is LPS preincubated with delipoproteinated NRS.
  • the acute phase response appears to incude a means for dealing with lipopolysaccharide endotoxins, and that means appears to be the human alpha 2 ,beta,-glycoprotein discussed and described herein, and its homologs in other animals.
  • NRS Reconstitution Assay Two examples of the NRS reconstitution assay for lipopolysaccharide binding protein (LBP) activity in the lapine (rabbit) system are shown in Figure 5. In control studies, no systematic dependence on the assay results were observed when smaller total assay volumes; i.e., 0.5 or 0.25 ml rather than the standrard 1 ml, were used. Addition of 0.05 percent CHAPS [an N-alkyl sulfobetaine derivative of a bile acid amide reported to have an empirical. formula of
  • Partial amino acid sequence data were obtained for two preparations of LBP, the mixture of 60.5 kD and 58 kD proteins obtained from Mono-Q chromatography and the 58 kD protein purified by SDS-PAGE. Initially, material collected from Mono-Q chromatography was sequenced. Since this material consists of 80-90 percent of the 60.5 kD protein, these data reflect the sequence of the major component recovered from the column. The amino acid residue sequence of the first
  • the 39 amino acid sequence was used to search for homologous sequences in the National Bioraedical Research Foundation protein sequence database using the Wordsearch program from the University of Wisconsin Genetics Computer Group. To distinguish between random and non-random matches found by the computer search, the Wordsearch program was submitted to a randomized sequence having the same composition as the peptide shown above. Those matches found with the LBP sequence were eliminated from consideration whose "quality scores" were not better than the matches found with the randomized sequence.
  • the Wordsearch program was also used to look for homology between the sequences of LBP and rabbit CRP [Wang et al., J. Biol. Chem. iZ :1 3610 (1982)], human serum amyloid P [Mantzouranis et al., J. Biol. Chem. 260:7752 (1985)], human serum amyloid a [Sipe et al.. Biochemistry 24:2931 (1985)], Syrian hamster female acute phase protein [Dowton et al.. Science 228:1206 (1985)], human alpha-1-antichymotrypsin precursor [Chandra et al. , Biochem 22:5055 (1983)], human alpha-1 acid glycoprotein [Dente et al..
  • rat antisera induced by introduction of whole, substantially purified lapine LBP were reactive with APRS and LBP-containing fractions of APRS isolated by Bio-Rex 70 and Mono-Q columns, but not with NRS or NRS fractions corresponding to the fractions isolated from APRS.
  • the antisera were then tested for their ability to immunoprecipitate LBP and simultaneously remove LBP activity from APRS.
  • both NRS and APRS were admixed with immune and non-immune rat sera. After precipitation, the supernates were collected to determine their ability to form C1.3 (Figure 13) , indicating the presence or absence of LBP activity, and the precipitates were saved for analysis by SDS-PAGE ( Figure 14) .
  • H-LPS and APRS were admixed, allowed to react and form a complex at 37 degrees C for 10 minutes, and the admixture was then cooled to zero degrees C before rat anti-LBP was added.
  • the H-LPS content of a portion of the immunoprecipitate was determined by liquid scintillation counting while the remainder of the precipitate was taken for SDS-PAGE. Since LPS may bind non-spe ⁇ ifically to immune precipitates [Ginsberg et al., J. Immunol. 120:317 (1978)], immunoprecipitation of rabbit C-reactive protein
  • 125I-ASD-LPS was shown to cosediment with underivatized LPS in a CsCl gradient, form Cl.3 with
  • Lane 1 of Figure 16 shows that 125 I-ASD-LPS photolysed in 20 mM EDTA, 150 mM NaCl, pH 7.4 does not yield a Coomassie blue stainable band (la) and the 125 ⁇ runs with the dye front (lb) .
  • Lanes 2 and 3 show that 125 I-ASD-LPS mixed with immunopurified anti-LPS labels immunoglubulin heavy chains if photolysed after admixing (lane 3) , but only very weakly if photolysed also before admixing (lane 2).
  • lanes 1-3 aliquots of the reaction mixtures were applied directly to the gels.
  • lanes 4-7 the reaction mixtures were immunoprecipitated with anti-LPS before application to the gel.
  • Lanes 4 and 5 show that 125 I-ASD-LPS, whether photolysed only after admixture with NRS (lane 5) or also photolysed before with NRS did not label any material immunoprecipitable with anti-LBP; i.e., lanes 4b and 5b are clear.
  • Lanes 6 and 7 show that 125 I-ASD-LPS admixed with APRS labels LBP strongly if photolysed after admixing with APRS (lane 7) , but labels LBP only weakly if also photolysed before admixing with APRS (lane 6) .V. V. MATERIALS AND METHODS
  • LPS Lipopolysaccharide Purification
  • the suspension was then centrifuged (5000 rpm for 15 minutes in a Sorvall GSA rotor) to form a bacterial pellet and an LPS-containing supernatant.
  • the pellet and supernatant were separated and the supernatant was filtered to remove any remaining bacteria or cellular debris.
  • the bacterial pellet was subjected to the same extraction procedure one or two more times with each resulting LPS-containing supernatant being admixed with the first. Petroleum ether and chloroform were then removed from the pooled supernatants by rotary evaporation at 30-40 degrees C.
  • To the resulting LPS-containing aqueous phenol solution was slowly admixed water until the LPS precipitated.
  • the admixture was subsequently centrifuged (3000 rpm for 10 minutes in a Sorvall GSA rotor) so as to form a LPS pellet and supernatant.
  • the LPS pellet was separated from the supernatant and washed two or three times with about 5 ml of 80 percent phenol (w/v in H.O) by suspension and centrifugation. The LPS pellet was then washed three times with 5 ml of ether by suspension and centrifugation to remove any remaining phenol, and dried jj vacuo. The dried Re595 was then admixed with sufficient 20 mM EDTA (pH 7.5) to dissolve the LPS upon sonic oscillation in a model W-375 sonicator from Heat Systems-Ultrasonics, Inc., Plainview, NY.
  • the LPS solution was dialyzed against 3 liters of sterile water for 72 hours, with a change of the dialysis bath every 12 hours, and then lyophilized. Fresh stock solutions of 5 mg/ml LPS were prepared by addition of the LPS to the appropriate buffer, followed by sonic oscillaiton at 25 degrees C.
  • Growth medium typically one liter in a three liter flask with baffle plates, was inoculated with 20 ml of a confluent (plateau) S_. minnesota Re595 culture and inoculated at 37 degrees C with vigorous agitation for about 8 hours.
  • Bacteria containing LPS were subsequently harvested by centrifuging the cultures to form a bacterial pellet and supernatant.
  • the bacterial pellet was separated from the supernatant and washed three times by resuspension in deionized water and repelleting by centrifugation.
  • the final washed pellet was resuspended in about 50 ml of deionized water and lyophlized, typically yielding about 4.5 g dry weight bacteria per liter of culture.
  • CA CA
  • Etiocholanolone was used to induce an acute phase response [McAdam et al. (1978) J. Clin. Invest. £1 ⁇ 390] in three human volunteers by intramuscular injection of 0.3 milligrams etiocholanolone per kilogram of volunteer body weight. Serum was collected at various times from 24 hours previous to etiocholanolone injection, to 120 hours after injection. Observation and quantitation of Re595-LPS complex formation in sera using CsCl gradients was as described in Tobias and Ulevitch (1983) J. Immunol. 131:1913.
  • Acute phase rabbit serum was prepared as described in Tobias and Ulevitch (1983) J. Immunol. 131:1913.
  • the newly identified gp60 material was purified from APRS as follows.
  • the primary purification step was ion-exchange chromatography on Bio-Rex 70 (Bio-Rad Laboratories, Richmond, CA) .
  • a 25 ml column of resin was equilibrated with 0.05 molar (M) sodium phosphate, 2 mM EDTA, pH 7.3 buffer (Pi/EDTA buffer) in the cold (about 4°C) . Thereafter, 200 ml of APRS was run through the column.
  • the column was then washed first with Pi/EDTA buffer, and then with 0.22 M NaCl, 0.05 M sodium phosphate, 2 mM EDTA as a pH 7.3 buffer until the ultraviolet absorption of the eluate at 280 nanometers was less than 0.1.
  • the desired glycoprotein-LPS binding (complex forming) activity primarily eluted at the end of the gradient with 1 M NaCl.
  • the active fractions were further purified by gel filtration using G-150 Sephadex (Pharmacia Fine Chemicals, Piscataway, NJ) in 5 mM sodium phosphate at pH 7.3 to remove low molecular weight contaminants.
  • the activity eluted from the gel filtration column was very close to the elution position of BSA.
  • the activity-bearing glycoprotein had an apparent relative molecular weight of about 60,000.
  • the acute phase glycoprotein reactant bound to DE-52 cellulose (Whatman, Inc., Clifton, NJ) equilibrated with 5 mM sodium phosphate pH 8.3 and eluted in active form with 1 M NaCl. Salt gradient elution from the column provided a further purification step.
  • the activity was recovered from a sucrose density gradient prepared with 5-20 percent sucrose and centrifugation for 2 hours at 45,000 RPM using a TV 865 rotor (DuPont Co., Instrument Products Biomedical Div., Newtown, CT) at an average value of 4 Svedbergs (S) .
  • Each gradient so prepared was divided into 8 fractions.
  • Each fraction so obtained was assayed for its ability to form a 1.3 g/cc complex using a CsCl density gradient method analogous to that described hereinafter using J H-LPS and APRS. These assays serve to confirm the roughly 60,000 molecular weight of the activity-containing glycoprotein.
  • An aliquot of an animal body sample to be assayed for LPS binding activity was first concentrated, as necessary, to provide a composition containing about 1 mg/ml of solids having a molecular weight greater than about 10,000 using an Amicon ultrafiltration apparatus with a YM 10 membrane
  • the resulting admixture was centrifuged for a time period of 16 hours at 45,000 RPM using a TV865 rotor.
  • the resulting density gradient was fractionated and the counts in fractions of varying densities were determined as is shown in Figure 1.
  • HDL High density lipoprotein
  • (depleted) serum from either normal or acute phase rabbits or humans, was prepared by methods well known in the art. Briefly, the density of 35 ml of serum was adjusted to about 1.24 g/cirr by admixing 13.2 g of KBr. The serum/salt admixture was then centrifuged to gradient equilibrium in a high gravitational field, i.e., about 113,000 x gravity [48-60 hours at 40,000 rpm in a 60 ti rotor (Beckman Instruments, Palo Alto, CA] . HDL, being less dense than the other components of the admixture, concentrates as a stable band with a yellowish color at the top of the gradient. The HDL band was separated from the gradient and discarded. The volume of the remaining serum/salt admixture was then adjusted to 35 ml and its density adjusted to 1.24
  • the centrifugation and separation procedure was repeated.
  • the resulting serum/salt admixture was then dialyzed against 0.9 percent saline to substantially remove KBr from the admixture.
  • the admixture was adjusted to its original volume (35 ml) by admixture of an appropriate amount of 150 mM NaCl.
  • 3 H-LPS were admixed with 15 ml of either (1) HDL deficient APRS; or (2) HDL deficient NRS. The admixtures were incubated for 10-30 minutes at 20 degrees C to allow binding of 3 H-LPS to any acute phase reactants in the admixtures.
  • Biosynthetically tritiated LPS ( H-LPS) and unlabelled LPS were isolated from Salmonella minnesota Re595 as described previously. (Tobias et al.. Infect. Immun. 50.:73 (1985); Galanos et al. Eur. J. Biochem. 9:245 (1969). Rabbit blood was collected either by bleeding from the median ear artery or by heart puncture, allowed to clot at 37 degrees C for 2-6 hours and at zero degrees C overnight, centrifuged to remove clot fragments and cells, and the serum was stored frozen without preservative.
  • Acute phase rabbit serum was collected 24 hours after induction of an acute phase response by subcutaneous injection of 1 ml of 3% (W/V) silver nitrate in distilled water. Serum collected from non-induced rabbits was tested for "normality" before being used as normal rabbit serum (NRS) . The initial assay used was immunodiffusion versus antiserum to rabbit c-reactive protein (CRP) . Sera assaying negative for CRP were further assayed as described below to ensure a sufficiently rapid rate of binding of LPS to HDL. These precautions were instituted after observing that more than 50% of a batch of newly acquired rabbits had readily detectable acute phase reactants in their sera.
  • Precipitates were collected by centrifugation and washed twice with 50 mM phosphate buffer, 150 millimolar (mM) NaCl, 0.1% Tween-20 [polyoxyethylene (20) sorbitan monolaurate] , pH 7.4. Unfractionated lipoproteins and deliproproteinated sera were prepared by ultracentrifugation. To 35 milliliters (ml) of serum were added 13.23 grams (g) of KBr, after which the serum was spun at 40,000 RPM in a 60 Ti (Be ⁇ kman Instruments, Fullerton, CA) rotor for 36-60 hours at 4 degrees.
  • lipoproteins and serum proteins were separately pooled and dialysed extensively against 10 mM HEPES, 150 mM NaCl, pH 7.4. Finally the delipoproteinated sera and the lipoproteins were brought to 75 percent and 25 percent of the original serum volumes, respectively, by dilution or concentration as required. Delipoproteinated sera and lipoproteins were recombined in a 3:1 ratio, respectively, to prepare lipoprotein reconstituted sera.
  • 125 I-ASD—LPS co-sediments with LPS in CsCl gradients 125I-ASD-LPS is quantitatively taken up by HDL and NRS and APRS, and ASD-LPS has the same mitogenicity as LPS when assayed with murine splenic B cells.
  • Photolysis of 125 I-ASD-LPS was accomplished using a Rayonet photochemical reactor (Southern N.E. Ultraviolet Co., Middletown, CT) equipped with General Electric F8T5.BLB lamps with a peak output at 370 nanometers (nm) . Reaction mixtures were exposed for 10 minutes on ice. METHODS:
  • the gradient was to be determined, and the H-LPS in each fraction determined.
  • the efficiency of measuring 3A-Re 595 LPS was found to be independent of the amount of CsCl in each vial.
  • the amount of radioactivity in the body of the gradient i.e., not bound to the HDL which floats at the gradient, was calculated as a,percentage of the radioactivity recovered in the entire gradient.
  • a logarithmic of thi-s percentage as a function of the time of removal of the aliquot from the LPS serum reaction mixture yielded the half time for the binding of LPS to HDL.
  • Reconstitution Assay for LBP Activity The basic method used during development of the purification procedure for LBP was a reconstitution assay in which fractions of acute phase serum were assayed for their ability to reconstitute "acute phase behavior" in NRS.
  • the screening assay used was to mix a sample of the material to be tested with 1.0 ml of NRS at 37 degrees C for 30 minutes. LPS and EDTA were then added to the concentrations given above, and the LPS-HDL binding reaction was allowed to proceed for ten minutes at 37 degrees before addition of CsCl and centrifugation. The 10 minute reaction time was chosen as a compromise between the times required for
  • LBP activity in purified fractions of APRS was quantitated in purified fractions of APRS.
  • the reconstitution assay just described was performed with a series of different amounts of the sample being assayed up to a maximum of 200 ul per ml NRS.
  • a plot of percent LPS as Cl.3 versus sample volume was made.
  • One LBP unit is defined as that amount of LBP activity that causes 50% of the recovered LPS to be recovered as Cl.3 in the above procedure.
  • LBP activity in APRS was assayed similarly, except that the final volume of the NRS-APRS mixture was held constant and the final plot was then (percent LPS as Cl.3) versus (percent APRS) .
  • the column was then washed with column equilibration buffer overnight (about 18 hours) or until the absorbance at 280 nm of the eluate was less than 0.2 absorbance units (AU) . Washing was continued with 220 mM NaCl in phosphate/EDTA again until the absorbance was below 0.2, followed by a linear gradient formed from 60 ml each of 220 mM and 500 mM NaCl in phosphate/EDTA. Finally, the column was washed with 1 M NaCl in phosphate/EDTA.
  • the second chromatographic step used high performance liquid chromatography (HPLC) (Perkin-Elmer) with a Mono-Q column (Pharmacia, Piscataway, NJ) as the adsorbent using the instructions provided with the pre-packed column. Unless otherwise noted, the flow rate was 1 ml per minute.
  • HPLC high performance liquid chromatography
  • the column was equilibrated with 20 mM diethanolamine buffer, pH 8.3. Injection of the sample was followed immediately by a 15 ml gradient of zero to 50 mM ammonium sulfate in 20 mM diethanolamine, pH 8.3. The gradient was then steepened, going in 15 minutes from 50 mM to 333 mM ammonium sulfate in the same buffer.

Abstract

Composition thérapeutique contenant une glycoprotéine pour le traitement d'un hôte animal, procédés, polypeptides et anticorps relatifs à ladite glycoprotéine. La composition contient une quantité efficace d'une glycoprotéine, laquelle (a) est présente dans un sérum pendant la phase aiguë, mais est essentiellement absente du sérum normal; (b) se lie au lipopolysaccharide sécrété par les bactéries gram-négatives in vitro dans le sérum de l'animal traité; (c) retarde in vitro la liaison du lipopolysaccharide à une lipoprotéine de densité élevée présente dans le sérum normal de l'hôte animal.
PCT/US1986/000936 1985-04-30 1986-04-28 Proteine de phase aigue modulant l'activite endotoxique de lipopolysaccharides, compositions et procedes WO1986006279A1 (fr)

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EP0279517A1 (fr) * 1987-01-21 1988-08-24 E.I. Du Pont De Nemours And Company Fixation non immunochimique de lipopolysaccharides et essais sandwichs à cet effet
EP0460058A1 (fr) * 1989-02-14 1991-12-11 Incyte Pharmaceuticals, Inc. Utilisation d'une proteine bactericide/accroissant la permeabilite ou d'analogues biologiquement actifs de cette proteine pour fabriquer un medicament pour traiter les affections liees a l'endotoxine
WO1992004908A1 (fr) * 1990-09-14 1992-04-02 Imtox Privatinstitut Für Immunbiologische Forschung Gmbh Medicament contenant du cd14
US5245013A (en) * 1985-04-30 1993-09-14 Richard Ulevitch Acute phase protein modulating endotoxic activity of lipopolysaccharides, assay methods and polypeptides
WO1994020532A1 (fr) * 1993-03-12 1994-09-15 Xoma Corporation Peptides biologiquement actifs issus de domaines fonctionnels de proteine bactericide/augmentant la permeabilite et utilisations de ladite proteine
WO1995005393A2 (fr) * 1993-08-18 1995-02-23 Morphosys Gesellschaft Für Proteinoptimierung Mbh Peptides liant et neturalisant les lipopolysaccharides
WO1995019372A1 (fr) * 1994-01-14 1995-07-20 Xoma Corporation Peptides bioroniquement actifs provenant de domaines fonctionnels d'une proteine bactericide augmentant la permeabilite, et leurs utilisations
WO1995020163A1 (fr) * 1994-01-24 1995-07-27 Xoma Corporation Procede pour determiner la quantite de lbp dans des fluides corporels
US5990082A (en) * 1997-10-22 1999-11-23 Xoma Corporation Uses of lipopolysaccharide binding protein
US6376462B1 (en) 1993-06-17 2002-04-23 Xoma Corporation Lipopolysaccharide binding protein derivatives
CN100396695C (zh) * 1993-03-12 2008-06-25 爱克索马技术有限公司 来自杀菌性通透性增强蛋白的功能区的生物活性肽及其应用

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Cited By (16)

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Publication number Priority date Publication date Assignee Title
US5245013A (en) * 1985-04-30 1993-09-14 Richard Ulevitch Acute phase protein modulating endotoxic activity of lipopolysaccharides, assay methods and polypeptides
EP0279517A1 (fr) * 1987-01-21 1988-08-24 E.I. Du Pont De Nemours And Company Fixation non immunochimique de lipopolysaccharides et essais sandwichs à cet effet
EP0460058A1 (fr) * 1989-02-14 1991-12-11 Incyte Pharmaceuticals, Inc. Utilisation d'une proteine bactericide/accroissant la permeabilite ou d'analogues biologiquement actifs de cette proteine pour fabriquer un medicament pour traiter les affections liees a l'endotoxine
EP0460058A4 (en) * 1989-02-14 1992-02-12 Invitron Corporation Use of bactericidal/permeability increasing protein or biologically active analogs thereof to treat lipopolysaccharide associated gram negative infections
WO1992004908A1 (fr) * 1990-09-14 1992-04-02 Imtox Privatinstitut Für Immunbiologische Forschung Gmbh Medicament contenant du cd14
WO1994020532A1 (fr) * 1993-03-12 1994-09-15 Xoma Corporation Peptides biologiquement actifs issus de domaines fonctionnels de proteine bactericide/augmentant la permeabilite et utilisations de ladite proteine
CN100396695C (zh) * 1993-03-12 2008-06-25 爱克索马技术有限公司 来自杀菌性通透性增强蛋白的功能区的生物活性肽及其应用
US6376462B1 (en) 1993-06-17 2002-04-23 Xoma Corporation Lipopolysaccharide binding protein derivatives
WO1995005393A2 (fr) * 1993-08-18 1995-02-23 Morphosys Gesellschaft Für Proteinoptimierung Mbh Peptides liant et neturalisant les lipopolysaccharides
WO1995005393A3 (fr) * 1993-08-18 1995-03-23 Morphosys Proteinoptimierung Peptides liant et neturalisant les lipopolysaccharides
US6384188B1 (en) 1993-08-18 2002-05-07 Dana-Farber Cancer Institute, Inc. Lipopolysaccharide-binding and neutralizing peptides
WO1995019372A1 (fr) * 1994-01-14 1995-07-20 Xoma Corporation Peptides bioroniquement actifs provenant de domaines fonctionnels d'une proteine bactericide augmentant la permeabilite, et leurs utilisations
CN1071759C (zh) * 1994-01-14 2001-09-26 爱克斯欧玛公司 来自杀菌性通透性增强蛋白的功能区的生物活性肽及其应用
WO1995020163A1 (fr) * 1994-01-24 1995-07-27 Xoma Corporation Procede pour determiner la quantite de lbp dans des fluides corporels
US6306824B1 (en) 1997-10-22 2001-10-23 Xoma Corporation Uses of lipopolysaccharide binding protein
US5990082A (en) * 1997-10-22 1999-11-23 Xoma Corporation Uses of lipopolysaccharide binding protein

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