CA2181815C - Method for quantifying lbp in body fluids - Google Patents

Abstract

The present invention provides a method for quantifying the presence of extracellular LBP in body fluids including blood in a subject comprising conducting an LBP immunoassay on plasma obtained from said subject.

Description

METHOD FOR QUANTIFYING LBP IN BODY FLUIDS
BACKGROUND OF THE INVENTION
The present invention relates to methods for determination of the presence of Lipopolysaccharide binding protein (LBP) in body fluid samples including blood samples.
Lipopolysaccharide (LPS) is a common component of the outer membrane of Gram-negative bacteria and is responsible for many of the pathologic effects associated with gram-negative bacterial infection and endotoxemia. Because of the association between bacterial infection and sepsis, attempts have been made to correlate serum/plasma levels of endotoxin with disease. Typically, endotoxin levels have been measured using the Limulus amebocyte lysate (LAL) assay, in which endotoxin initiates a coagulation cascade that can be measured physically, turbidimetrically, or spectrophotometricaliy. Despite these attempts, however, no reliable correlations between endotoxin levels and sepsis severity or outcome have been identified. This is most likely due to the fact that (i) endotoxin levels in septic patients are very low (> 10 pg/L), (ii) several serum proteins interfere with the proteolytic LAL cascade, (iii) endotoxin, once in contact with blood, can be "detoxified" by interaction with a variety of blood components, including high-density lipoprotein (HDL) and low-density lipoprotein (LDL) and (iv) endotoxin from different gram-negative organisms varies in its ability to trigger the LAL
cascade. Thus, the absolute levels of endotoxin in a patient sample may not correspond to the actual concentrations of bioactive endotoxin present in vivo.
Two related proteins have been identified in humans and other animals that bind LPS with high affinity. These two proteins, Lipopolysaccharide binding protein (LBP), and bactericidal/permeability increasing protein (BPI) have roughly the same molecular weight and share 45% amino acid homology, yet exhibit distinct physiological differences. LBP is a 60 kD glycoprotein W095l20163 T ~, ~; ;;: " i~ PC1'IUS95100982 synthesized in the liver, while BPI is found in the azurophilic granules of neutrophils. LBP is found in the serum of normal humans at levels of 5-10 ~g/mL but can reach levels of 50-100 ug/mL in septic patients. Schumann et al., Science, 249:1429 (1990) disclose the amino acid sequences and encoding cDNA
of both human and rabbit LBP. Like BPI, LBP has a binding site for lipid A and binds to the LPS from rough (R-) and smooth (S-) form bacteria. Unlike BPI, LBP does not possess significant bactericidal activity. BPI has been observed to neutralize and inhibit the production of TNF resulting from interaction of LBP
with LPS and CD14 on monocytes and macrophages. Marta et al., J. lmmunol.
148: 532 (1992), Weiss et al., J. Clin. Invest. 90: 1122 (1992). In contrast, LBP
is observed to enhance LPS-induced TNF production. Wright et al., Science, 249:1131 (1990). Thus, in contrast to BPI, LBP has been recognized as an immunostimulatory molecule. See, e.g., Seilhamer, PCT International Application VJO 93106228 which discloses a variant form of LBP which it terms LBP-~. Also of interest to the present invention are Ulevitch, PCT
International Application WO 91101639 which discloses, among other things, anti-LBP
antibodies as an anti-sepsis therapeutic agent and U.S. Patent No. 5,245,013 which relates to LBP and discloses antibodies which immunoreact with a polypeptide having homology to LBP.
LBP has been characterized in the art as an "acute phase protein", that is one of many plasma proteins (such as C-reactive protein, fibrinogen and serum amyloid A) that increase in concentration in response to infectious and non-infectious tissue destructive processes. As such, it would be anticipated that LBP
levels would be elevated in samples from patients suffering from a number of autoimmune diseases such as rheumatoid arthritis and lupus erythematosus.
Of interest to the present invention are disclosures related to the assaying of BPI activity in subjects. von der Mohien et al., Abstract, 13th International Symposium on Intensive Care and Emergency Medicine, Brussels (March 1993) discloses the results of assays for serum levels of BPI in patients with gram-negative sepsis and healthy subjects. The abstract disclosed that no BPI was detectable under the conditions of the assay in the serum of healthy subjects while circulating BPI was detected in all septic patients. Also of interest is the disclosure of co-owned U.S. Patent No. 5,466,581. Those patent applications disclose that levels of BPI in blood plasma samples correlate with the presence or absence of sepsis while levels of BPI in blood serum samples do not. The patent applications teach that levels of BPI present in serum are not representative of endogenous extracellular levels of BPI in circulating blood while levels of BPI in plasma are.
Also of interest to the present invention are the disclosures of Leturcq et al., Keystone Tahoe Endotoxin Conference, March, 1-7, 1992 (Abstract) in which the generation of monoclonal antibodies to human LBP is reported. Also reported is the screening of normal human serum samples for the presence of LBP. LBP levels for normal serum samples were reported to range from 1 Ng/mL to 24 Ng/mL with an average of 7 Ng/mL. Further of interest is the disclosure of Richard Ulevitch at the American Society for Microbiology General Meeting in Atlanta, Georgia May 16-21 (1993) at which data was presented on LBP and soluble CD14 levels in the serum of septic and healthy individuals.
The average soluble CD14 and LBP concentrations in the serum of healthy adults were 1 NgImL and 7 Ng/mL respectively. The average soluble CD14 and LBP concentrations in the serum of septic patients were reported to be 2 NgImL
and 55 Ng/mL respectively.
Geller et al., Arch. Surg., 128:22-28 (1993) disclose experiments in which the induction of LBP mRNA was studied in three models known to induce acute phase responses: (1) LPS injection; (2) Corynebacterium parvum injection; and (3) turpentine injection. The publication reports that LBP mRNA
is induced during hepatic inflammation and suggest that LBP is an acute-phase protein important in regulating the in vivo response to endotoxin.
Gallay et al., Infect. Immun., 61:378-383 (1993) disclose that an acute phase response in mice injected with silver nitrate induced LBP synthesis, and that LBP levels increase approximately 10-fold over normal levels after an acute-phase response.
There exists a desire in the art for methods for determining the exposure of subjects to endotoxin and for distinguishing the effects of exposure .
to endotoxin from other acute phase physiologic responses. Also desired are methods for diagnosing the presence or severity of gram-negative sepsis in a subject and for predicting the prognosis of a subject suffering from sepsis.
The present invention provides methods for determining exposure of a subject to endotoxin by assaying for LBP. The invention further provides methods for screening for exposure to gram-negative bacterial endotoxin in an acute phase response in humans by assaying for LBP. Specifically, the method comprises the steps of determining the concentration of LBP in a sample of body fluid from the subject and correlating the concentration of LBP with a standard indicative of the exposure to endotoxin. Such standards can include a subjective standard for a given subject determined by LBP levels of that subject fln a pretreatment state such as prior to undergoing surgery. Exposure to endotoxin as a consequence of such surgery can be determined by comparing post-surgical LBP levels with the standard established prior to surgery for that subject.
Where access to a pretreatment standard level of LBP is not available for a given individual, objective standards based upon population or subpopulation averages may be applied for comparison. One such standard can be a concentration greater than approximately 15 ~.glmL in human plasma or serum, as determined herein for LBP values in subjects suffering from numerous disease states. Subjects exhibiting LBP levels above that standard could presumptively be diagnosed as suffering from exposure to endotoxin while those having levels below that standard would not be. It is clear that alternative standards could be established depending upon the desired sensitivity and selectivity of an assay method and upon the subpopulation in which a given subject falls. For example, standards might be established at different levels for different ages, genders, ethnicities and W O 95120163 PCT'1US95/00982 2~~~~i~
underlying health conditions of various subpopulations. Moreover, it should be understood that standard levels will differ according to the identity of the particular body fluid which is assayed.
The invention furttler provides methods for diagnosing the presence 5 or severity of sepsis in a subject comprising the steps of determining the concentration of LBP in a sample of body fluid from the subject and correlating She concentration of LBP with a standard indicative of the presence or severity of sepsis. The invention further provides methods for predicting the prognosis of a subject suffering from sepsis comprising the steps of determining the concentration of LBP in a sample of body fluid from the subject and correlating the concentration of LBP with a standard indicative of the prognosis of a subject suffering from sepsis.

Fig. 1 depicts the dose-response curves for rLBP, rLBPu, rBPI and rBPI~ in LBP sandwich assays;
Fig. 2 depicts LBP levels (mean t standard error) in the plasma of healthy human subjects and human subjects suffering from various disease StaiES;
Fig. 3 depicts LBP levels (mean f standard error) in healthy subjects treated with LPS;
Fig. 4 depicts comparative survival in suspected glacn-negative sepsis patients classified as havhng either high or low levels of plasma LBP;
and Figs. Sa-Sc depict LBP, C-reactive protein (CRP) and fibrinogen levels (mean t standard error) in healthy, rheumatoid arthritic and septic subjects.
DETA>?.ED DESCRIPTION OF THE INVENT'fON
The present invention relates to methods for quantifying the presence of LBP in body fluids including blood. While the assay can be used to determine the presence and quantity of LBP which has been administered therapeutically, it is particularly useful for quantifying the presence of endogenous LBP in circulating blood as an indication of exposure of a subject to endotoxin. Moreover, quantifying the presence of LBP is contemplated to be useful in diagnostic and prognostic methods for evaluating gram- negative sepsis patients.
The present invention provides a sandwich ELISA assay for human LBP
which exhibits high assay sensitivity, high specificity, and excellent reproducibility. As used herein "LBP" quantitated according to assay methods includes native LBP, recombinant LBP, LBP fragments and analogs as well as other LBP proteins and protein products.
The amino acid and nucleotide sequence of recombinant LBP are set out in co-owned and copending PCT Application Serial No. PCT/US94/06931 published as W095/00641 as shown in SEQ ID NOS: 1 and 2 herein. A
recombinant LBP amino-terminal fragment is characterized by the amino acid sequence of the first 197 amino acids of the amino-terminus of LBP as set out in SEQ ID NOS: 3 and 4 the production of which is described in co-owned U.S.
Patent No. 5,731,415. Such LBP protein products may be readily quantified using assays including immunological assays and bioassays in the subnanogram per mL range. Immunological assays capable of quantifying LBP
are preferably carried out by enzyme linked immunosorbant (ELISA) sandwich assays but competitive assays and immunological assays utilizing other labelling formats may also be used. Preferred assays of the invention utilize anti-LBP antibodies, including monoclonal antibodies and affinity-purified rabbit polyclonal antibodies. Rabbit polyclonal anti-LBP antibodies may be prepared according to conventional methods using LBP as an immunogen. Non-immunological methods may also be used to assay for LBP. As one example, Ulevitch et al., U.S. Patent No. 5,245,013 disclose assay methods comprising binding of LBP to LPS and separating the complex by a centrifugation density gradient method. As another example, Geller et al., Arch. Sung. 728:22-28 (1993) disclose LBP bioactivity assays in which IL-6 and TNF upregulation are measured.

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Body fluids which can be assayed for the presence of LBP include whole blood with blood serum and blood plasma being preferred. Because LBP
is a serum protein it is contemplat~;d that it could be excreted and that analysis of LBP levels nn urine may provide diagnostic and prognostic utility. The LBP
immunoassays of the invention may also be used to determine the concentration of LBP in other body fluids including, but not limited to lung lavages, vitreous fluid, crevicular fluid, cerebralspinal fluid, saliva and synovial fluid.
Because LBP has been characterized as an "acute phase protein"
it would be expected that LBP leva;ls would be elevated in subjects suffering from autoimmune diseases. As one as7xct of the present invention it has been found that LBP levels are not generally elevated over normal in subjects suffering from acute lymphoblastic leukemia (ALI,), acute graft versus host disease (aGvI~), chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL), type 1 diabetes, aplastic anemia (AA), Crohn's Disease, psoriasis, rheumatoid arthritis (RA), sclerodetma, and systemic lupus erythematosus (SLR.
Certain subjects tentatively identified as suffering from gram-negative sepsis but ultimately identified as suffering from gram-positive sepsis also had elevated LBP levels. It is noted that trenslocation of bacteria and/or endotoxin from the gut into the t>loodstream can occur in any infection. Thus, infections due to gram-positive bacteria or fungi may also lead to the presence of endotoxin or gram-negative bacteria in the blood and, therefore elevated levels of LBP.
The present invention is based in part upon the observation that serum and plasma levels of LBP directly correlate with a subject's exposure to biologically active LPS. Ndoreover, LBP levels appear to correlate with survival in suspected gram-negative sepsis patients. For example, subjects with levels of circulating LBP below 27.3 uglmL (the median value for 58 subjects suffering from gram-negative sepsis) tended to have a greater 14 day survival than did those subjects with levels of LBP above that median. Further, for example, when a plasma LBP threshold level was set at 46 ~.glmL, those subjects having a pretreatment LBP plasma level less than 46 ~.g/mL had a significantly greater survival rate (p=0.004) over a 27 day period than did those subjects having a pretreatment plasma LBP level greater than 46 Ng/mL.
It is further contemplated by the invention that elevated levels of LBP
may result from exposure to larger amounts of endotoxin, and may therefore be diagnostic of greater infection and/or endotoxemia severity. Elevated levels of LBP may also be used to indicate the suitability of using antibiotics directed against gram-negative bacteria or other therapeutic agents targeted directly to endotoxin such as BPI or anti-endotoxin antibodies including the monoclonal antibody E5.
Other aspects and advantages of the present invention will be understood upon consideration of the following illustrative examples. Example relates to the preparation of affinity purified rabbit anti-BPI antibodies;
Example 2 relates to the biotin labeling of such antibodies; and Example 3 relates to ELISA procedures utilizing such antibodies. Example 4 relates to the comparative immunoreactivity of rLBP, rLBP25, rBPI AND rBPI~. Example 5 relates to the measurement of rLBP spiked into pooled human plasma; and Example 6 relates to the comparison of LBP levels in human plasma and serum. Example 7 relates to the clinical correlations of endogenous LBP
immunoreactivity with sepsis and other disease states in human plasma; and Example 8 relates to the effect of LPS administration on endogenous LBP
levels in healthy subjects. Example 9 relates to clinical correlations between plasma LBP levels and survival in suspected gram-negative sepsis patients;
and Example 10 relates to clinical correlations of acute phase proteins in healthy, rheumatoid arthritic and septic patients.
Example 1 PREPARATION OF AFFINITY PURIFIED RABBIT ANTI-rLBP ANTIBODY
According to this example affinity purred rabbit anti-rLBP antibody was prepared. Specifically, rLBP (20 mg) produced according to co-owned U.S.
Patent No. 5,731,415 was coupled to 10 mL of cyanogen bromide-activated Sepharose 4B (Sigma Chemical Co., St Louis, MO) in 0.2 M bicarbonate, pH

8.6, containing 0.5 NaCI. Approximately 94% of the rLBP was coupled to the resin.
Pooled antisera (125 mL) from two rabbits, immunized initially with rLBP25 produced according to the methods of International PCT Publication WO 95/00641 filed June 17, 1994 and thereafter with rLBP, were diluted with an equal volume of phosphate buffered saline, pH 7.2 (PBS). A portion (50 mL) of the diluted antisera was passed through the 10 mL rLBP-Sepharose~ column; the column was then washed with PBS and bound antibodies were eluted with 0.1 M glycine, pH 2.5.
Collected fractions were immediately neutralized with 1 M phosphate buffer, pH 8Ø Peak fractions were identified by measuring absorbance at 280 nm according to the method of Harlow et al., Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory Press, New York, p. 312 (1988). After several sequential column cycles, the affinity purified rabbit anti-LBP antibody was dialyzed against PBS-azide pH 7.2.
Example 2 PREPARATION OF BIOTIN LABELED RABBIT ANTI-rLBP ANTIBODY
In this example twenty milligrams of affinity purified rabbit anti-rLBP
antibody produced according to the method of Example 1 was incubated with 2 mg of biotinamidocaproate N-hydroxysuccinimide ester (Sigma Chemical Co., St. Louis, MO) in 11 mL of 0.1 M sodium bicarbonate pH 8.3 for two hours at room temperature. Unconjugated biotin was removed and the alkaline buffer exchanged by fractionating the reaction mixture on a PD-10 column (Pharmacia Biotech Inc., Piscataway, NJ) equilibrated with PBS containing 0.1% sodium azide.

Example 3 ELISA PROCEDURE

Fifty microliters of affinity purified rabbit anti-rLBP antibody (2 Ng/mL in PBS) were incubated overnight at 2-8°C (or alternatively, 1 hour at 37°C) in the wells of Immulon~ 2 (Dynatech Laboratories Inc., Chantilly, VA) microtiter plates. The antibody solution was removed and 200 NL of 1 % non-fat milk in 10 PBS (blocking agent) was added to all wells. After blocking the plates for 1 hour at room temperature, the wells were washed 3 times with 300 NL of wash buffer (PBS/0.05% Tween-20).
Standards, samples and controls were diluted in triplicate with PBS
containing 1 % bovine serum albumin, 0.05% Tween 20 (PBS-BSAITween) and 10 units/mL of sodium heparin (Sigma Chemical Co., St. Louis, MO) in separate 96-well plates. rLBP or rLBP25 standard solutions were prepared as serial two-fold dilutions from 100 to 0.012 nglmL. Each replicate and dilution of the standards, samples and controls (50 pL) was transferred to the blocked microtiter plates and incubated for 1 hour at 37°C. After the primary incubation, the wells were washed 3 times with wash buffer. Biotin-labeled rabbit anti-LBP
antibody was diluted 1/2000 in PBS-BSAITween~ and 50 NL was added to all wells. The plates were then incubated for 1 hour at 37°C. Subsequently, all wells were washed 3 times with wash buffer. Alkaline phosphatase-labeled streptavidin (Zymed Laboratories Inc., San Francisco, CA) was diluted 112000 in PBS-BSA/Tween and 50 NL was added to all wells. After incubation for 15 minutes at 37°C, all wells were washed 3 times with wash buffer and 3 times with deionized water and the chromogenic substrate p-nitrophenylphosphate (1 mg/mL in 10°r6 diethanolamine buffer) was added in a volume of 50 pL to all wells. Color development was allowed to proceed for 1 hour at room temperature, after which 50 NL of 1 N NaOH was added to stop the reaction.
The absorbance at 405 nm was determined for all wells using a Vmax Plate Reader (Molecular Devices Corp., Menlo Park, CA).

WO 95120163 i ~ ' PCT/US95/00982 s:~ : ~. .

The mean absorbance at 405 nnl (A4~) for all samples and standards (in triplicate) were corrected for background by subtracting the mean A,~ of wells receiving only sample dilution buffer (no LBP) in the primary incubation step. A standard curve was then plotted as A,~ versus ng/mL of rLBP or rLBP~. The linear range was selected, a lunear regression analysis was performed and concentrations were determined for samples and controls by interpolation from the standard curve.
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COMPARATIVE IDwILTNOREACTIVITY OF rLBP, rLBP,s, rBPI AND rBPI,~
In this example, the immunoreactivity of rLBP, rLBPu, rBPI and rBPI~ were compared in the BPI sandwich ELISA to determine possible immunologic cross-reactivity. Despite considerable sequence homology ~~~n ~P and BPI (see, e.g., Schumann et al., Science, 249:1429 (1990), the results illustrated in Fig. 1 show that, on a mass basis, rBPI~, produced a signal which was approximately 3 orders of magnitude lower than that of rLBP~ and rLBP, while rBPI produced a signal that was approximately 5 orders of magnitude lower than that of rLBP and rLBP~. For example, a °°n~n~~on of 100,000 ng/mL (100 WgImL) of rBPI or 400 ag/mI, rBPI~, generated a signal which was equal to that produced by 0.8 ng/mL of rLBP or 0.4 nglmL of rLBPu. These results demonstrate minimal cross-reactivity of the antibody with BPI and confirm the specificity of the assay for LBP.
Ex le MEASUREMENT OF rLBP SPACED INTO
POOLED TiLTMAN PLASI1~LA
In this example, the recovery of rLBP in human blood fluids was evaluated by examining pooled human plasma spiked with different concentrations of rLBP and then frozen and thawed prior to measurement in the sandwich ELISA. Recovery of spiked LBP was defined as the amount of LBP measured in spiked human plasma samples minus the concentration in the W095/20163 , ' PC1'IUS95/00982 4. .. s a.
t V. ~.1 .'8..:

unspiked control, divided by the actual amount spiked in the sample. The fraction recovered was multiplied by 100 and the results were expressed as a percentage of the input concentration. Recovery of different concentrations of rLBP spiked into pooled human plasma samples averaged 6896 and ranged ' from 59 96 at 42 ~cgl mL to 78 9b at 168 tcgl mL. Table I summarizes the recovery data for each LBP spiked plasma sample.
TABLE I
Recovery of rLBP Spiked into Pooled Citrated Human Plasma Amount Spiked Amount Measured Amount Recovered Percent ~''g/mI') (l;g~mI-) (Ecg/mL) Recovery 0 2.47 ___ ___ 10.5 9.85 7.38 70 ~
21 16.1 13.63 659b 42 27.3 24.83 599b 84 60.8 58.33 699b 168 133 130.53 78 ~
Mean Recovery 68 96 COMPARISON OF PLASMA AND SERUM LBP LEVELS
According to this example concentrations of LBP in the serum and plasma of healthy subjects were assayed and compared utilizing the sandwich ELISA assay according to Example 3. Plasma concentrations of LBP were found to be essentially the same as serum concentrations for LBP
when the plasma volume was corrected for dilution (dividing by a factor of , 0.85) resulting from the addition of anticoagulant. Plasma concentrations in normal human subjects were found to be 3.1 ~cglmL (S.D. 0.9 tcg/mL) or 3.7 ~'g/mL (S.D. 1.1 wI/mL) corrected, compared with 3.7 ~g/mL (S.D. 0.9 ~cg/mL) for serum.

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Example 77 CLINICAL CORRELATIONS OF
ENDOGENOUS LBP INlTviIINOREACTIVITY IN HUMAN PLASMA
In this example endogenous E.BP immunoreactivity was measured in human plasma or semen samples collected from a variety of subjects suffering from gram-negative sepsis and a variety of other clinical conditions. Specifically, plasma samples of healthy individuals (30 subjects) and individuals diagnosed with gzam-negative sepsis (363 subjects) were assayed for LBP levels. Serum samples of individuals with acute lymphoblastic leukemia (ALL) (6 subjects); acute graft versus host disease (aGvHD) (8 subjects); chronic lymphocytic leukemia (CLL) (9 subjects);
cutaneous T-cell lymphoma (CTCL) (12 subjects); type 1 diabetes (13 subjects); aplastic anemia (AA) (16 subjects); Crohn's Disease (8 subjects);
psoriasis (13 subjects); rheumatoid arthritis (RA) (86 subjects); scleroderma (4 subjects), and systemic lupus er;jrthematosus (SLE) (10 subjects) were assayed for LBP levels. The results are shown in Fig. 2.
While LBP levels among subjects diagnosed as suffering from gram-negative sepsis were elevated it was found that LBP levels are not elevated over normal in subjects suffering from acute lymphoblastic leukemia, acute graft versus host disease, chronic lymphocytic leukemia, cutaneous T-cell lymphoma, type 1 diabetes, aplastic anemia, Crohn's Disease, psoriasis, rheumatoid arthritis, scleroderm,a, and systemic lupus erythematosus (SLE).
Accordingly, the LBP assay of the invention is valuabbe for distinguishing conditions associated with endotoxin from other acute phase conditions (such as RA, SLE and the like).
THE EFFECT OF LPS ADMINISTRATION ON
ENDOGENOUS LBP LEVELS IN HEALTHY SUBJECTS
In this example, the effect of LPS administration on endogenous LBP immunoreactivity in healthy human subjects was determined.

Specifically, healthy subjects were monitored utilizing the LBP sandwich assay for changes in LBP plasma levels at various time points after intravenous administration of 4 ng/kg LPS (16 subjects) or in control subjects (2) not receiving LPS. The results illustrated in Fig. 3 show the change in mean plasma LBP concentration with time. For those subjects treated with LPS LBP levels began to rise about 6 hours after LPS administration. Peak LBP plasma levels were observed in most subjects between 10 to 12 hours after the LPS
administration. The average increase from baseline to peak LBP level was approximately 3-fold. Over this time period the mean LBP levels in control subjects remained within normal range (approximately 5 Ng/mL).
It is contemplated that additional analysis will illustrate the correlation of LBP levels in body fluids with the symptoms of exposure to endotoxin and that LBP levels will be diagnostic and prognostic of disease states resulting from exposure to endotoxin.
It is contemplated that additional analysis will illustrate the correlation of LBP levels with symptoms of bacterial infections, endotoxemia and sepsis including conditions associated with sepsis including disseminated intravascular coagulation (DIC) and adult respiratory distress syndrome CARDS).
Exam~~le 9 CLINICAL CORRELATIONS BETWEEN PLASMA LBP LEVELS AND
SURVIVAL IN SUSPECTED GRAM-NEGATIVE SEPSIS PATIENTS
Correlations between plasma LBP levels and survival in suspected gram-negative sepsis patients were compared using data obtained from the septic subjects described in Example 7. In this case, a standard LBP
concentration was set at 46 Ng/mL and patients with suspected gram-negative sepsis were classified as having either high (>46 Ng/mL) or low (<46 pg/mL) LBP plasma levels as measured in pretreatment samples. As shown in the data presented in Fig. 4, those subjects having low pretreatment plasma levels of LBP had a significantly greater survival rate (p=0.004) over a 27 day period than did those subjects having a high pretreatment plasma LBP
level. These data show the utility of assaying LBP levels and comparing them W095I20163 ~ PCTIUS95/00982 t: ~'a . . , to a standard LBP value for predicting the prognosis of subjects suffering from sepsis.
Exam Ip a 10 HFAT.TFIY, RIiEUMAT0T11 ARTfiRITIC, AND SEPTIC PATIENTS
Plasma levels of LBP, C-reactive protein (CRP) and fibrinogen were measured in small groups of healthy, rheumatoid arthritic and septic patients with the results shown in Figs. 5a (LBP levels), 5b (CRP levels) and 10 Sc (fibrinogen levels). The results show that relative to healthy subjects, mean fibrinogen levels were elevated approximately 2.5 fold for both rheumatoid arthritic and septic subjects. Relative to healthy subjects, mean CRP levels were found to be elevated approx'unately 40-fold for rheumatoid arthritic subjects and 200-fold for septic subjects. In contrast, and consistent with the 15 results nn Example 7, mean LBP levels were only slightly increased (less than 2-fold) for rheumatoid arthritis subjects while the mean LBP levels were increased by more than 6 fold for septic subjects.
Numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing description of the presently preferred embodiments thereof.
Consequently, the only limitations which should be placed upon the scope of the present invention are those which appear in the appended claims.

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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: XOMA
(ii) TITLE OF INVENTION: METHOD FOR QDANTIFYING L8P IN BODY FLUIDS
(iii) NUMBER OF SEQUBNCES: 4 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSfiE: Marshall, 0'Toole, Gerstein, Murray & Borun (B) STREET: 6300 Sears Tower, 233 South blacker Drive (C) CITY: Chicago (D) STATE: Illinois (E) COUNTRY: United States of America (F) ZIP: 60606-6402 (v) COMPUTER RBADABLB FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPDTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(8) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY INFORMATION:
(A) NAME: Sharp, Jeffrey S.
(B) REGISTRATION NOMBER: 31,879 (C) REFERENCE/DOCKET NUMBER: 27129/31843 (ix) TELECOMMUNICATION INFORMATIC
(A) THLEPHONE: 312/474-6300 (BJ TELEFAX: 312/474-0448 (C) TELEX: 25-3856 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCfi CHARACTERISTICS:
(A) LENGTH: 1443 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY; CDS
(B) LOCATION: 1..1443 (ix) FEATURE:
(A) NAME/KEY: mat~eptide (8) LOCATION: 76..1443 (ix) FEATURE:
(A) NAME/KEY: mist feature WO 95!20163 PCTIUS95/00982 2181815;
~, m (D) OTH&R INFORMATION: "rLBP"
(xi) S$QDENCH D&SCRIPTION: S8~ ID NO:1:

AGA ATA TTG
GCC

Met Gly A1aLeuAla ArgAlaLeu ProSerIle LeuLeuAla LeuLeu -2s -20 -is -10 Leu Thr SerThrPro GluAlaLeu G7.yAlaAen ProGlyLeu ValAla Arg Ile ThrAspLys GlyLeuGln TyrAlaAla GlnGluGly LeuLeu to is ao Ala Leu GlnSerGlu LeuLeuArg I7.eThrLeu ProAspPhe ThrGly 25 30 3s Asp Leu ArgIlePro HisValGly ArgGlyArg TyrGluPhe HieSer 40 4s 50 s5 Leu Asn IleHisSer C~sGluLeu LeuHieSer AlaLeuArg ProVal 60 6s 70 CCT GGC CAGGGCCTG AGTCTCAGC A7.'CTCCGAC TCCTCCATC CGGGTC 336 Pro Gly GlnGlyLeu SerLeuSer I7:eSerAsp SerSerIle ArgVal 75 80 8s Gln Gly ArgTrpLys ValArgLys SerPhePhe LysLeuGln GlySer Phe Asp ValSerVal LysGlyIle SerIleSer ValAsnLeu LeuLeu lOS 110 115 Gly Ser GluSerSer GlyArgPro ThrValThr AlaSerSer CysSer 120 12s 130 13s AGT GAC ATCGCTGAC GTGGAGGTG G7aCATGTCG GGAGACTTG GGGTGG 528 Ser Asp IleAlaAsp ValGluVal AspMetSer GlyAspLeu GlyTzp CTG TTG AACCTCTTC CACAACCAG ATTGAGTCC AAGTTCCAG AAAGTA s76 Leu Leu AsnLeuPhe HisAsnGln IleGluSer LysPheGln LysVa1 lss 160 lss Leu Glu SerArgIle CysGluMet I1eGlnLys SerValSer SerAsp CTA CAG CCTTATCTC CAAACTCTG CCAG':'.'ACA ACAGAGATT GACAGT 672 Leu Gln ProTyrLeu GlnThrLeu ProVaiThr ThrGluIle AepSer TTC GCC GACATTGAT TATAGCTTA GTGGAAGfC CCTCGGGCA ACAGCC 720 Phe A1a AspIleAsp TyrSerLeu Va1GluAis ProArgAla ThrAla aoo 2os zlo 21s ATG GAA TTT AAC

Gln LeuGluVal MetPhe Lys Gly Ile His Arg His Met Glu Phe Aen CGT CCAGTTACC CTCCTT GCT GCA ATG CTf CCT GAA 816 TCT GTC AGC GAG

Arg ProValThr LeuLeu Ala Ala Met Leu Pro Glu Ser Val Ser Glu CAC AAAATGGTC TACTTT.GCC ATC GAT GTC TTC ACG 864 AAC TCG TAT AAC

His LysMetVal TyrPhe Ala Ile Asp Val Phe Thr Asn Ser Tyr Asn AGC TAT AAC ATC

Ala LeuValTyr HisGlu Glu Gly I.eu Phe Ser Thr Ser Tyr Asn Ile GAG ATC CTG AAG

Asp MetIlePro ProAsp Ser Asn Arg Thr Thr Ser Glu Ile Leu Lys CGA AGG TAC ATG

Phe ProPheVal ProArg Leu Ala Leu Pro Asn Asn Arg Arg Tyr Met GAA GCT CTC TTC

Leu LeuGlnGly SerVal Pro Ser Pro Leu Asn Ser Glu Ala Leu Phe GGG ATG ATA TTT

Pro AsnLeuSer ValAsp Pro Tyr Glu Asp Ala Val Gly Met Ile Phe CTG GTC CGG GTG

Leu ProSerSer SerLys Glu Pro Phe Leu Ser Ala Leu Val Arg Val AAT AAT AGC ACT

Thr ValSerAla ThrLeu Thr Phe Thr Lys Ile Gly Asn Asn Ser Thr CTG GAA AAA AAA

Phe LysProGly LysVal Lys Val Leu Glu Ser Val Leu Glu Lys Lys CTA GCG CTC TAC

Gly PheAenAla GluLeu Leu Glu Leu Aen Tyr Ile Leu Ala Leu Tyr CTT ACCTTCTAC .CCCAAG TTC AAT AAG GCC GAA TTC 1344 AAC GAT TTG GGC

Leu ThrPheTyr ProLys Phe Asn Lys Ala Glu Phe Asn Asp Leu Gly CTT CTC GAC CTG

Pro ProLeuLeu LysArg Val Gln Tyr Leu Gly Gln Leu Leu Asp Leu CAT GCC GTC ATG

Ile LysAspPhe LeuPhe Leu G1y Asn Gln Tyr Arg His Ala Val Met Val -~,. , , (2) INFORMATION FOR SEQ ID N0:2:
(i) SfiQUSNCfi CHARACTBRISTICS:
- (A) LHNGTH: 481 amino acids (S) TYPfi: amino acid (D) TOPOLOGY: linear (ii) MOLfiCULE TYPfi: protein ( ix) FSATORfi (A) NAMfi/KfiY: miac featu~.re (D) OTHfiR INFORMATION: "rLBP"
(xi) SfiQOBNCfi DfiSCRIPTION: SEQ ID N0:2:
Met Gly Ala Leu Ala Arg Ala Leu Pro Ser Ile Leu Leu Ala Leu Leu Leu Thr Ser Thr Pro Glu Ala Leu Gly Ala Rsn Pro Gly Leu Val Ala Arg Ile Thr Asp Lye Gly Leu Gln Tyr Ala Ala Gln Glu Gly Leu Leu Ala Leu Gln Ser Glu Leu Leu Arg Ile Thr Leu Pro Asp Phe Thr Gly Asp Leu Arg Ile Pro His Va1 Gly Arg Gly Arg Tyr Glu Phe His Ser Leu Aan Ile Hie Ser Cya Glu Leu Leu Hie Ser Ala Leu Arg Pro Val Pro Gly Gln Gly Leu Ser Leu Ser Il.e Ser Asp Ser Ser Ile Arg Val Gln Gly Rrg Trp Lys Val Arg Lys Ser Phe Phe Lys Leu Gln Gly Ser Phe Aap Val Ser Val Lye Gly Ile Ser Ile Ser Val Aan Leu Leu Leu Gly Ser Glu Ser Ser Gly Arg Pro Thr Val Thr Ala Ser Ser Cars Ser Ser Asp Ile Rla Asp Val Glu VaI Asp Met Ser G1y Asp Leu Gly Trp Leu Leu Asn Leu Phe His Asn Gln 27.e Glu Ser Lya Phe Gln Lys Val Leu Glu Ser Arg Ile Cys Glu Met I1e Gln Lys Ser Val Ser Ser Asp Leu Gln Pro Tyr Leu Gln Thr Leu Pro Va1 Thr Thr Glu Ile Asp Ser Phe Ala Asp Ile Asp Tyr Ser Leu Val Glu Ala Pro Arg Ala Thr Ala Gln Met Leu Glu Val Met Phe Lys Gly Glu Ile Phe His Arg Asn His WO 95/20163 ~ 1 8 1 8 1 5 PCTIUS95I00982 r ':1 '. 3 H
i . L
Arg Ser Pro Val Thr Leu Leu Ala Ala Val Met Ser Leu Pro Glu Glu His Asn Lye Met Val Tyr Phe Ala Ile Ser Asp Tyr Val Phe Asn Thr aso 2ss aso Ala Ser Leu Val Tyr His Glu Glu Gly Tyr Leu Asn Phe Ser Ile Thr Asp Glu Met Ile Pro Pro Asp Ser Aen Ile Arg Leu Thr Thr Lys Ser Phe Arg Pro Phe Val Pro Arg Leu Ala Arg Leu Tyr Pro Asn Met Asn 300 30s 310 Leu Glu Leu Gln Gly Ser Val Pro Ser Ala Pro Leu Leu Aen Phe Ser Pro Gly Asn Leu Ser Val Asp Pro Tyr Met Glu Ile Asp Ala Phe Val 330 33s 340 Leu Leu Pro Ser Ser Ser Lys Glu Pro Val Phe Rrg Leu Ser Val A1a 345 350 ass Thr Asn Val Ser Ala Thr Leu Thr Phe Asn Thr Ser Lys Ile Thr Gly 360 365 370 37s Phe Leu Lys Pro Gly Lys Val Lys Val Glu Leu Lys Glu Ser Lys Val 380 38s 390 Gly Leu Phe Asn Ala Glu Leu Leu Glu Ala Leu Leu Aen Tyr Tyr Ile Leu Asn Thr Phe Tyr Pro Lys Phe Asn Asp Lys Leu Ala Glu Gly Phs 410 41s 420 Pro Leu Pro Leu Leu Lys Arg Val Gln Leu Tyr Asp Leu Gly Leu Gln 425 430 43s Ile His Lys Asp Phe Leu Phe Leu Gly Ala Asn Val Gln Tyr Met Arg 440 445 450 45s Val (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQBENCE CHARACTERISTICS:
(A) LENGTH: 591 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..s91 (ix) FEATURE:
(A) NAME/KEY: misc_feature (D) OTHER INFORMATION: ~~rLBP25~~
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

AAC AGG AAG CTG
ATC
ACC

AlaAsn ProGlyLeu ValAla ArgI1eThr AspLysGly LeuGlnTyr AlaAla GlnGluGly LeuLeu AlaLeuGln SerGluLeu LeuArgIle ThrLeu ProAspPhe ThrGly AspLeuArg IleProHis ValGlyArg GlyArg TyrGluPhe HisSer LeuAenIle HisSerCye GluLeuLeu HisSer AlaLeuArg ProVal ProG1yGln GlyLeuSer LeuSerIle 65 70 75 g0 SerAsp SerSerIle ArgVal GlnG7.yArg TrpLysVal ArgLysSer PhePhe LysLeuGln GlySer PheAspVal SerValLys GlyIleSer IleSer ValAsnLeu LeuLeu GlySerGlu SerSerGly ArgProThr ValThr AlaSerSer CysSer SerAspIle AlaAspVal GluValAsp MetSer G1yAspLeu GlyTrp LeuLeuAen LeuPheHis AsnGlnIle GluSer LysPheGln LysVal LeuG7.uSer RrgIleCys GluMetIle CAGAAA TCGGTGTCC TCCGAT CTAC11GCCZ'TATCTCCAA ACTCTGCCA 576 GlnLys SerValSer SerRsp LeuGlnPro TyrLeuGln ThrLeuPro ValThr ThrGluIle (2)INFORMAT20N FOR SEQID
N0:4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 197 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/ICEY: misc feature W095120163 X1.81815 PCTIUS95100982 ~r .;
.a (D) OTHER INFORMATION: '~rLBP25"
(xi) SHQLTENCE DESCRIPTION: SEQ ID N0:4: ' Ala Asn Pro Gly Leu Val Ala Arg Ile Thr Asp Lys Gly Leu Gln Tyr Ala Ala Gln Glu Gly Leu Leu Ala Leu Gln Ser Glu Leu Leu Arg Ile Thr Leu Pro Asp Phe Thr Gly Asp Leu Arg Ile Pro His Val Gly Arg Gly Arg Tyr Glu Phe His Ser Leu Aen Ile His Ser Cys Glu Leu Leu His Ser Ala Leu Arg Pro Val Pro Gly Gln Gly Leu Ser Leu Ser Ile Ser Asp Ser Ser Ile Arg Val Gln Gly Arg Trp Lys Val Arg Lys Ser Phe Phe Lys Leu Gln Gly Ser Phe Asp Val Ser Val Lys Gly Ile Ser Ile Ser Val Asn Leu Leu Leu Gly Ser Glu Ser Ser Gly Arg Pro Thr -Val Thr Ala Ser Ser Cys Ser Ser Asp Ile Ala Asp Val Glu Val Asp Met Ser Gly Asp Leu Gly Trp Leu Leu Asn Leu Phe His Asn Gln Ile 14s iso lss lso Glu Ser Lye Phe Gln Lys Val Leu Glu Ser Arg Ile Cys Glu Met Ile Gln Lys Ser Val Ser Ser Asp Leu Gln Pro Tyr Leu Gln Thr Leu Pro Val Thr Thr Glu Ile -

Claims (23)

THE EMBODIMENTS OF THE INVENTION FOR WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for diagnosing exposure of a subject to endotoxin comprising the steps of determining the concentration of lipopolysaccharide binding protein (LBP) in a sample of body fluid from the subject and correlating the concentration of LBP with a standard indicative of the exposure to endotoxin, wherein a LBP concentration above the standard is presumptively diagnostic of the subject suffering from exposure to endotoxin, while a concentration below the standard is not.

2. The method according to Claim 1, wherein the standard is a concentration greater than the LBP concentration for said subject assayed during a period of pretreatment.

3. The method according to Claim 1, wherein the exposure to endotoxin is indicative of an infection or endotoxin disease state.

4. The method according to Claim 3, wherein the exposure to endotoxin is indicative of sepsis, including conditions associated with sepsis.

5. The method according to Claim 3, wherein an elevated level of lipopolysaccharide binding protein is indicative of greater infection and/or endotoxemia severity.

6. The method according to Claim 5, wherein an elevated level of lipopolysaccharide binding protein may be used to indicate the suitability of using antibiotics directed against gram-negative bacteria or other therapeutic agents targeted directly to endotoxin.

7. The method according to Claim 3, wherein the standard is a concentration greater than the LBP concentration for said subject assayed during a period prior to exposure to endotoxin.

8. The method according to Claim 4, wherein the standard is a concentration greater than the LBP concentration for said subject assayed during a period prior to suffering from sepsis or another condition associated with sepsis.

9. A method for determining the prognosis of a subject comprising the steps of determining the concentration of lipopolysaccharide binding protein in a sample of body fluid from the subject and correlating the concentration of lipopolysaccharide binding protein with a standard indicative of a disease state resulting from exposure to endotoxin, wherein an elevated concentration of lipopolysaccharide binding protein compared to said standard is indicative of said exposure to endotoxin and wherein the greater the concentration of lipopolysaccharide binding protein above the standard the more adverse said prognosis.

10. The method according to Claim 9, wherein the exposure to endotoxin is indicative of sepsis, for determining the prognosis of a subject comprising the steps of determining the concentration of lipopolysaccharide binding protein in a sample of body fluid from the subject and correlating the concentration of lipopolysaccharide binding protein with a standard indicative of sepsis, including conditions associated with sepsis.

11. The method according to Claim 9, wherein the standard is a concentration greater than the LBP concentration for said subject assayed during a period prior to exposure to endotoxin.

12. The method according to Claim 10, wherein the standard is a concentration greater than the LBP concentration for said subject assayed during a period prior to suffering from sepsis or another condition associated with sepsis.

13. A method for determining exposure to endotoxin of a human subject who has undergone surgery comprising the steps of determining the concentration of lipopolysaccharide binding protein (LBP) in a sample of body fluid from the subject and correlating the concentration of lipopolysaccharide binding protein with a standard indicative of the exposure to endotoxin, wherein the standard is a LBP
concentration of the subject determined prior to undergoing surgery, wherein an increase in the concentration of LBP compared to the standard is indicative of an exposure to endotoxin.

14. The method according to any one of Claims 1 to 13, wherein said sample is a blood sample.

15. The method according to any one of Claims 1 to 13, wherein said sample is a plasma or serum sample.

16. The method according to any one of Claims 1 to 15, wherein the concentration of lipopolysaccharide binding protein is determined by means of an immunoassay.

17. The method according to Claim 16, wherein the immunoassay is a sandwich immunoassay.

18. A method for screening for exposure to gram-negative bacterial endotoxin in an acute phase response in humans comprising:
(a) measuring the level of lipopolysaccharide binding protein in blood circulation of a human subject, and (b) comparing the level of lipopolysaccharide binding protein determined in step (a) to levels of lipopolysaccharide binding protein in blood circulation in normal subjects, elevated levels of lipopolysaccharide binding protein being indicative of an acute phase response involving gram-negative bacterial endotoxin.

19. The method according to Claim 18, wherein the acute phase response is associated with sepsis.

20. A method for screening for gram-negative bacterial endotoxin involvement in an acute phase response in humans comprising:
(a) contacting a blood sample from a subject exhibiting an acute phase response with a specific binding partner for lipopolysaccharide binding protein, (b) determining the level of lipopolysaccharide binding protein in said blood sample, and (c) comparing the level of lipopolysaccharide binding protein determined in step (b) to levels of lipopolysaccharide binding protein in circulation in normal subjects, elevated levels of lipopolysaccharide binding protein being indicative of an acute phase response involving gram-negative bacterial endotoxin.

21. The method according to Claim 20, wherein the acute phase response is associated with sepsis.

22. A kit for diagnosing exposure of a subject to endotoxin, said kit comprising an antibody that specifically binds lipopolysaccharide binding protein (LBP) and a standard indicative of the exposure to endotoxin, wherein LBP concentration in a sample of body fluid from the subject above said standard is presumptively diagnostic of the subject suffering from exposure to endotoxin, while a concentration below the standard is not.

23. A kit for use in the method according to claims 1 to 21, said kit comprising an antibody that specifically binds lipopolysaccharide binding protein (LBP) and instructions for use.