Patent Application: US-14662098-A

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:
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 u . s . patent application ser . no . 08 / 029 , 510 filed jun . 17 , 1993 as shown in seq id nos : 1 and 2 herein . a recombinant lbp amino - terminal fragnent is chracterizd 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 and copending u . s . patent application ser . no . 08 / 079 , 510 filed jun . 12 , 1993 the disclosure of which is incorporated herein . 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 polycelonal 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 . pat . no . 5 , 245 , 013 disclose assay methods composing binding of lbp to lps and separating the complex by a centrifugation density gradient method . as another example , geller et al ., arch . surg . 128 : 22 - 28 ( 1993 ) disclose lbp bioactivity assays in which il - 6 and tnf upregulation are measured . 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 contemplated that it could be excreted and that analysis of lbp levels in 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 elavages , 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 levels would be elevated in subjects suffering from autoimmune diseases . as one aspect of the present invention it has been found that lbp levels are not generally elevated over normal in subjects suffering from acute lymphoblastic leukemia ( all ), acute graft versus host disease ( agvhd ), chronic lymphocytic leukemia ( cll ), cutaneous t - cell lymphoma ( ctcl ), type 1 diabetes , aplastic anemia ( aa ), crohn &# 39 ; s disease , psoriasis , rheumatoid arthritis ( ra ), scleroderma , and systemic lupus erythematosus ( se ). 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 translocation of bacteria and / or endotoxin from the gut into the bloodstream 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 &# 39 ; s exposure to biologically active lps . moreover , lbp levels appear to correlate with survival in suspected gram - negative sepsis patients . for example , subjects with levels of circulating lbp below 27 . 3 μg / ml ( 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 μg / ml , those subjects having a pretreatment lbp plasma level less than 46 μg / 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 μg / 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 antie - ndotoxin 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 1 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 , rlbp 25 , rbpi and rbpi 23 . 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 . according to this example affinity purified rabbit anti - rlbp antibody was prepared . specifically , rlbp ( 20 mg ) produced according to co - owned and copending u . s . patent application ser . no . 08 / 079 , 510 filed jun . 17 , 1993 , the disclosure of which is hereby incorporated by reference 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 nacl . approximately 94 % of the rlbp was coupled to the resin . pooled antisera ( 125 ml ) from two rabbits , immunized initially with rlbp 25 produced according to the methods of u . s . patent application ser . no . 08 / 079 , 510 filed jun . 17 , 1993 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 8 . 0 . 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 . 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 , n . j .) equilibrated with pbs containing 0 . 1 % sodium azide . fifty microliters of affinity purified rabbit anti - rlbp antibody ( 2 μg / 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 μl of 1 % non - fat milk in 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 μl 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 - bsa / tween ) and 10 units / ml of sodium heparin ( sigma chemical co ., st . louis , mo .) in separate 96 - well plates . rlbp or rlbp 25 standard solutions were prepared as serial two - fold dilutions from 100 to 0 . 012 ng / ml . each replicate and dilution of the standards , samples and controls ( 50 μl ) 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 - bsa / tween and 50 μl 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 , calif .) was diluted 1 / 2000 in pbs - bsa / tween and 50 μl was added to all wells . after incubation for 15 minutes at 37 ° c ., all wells were washed 3times with wash buffer and 3 times with deionized water and the chromogenic substrate p - nitrophenylphosphate ( 1 mg / ml in 10 % diethanolamine buffer ) was added in a volume of 50 μl to all wells . color development was allowed to proceed for 1 hour at room temperature , after which 50 μl 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 , calif .). the mean absorbance at 405 nm ( a 405 ) for all samples and standards ( in triplicate ) were corrected for background by subtracting the mean a 405 of wells receiving only sample dilution buffer ( no lbp ) in the primary incubation step . a standard curve was then plotted as a 405 versus ng / ml of rlbp or rlbp 25 . the linear range was selected , a linear regression analysis was performed and concentrations were determined for samples and controls by interpolation from the standard curve . in this example , the immunoreactivity of rlbp , rlbp 25 , rbpi and bpi 23 were compared in the bpi sandwich elisa to determine possible immunologic cross - reactivity . despite considerable sequence homology between lbp and bpi ( see , e . g ., schumann et al ., science , 249 : 1429 ( 1990 ), the results illustrated in fig1 show that , on a mass basis , rbpi 23 produced a signal which was approximately 3 orders of magnitude lower than that of rlbp 25 and rlbp , while rbpi produced a signal that was approximately 5 orders of magnitude lower than that of rlbp and rlbp 25 . for example , a concentration of 100 , 000 ng / ml ( 100 μg / ml ) of rbpi or 400 ng / ml rbpi 23 generated a signal which was equal to that produced by 0 . 8 ng / ml of rlbp or 0 . 4 ng / ml of rlbp 25 . these results demonstrate minimal cross - reactivity of the antibody with bpi and confirm the specificity of the assay for lbp . 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 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 68 % and ranged from 59 % at 42 μg / ml to 78 % at 168 μg / ml . table i summarizes the recovery data for each lbp spiked plasma sample . 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 μg / ml ( s . d . 0 . 9 μg / ml ) or 3 . 7 μg / ml ( s . d . 1 . 1 μl / ml ) corrected , compared with 3 . 7 μg / ml ( s . d . 0 . 9 μg / ml ) for serum . in this example endogenous lbp immunoreactivity was measured in human plasma or serum 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 gram - 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 ); a plastic anemia ( aa ) ( 16 subjects ); crohn &# 39 ; s disease ( 8 subjects ); psoriasis ( 13 subjects ); rheumatoid arthritis ( ra ) ( 86 subjects ); scleroderma ( 4 subjects ), and systemic lupus erythematosus ( sle ) ( 10 subjects ) were assayed for lbp levels . the results are shown in fig2 . 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 &# 39 ; s disease , psoriasis , rheumatoid arthritis , scleroderma , and systemic lupus erythematosus ( sle ). accordingly , the lbp assay of the invention is valuable 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 fig3 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 μg / 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 dic and ards . 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 μg / ml and patients with suspected gram - negative sepsis were classified as having either high (& gt ; 46 μg / ml ) or low (& lt ; 46 μg / ml ) lbp plasma levels as measured in pretreatment samples . as shown in the data presented in fig4 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 to a standard lbp value for predicting the prognosis of subjects suffering from sepsis . clinical correlations of acute phase proteins in healthy , rheumatoid arthritic , 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 fig5 a ( lmp levels ), 5 b ( crp levels ) and 5 c ( 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 approximately 40 - fold for rheumatoid arthritic subjects and 200 - fold for septic subjects . in contrast , and consistent with the results in 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 . atg ggg gcc ttg gcc aga gcc ctg ccg tcc ata ctg ctg gca ttg ctg 48 ctt acg tcc acc cca gag gct ctg ggt gcc aac ccc ggc ttg gtc gcc 96 agg atc acc gac aag gga ctg cag tat gcg gcc cag gag ggg cta ttg 144 arg ile thr asp lys gly leu gln tyr ala ala gln glu gly leu leu gct ctg cag agt gag ctg ctc agg atc acg ctg cct gac ttc acc ggg 192 ala leu gln ser glu leu leu arg ile thr leu pro asp phe thr gly gac ttg agg atc ccc cac gtc ggc cgt ggg cgc tat gag ttc cac agc 240 asp leu arg ile pro his val gly arg gly arg tyr glu phe his ser ctg aac atc cac agc tgt gag ctg ctt cac tct gcg ctg agg cct gtc 288 leu asn ile his ser cys glu leu leu his ser ala leu arg pro val cct ggc cag ggc ctg agt ctc agc atc tcc gac tcc tcc atc cgg gtc 336 cag ggc agg tgg aag gtg cgc aag tca ttc ttc aaa cta cag ggc tcc 384 ttt gat gtc agt gtc aag ggc atc agc att tcg gtc aac ctc ctg ttg 432 ggc agc gag 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ctg cag agt gag ctg ctc agg atc 96 acg ctg cct gac ttc acc ggg gac ttg agg atc ccc cac gtc ggc cgt 144 ggg cgc tat gag ttc cac agc ctg aac atc cac agc tgt gag ctg ctt 192 gly arg tyr glu phe his ser leu asn ile his ser cys glu leu leu cac tct gcg ctg agg cct gtc cct ggc cag ggc ctg agt ctc agc atc 240 tcc gac tcc tcc atc cgg gtc cag ggc agg tgg aag gtg cgc aag tca 288 ttc ttc aaa cta cag ggc tcc ttt gat gtc agt gtc aag ggc atc agc 336 att tcg gtc aac ctc ctg ttg ggc agc gag tcc tcc ggg agg ccc aca 384 gtt act gcc tcc agc tgc agc agt gac atc gct gac gtg gag gtg gac 432 atg tcg gga gac ttg ggg tgg ctg ttg aac ctc ttc cac aac cag att 480 met ser gly asp leu gly trp leu leu asn leu phe his asn gln ile gag tcc aag ttc cag aaa gta ctg gag agc agg att tgc gaa atg atc 528 glu ser lys phe gln lys val leu glu ser arg ile cys glu met ile cag aaa tcg gtg tcc tcc gat cta cag cct tat ctc caa act ctg cca 576 ala asn pro gly leu val ala arg ile thr asp lys gly leu gln tyr gly arg tyr glu phe his ser 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