Patent Application: US-90473710-A

Abstract:
the present invention relates to a method of detecting the presence of cancer by measuring the level of enzyme activity and autoantibodies in the blood of an individual . in particular the present invention relates to methods for measurement of activated camp - dependent protein kinase a activity and antibodies to pka , a kit for activated pka activity measurement , and the use of the measured levels of these analytes for determining the presence of cancer .

Description:
serum samples from patients with early and late - stage cancer were obtained from promeddx , llc . in one embodiment blood samples from prostate cancer patients and normal controls presumably without cancer were assayed for activated pka activity . in this assay activated pka in samples was mixed with a defined peptide used as a substrate . phosphorylation of the peptide was detected using biotinylated phosphoserine antibody , which was in turn was detected in an elisa format using peroxidase - conjugated to streptavidin . detection of the bound peroxidase was established using a color - producing peroxidase substrate . bovine pka catalytic unit was used at varying concentrations to develop a standard activity curve . the detail of the assay protocol is described below . a . mesacup protein kinase assay kit ( mbl code no . 5230 ) b . atp : 10 mm in water i . dissolve 60 mg atp ( sigma prod . no . a2383 ) in 1 . 0 ml water ii . determine the absorbance of a 1 / 1000 dilution in pbs at 259 nm iii . store at − 20 ° c . iv . immediately before use dilute to 10 mm based on the absorbance and the molar extinction coefficient ( e259 , ph 7 = 15 , 400 ) c . pki inhibitor : 0 . 5 mm in water ( santa cruz prod . no . sc - 201160 ) i . dissolve 1 mg in 1 . 0 ml water ii . store at − 20 ° c . d . pka diluent : 25 mm kh2po4 , 5 mm edta , 150 mm nacl , 50 % ( w / v ) glycerol , 1 mg / ml bsa , 5 mm β - mercaptoethanol , ph 6 . 5 e . pka catalytic subunit standard : i . dissolve bovine pka ( sigma prod . no . p2645 ) in cold pka diluent to a final concentration of 1 μg / ml ii . store at − 20 ° c . i . thaw serum samples ii . centrifuge 10 minutes at 16 , 000 × g iii . collect the clear supernatant iv . mix 0 . 0108 ml supernatant with 0 . 0012 ml diluent in a dilution plate , two wells per sample v . incubate one hour at room temperature i . prepare serial ½ dilutions of pka 20 - 0 . 4 ng / ml in pka diluent ii . dispense 0 . 012 ml per well of a dilution plate i . 25 mm tris - hcl , ph 7 . 0 ii . 3 mm mgcl2 iii . 1 mm atp iv . 0 or 0 . 5 um pki d . add 0 . 108 ml reaction buffer ( with or without pki ) to each sample or calibrator well of the dilution plate e . pre - incubate five minutes at 25 ° c . f . transfer 0 . 100 ml per well to assay plate g . incubate 20 minutes at 25 ° c . with shaking at 750 rpm h . add 0 . 100 ml kit stop solution per well i . wash three times with kit wash buffer j . add 0 . 100 ml kit biotinylated anti - phosphoserine per well k . incubate 60 minutes at 25 ° c . with shaking at 750 rpm l . wash three times with kit wash buffer m . add 0 . 100 ml kit peroxidase - conjugated streptavidin per well n . incubate 60 minutes at 25 ° c . with shaking at 750 rpm o . wash three times with kit wash buffer p . add 0 . 100 ml peroxidase substrate solution per well q . incubate 60 minutes at 25 ° c . with shaking at 750 rpm r . add 0 . 100 ml kit stop solution per well s . shake briefly until well mixed t . read absorbance at 450 nm a . plot absorbance versus concentration of the calibration curve b . perform a least squares linear regression on the data to determine the slope and intercept c . calculate kinase concentration in the samples the activity levels of activated pka in blood from the prostate cancer patients were lower ( below 4 ng / ml ) compared to those for samples from age and sex - matched controls ( fig1 ). blood samples from breast cancer patients and from age and sex - matched normal controls presumably without cancer were analyzed for activated pka activity using the same protocol as used in embodiment 1 . the activity levels of activated pka in blood from breast cancer patients were lower ( below 4 ng / ml ) than those for samples from the normal controls ( fig2 ). the same prostate cancer patient samples and related control samples used in embodiment 1 were tested for anti - pka antibodies as described below . a . maxisorp 96 well polystyrene plates ( nunc prod . no . 439454 ) b . pka diluent : 25 mm kh 2p04 , 5 mm edta , 150 mm nacl , 50 % ( w / v ) glycerol , 1 mg / ml bsa , 5 mm β - mercaptoethanol , ph 6 . 5 c . protein kinase a ( sigma prod . no . p2645 ) i . dissolve in pka diluent and dilute to 50 μg / ml ii . store at − 20 ° c . d . coating buffer : 10 mm sodium phosphate , 150 mm sodium chloride , ph 7 . 4 e . coating wash buffer : 20 mm hepes , 150 mm sodium chloride , 30 mm sucrose , ph 7 . 0 with 0 . 1 % bsa f . blocker casein ( pierce prod . no . 37532 ) g . assay buffer : 10 mm sodium phosphate , 150 mm sodium chloride , ph 7 . 4 with 0 . 25 % bsa and 0 . 1 % tween 20 h . assay wash buffer : 10 mm citrate , 150 mm sodium chloride , ph 5 . 1 with 0 . 1 % tween 20 i . detection antibody : peroxidase - conjugated donkey anti - human igg ( h + l ) ( jackson prod . no . 709 - 035 - 149 ), 0 . 8 mg / ml in 10 mm sodium phosphate , 250 mm sodium chloride , ph 7 . 6 with 15 mg / ml bovine serum albumin j . stop solution : 0 . 2 m h2s04 in water k . hrp substrate solution ( sigma prod . no . t8665 ) i . dilute pka to 0 . 5 μg / ml in coating buffer (+ pka wells ) ii . dilute pka diluent 1 / 100 in coating buffer (− pka wells ) iii . add 0 . 100 ml either + pka or − pka solution per well iv . seal and incubate overnight at 4 ° c . v . aspirate to remove the coating solutions vi . add 0 . 200 ml blocking solution per well vii . incubate one hour at room temperature viii . wash three times with coating wash buffer ix . use immediately i . thaw serum samples ii . centrifuge 10 minutes at 16 , 000 × g iii . collect the clear supernatant iv . dilute 1 / 500 in assay buffer c . add 0 . 100 ml diluted sample each to the + pka and − pka wells d . incubate two hours at 25 ° c . with shaking at 750 rpm e . wash three times with assay wash buffer f . dilute the detection antibody 1 / 20 , 000 in assay buffer g . add 0 . 100 ml per well h . incubate one hour at 25 ° c . with shaking at 750 rpm i . wash three times with assay wash buffer j . add 0 . 100 ml hrp substrate solution per well k . incubate 20 minutes at 25 ° c . with shaking at 750 rpm l . add 0 . 100 ml stop solution per well m . shake briefly until well mixed n . read absorbance at 450 nm the levels of anti - pka antibodies in the prostate cancer patient serum relative to the levels of activated pka activity ( anti - pka antibody / activated pka activity ) were higher than those for samples from age and sex - matched controls ( fig3 ). the activated pka activities for these samples also were low relative to those of matched controls ( fig1 ). these observations using the same samples used in embodiment 1 indicate that the results of both assays together can be used to detect the presence of cancer . in this embodiment blood samples from 24 patients with prostate , breast , colon , or lung cancer and 24 age - and sex - matched normal controls presumably without cancer were assayed for activated pka activity . twenty of twenty - four cancer patients had low activated pka activity , while twenty of twenty - four normal controls had high activity . the calculated sensitivity of the assay was 0 . 83 and the assay specificity was 0 . 83 . receiver operating characteristic analysis of the results indicated that there was a 0 . 833 correlation of low activated pka activity with the presence of cancer at a cutoff value of 3 . 8 ng / ml of activated pka activity ( fig4 ). the measurement of the levels of activated pka activity can be used to determine the presence of cancer in individuals . in addition the measurement of levels of activated pka activity and anti - 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