Patent Application: US-201013318899-A

Abstract:
the present invention is directed to a method for early , non - invasive , rapid , efficient , reliable and accurate diagnose of alzheimer &# 39 ; s disease . the present invention particularly addresses obtaining blood samples , and stabilizing platelets from healthy persons and patients with probable cognitive impairment and / or alzheimer &# 39 ; s disease ; extracting proteins from the platelets ; identifying both monomeric and oligomeric tau proteins in the platelets with at least two monoclonal antibodies against the tau proteins , quantifying the amounts of the identified tau proteins , and comparing the amounts and protein profiles of the tau molecular species in the platelets of the healthy person and the patient .

Description:
the present invention comprises a method and a kit intended for early , non - invasive , rapid , efficient , reliable and accurate diagnostic determination of a biomarker in biological samples from patients suspected susceptible or afflicted with neurodegenerative diseases or tau disorders , preferably ad . the proposed method and kit identifies a biomarker that corresponds to altered tau protein , preferably a heavy variant of the tau protein , which has a molecular weight of approximately 140 kda present in biological samples . this protein is found in biological fluids , preferably blood . in one form the invention comprises a method and a kit allowing the detection of heavy variant of the tau protein with a molecular weight of approximately 140 kda in platelets . the method of the invention comprises the identification of tau proteins in platelets with at least two monoclonal antibodies to tau , where at least one of them is directed to hyperphosphorylated variants of tau protein . the method of diagnosis includes obtaining and stabilizing platelets , extracting the proteins , then the analysis of platelet proteins electrophoresis pattern in 1 - d and 2 - d gels and immunoassay . preferred immunoassay is performed with antibodies against different variants of tau ( total tau , hyperphosphorylated tau or heavy type tau ), for example tau5 antibodies , at8 and phf1 . the proposed diagnostic method involves collection of blood samples in tubes with anticoagulants regularly used in the state of the art , preferably k2edta separating the platelets by differential centrifugation , stabilization of platelets and the extraction of proteins in the presence of proteinase inhibitors , preferably by the addition of cold ripa and a mixture of proteinase inhibitors , then centrifugation at 1500 g for at least 10 minutes at 4 ° c . the concentration of proteins will be determined by the method of bradford . optionally , the sample can be frozen in tubes at about − 80 ° c . without preservatives . the proteins are separated by sds - page polyacrylamide gels in 10 % gels and then bi - dimensional ( 2d ) to separate the variants of tau . proteins are transferred to a nitrocellulose membrane and analyzed by immunodetection ( western blot ) with an antibody that recognizes total tau ( tau5 ) and at8 and phf1 antibodies for hyperphosphorylated tau variants . then , a registration method , preferably chemiluminescence with a horseradish ( hrp ) conjugated antibody against the first antibody is used . five different groups of subjects older than 61 years were recruited , which showed the following characteristics : 1 ) mild alzheimer ; 2 ) moderate alzheimer ; 3 ) advanced alzheimer , 4 ) mild cognitive impairment ( mci ) and 5 ) control subjects . the subjects underwent a multi - stage procedure , with criteria including i ) aged 65 - 75 years , ii ) patients with alzheimer &# 39 ; s disease must comply nincds - adrda criteria ( dubois et al , 2007 ) and the mci group agreed with the approach of peterson ( maccioni et al , 2006 ), iii ) be free of medical illnesses , neurological or psychiatric that may affect participation in the study or treatment outcome , and iv ) be willing to participate after informed consent procedure of the study . the evaluation included brain imaging using computerized axial tomography or mri , to exclude vascular lesions of the brain , neoplasms , subdural hematomas and other conditions that could explain the dementia or interfere with the study . at the time of recruitment , patients were grouped by gender , educational level and demographic aspects . the control subjects were selected with a distribution of gender and similar age . the subjects of the study were from a region where 41 % are amerindian ancestry and 59 % of caucasian ancestry ( santiago , chile ). the sample size ( n ) was determined using the program g * power 3 . 0 . 10 ( franz faul , universität kiel , germany , http :// www . psycho . uni - duesseldorf . de / abteilungen / aap / gpower3 /) with an f = 0 . 25 ( cohen , 1988 ), a power of 0 . 95 and a significance of 0 . 05 . we calculated a sample size of 305 subjects , with 61 subjects per group . the subjects in the study underwent a neuropsychological assessment . the neuropsychological evaluation consisted in the examination of folstein and colleagues minimental state ( mmse ), neuropsychiatric inventory ( npi ) and alzheimer &# 39 ; s disease assessment scale ( adas - cog ). statistical comparisons of age , education and cognitive measures were performed using one - way analysis of variance ( anova ) with each measure as the dependent variable comparing the groups . comparisons of pair - wise between adjacent groups were made by tukey &# 39 ; s honestly significant difference at a significance level set at 0 . 05 . blood samples ( 5 ml per patient ) were obtained by venous puncture early in the morning after 8 - 10 hours of fasting . platelets were immediately separated . a portion of each sample was preserved in polypropylene tubes without preservative , frozen in dry ice and stored at − 80 ° c . platelets separated from fresh blood samples under conditions to prevent their activation . venous blood was extracted into edta tubes ( bd vacutainer k2e , 10 . 8 mg ) and kept at room temperature for processing during the day . platelets were separated by differential centrifugation according to rao ( 1988 ) with modifications involving spin at room temperature . the first centrifugation was run at 200 g for 10 minutes to obtain platelet - rich plasma , which is then centrifuged at 200 g for 10 minutes to remove the contamination of red blood cells and lymphocytes . the third stage centrifugation runs at 1600 g for 10 minutes to obtain a platelet pellet . platelet poor plasma was removed and platelet pellet was carefully resuspended in 0 . 83 % nh4cl at room temperature to lyse red blood cells remaining . the following centrifugation is performed at 1500 g for 10 minutes and supernatant was extracted . the platelet pellet was washed twice with phosphate buffer solution ( pbs ) and again centrifuged at 1500 g for 10 minutes . the purity of separated platelets was determined by romanowsky staining platelets were kept on ice to prevent proteolysis . platelet proteins were extracted by the addition of cold ripa and a mixture of proteinase inhibitors , then centrifuged at 1500 × g for 10 minutes at 4 ° c . the concentration of protein is determined by the method of bradford . electrophoresis was run at 1 and 2 dimensions . it ran a denaturant polyacrylamide gel ( sds - page ) according to the method of laemmli ( laemmli , 1970 ). a total of 100 mg of platelets protein was charged per well on 10 % acrylamide gels . the gel electrophoresis was performed for 2 - dimensional analysis of tau variants according to o &# 39 ; farrell ( 1975 ). a total of 200 mg of platelet protein was loaded per gel . strips of immobilized ph gradients biorad ( ph 3 - 10 ) was used for separation of proteins in the first dimension and 10 % sds - page was performed for the second dimension . the proteins separated in 1d and 2d gels were transferred to nitrocellulose membranes . each membrane was blocked with 5 % non fat dry milk . the immobilized proteins were tested with specific antibodies against tau protein such as tau5 to identify tau variants , at8 and phf1 to identify hyperphosphorylated tau variants and anti human β - actin as protein load control . all antibodies were tested in the same membrane , one by one , followed by membrane antibody stripping and re - test . an hrp conjugated antibody was used as second antibody . the visualization of antigens was performed by chemiluminescence using a kit of reagents for ecl western blotting detection ( amersham ) according to manufacturer &# 39 ; s instructions . the luminescence was recorded on film detection tcl . the protein bands of the film were scanned and digitized to quantify the intensity using the program image 1 . 40 ( nih ). the intensity of the band of protein of interest was divided by the intensity of the band that represents the control ( β - actin ) to calculate the relative amount ( maccioni et al , 2006 ). the results were plotted and the difference between normal subjects and alzheimer patients is shown in fig1 and 2 . it is appreciated that in healthy persons there is no difference in the profile of tau at high or low molecular weight , whereas in alzheimer &# 39 ; s patients the difference is significant and clear . example of the platelet biomarker kit for early diagnosis of alzheimer &# 39 ; s disease considering that we have developed the technology to generate a non - invasive blood biomarker tool for early diagnosis of alzheimer &# 39 ; s disease by analysis of tau protein variants in the platelets , using immunoblot techniques and densitometric analysis , we propose to carry out the packaging of the materials needed for this type of analysis , along with detailed instructions for performing all the steps required . this will be introduced in the form of a kit based on the technology of this patent document , intended for use in clinical laboratory . the proposed kit corresponds to a group of materials and solutions , which in turn are divided into separate , properly identified sets of supplies corresponding to the elements needed to perform each sequential step required for the described technology , which can be detailed as follows : a ) collection and processing of peripheral venous blood samples for platelet isolation and extraction of total platelet proteins b ) electrophoresis of total proteins in acrylamide / bisacrylamide gels and transfer to nitrocellulose membranes c ) immunoblots with specific antibodies against tau protein d ) scanning and densitometric analysis of platelets tau below is a sample of the instructions provided and the steps necessary for implementing the kit . a 5 cc sample of venous blood is obtained in a tube with k2edta ( bd vacutainer ™) the sample stored at room temperature should be processed as soon as possible ( within one hour for best results ). the sample is centrifuged in a clinical centrifuge at 200 g for 10 minutes at room temperature and a supernatant platelet - rich plasma ( prp ) is obtained . the rest of the sample is discarded and the prp is centrifuged at 1600 g for 10 minutes to obtain a platelets pellet . 150 μl of radioimmunoprecipitation buffer ( ripa ) and 3 μl of protease inhibitor cocktail are added and the pellet is resuspended . the mix is centrifuged at 1500 g for 10 minutes and the pellet is discarded . the samples ( supernatant ) can be stored at − 20 ° c . until further analysis . platelets total protein separation is performed in a minigel according to the technique of laemmli ( laemmli , 1970 ). 50 mcg of the sample protein are loaded in a 10 % polyacrylamide - bisacrylamide gel and electrophoresis is performed under denaturing conditions ( sds - page ) at 100 mv for 90 minutes . the sample is transferred to a nitrocellulose membrane in a tank for an hour at 100 mv . the membranes are blocked with 3 % bovine serum albumin ( bsa ) and then incubated with the antibody tau 5 ( 2 μg / ml ), and with a secondary hrp - conjugated anti mouse antibody to recognize the tau protein variants . a chemiluminescence reaction is carried out with supersignal west femto maximum sensitivity substrate ( thermo scientific ). the signal is detected on photographic plate , digitized in a scanner and the intensities of the bands are analyzed with imagej 1 . 40 software ( national institutes of health , usa ). the ratio between tau forms of high molecular weight & gt ; 70 kda and forms of & lt ; 70 kda is evaluated ( this ratio is increased in patients with alzheimer &# 39 ; s disease ). the kit is intended for the analysis of 18 samples and includes : 25 vacutainer ™ edta tubes 2 ml eppendorf tubes for molecular biology neptune ™ plastics ( 80 tubes ) a bottle with 10 ml 10 × red blood cells lysis buffer ( 1 . 5 m nh 4 cl , 100 mm nahco 3 , 10 mm na 2 edta ). 10 ml ripa solution ( 5 . 0 mm tris - hcl ph 7 . 5 , 1 . 5 mm nacl , 10 % np - 40 , 10 % deoxycholate , 20 mm edta ph 8 . 0 , 500 mm naf , 1 % sds ) 60 ul protease inhibitors cocktail ( sigma ) two 10 % acrylamide / bisacrylamide gels ( 0 . 015 % sds ) 1 ml 5 × loading buffer ( 60 mm tris - hcl ph 6 . 8 , 25 % glycerol , 14 . 4 mm 2 - mercapto ethanol , 0 . 1 % bromophenol blue , 2 % sds ) 2 eppendorf tubes with 10 μl of molecular weight marker 10 to 250 kda ( pageruler ™ plus prestained protein ladder from fermentas ) 1 package to reconstitute 2 liters of run buffer ( 0 . 39 m glycine , 1 . 18 m tris base ) sds ( 2 ml ) to reconstitute 20 ml of 10 % sds technical grade methanol ( 200 ml ) a package to reconstitute 2 liters of phosphate buffered saline ( pbs ) ( 270 mm nacl , 5 . 37 mm kcl , 10 . 14 mm na 2 hpo 4 , 1 . 76 mm kh 2 po 4 ) 2 nitrocellulose membranes of 8 × 6 cm , ( amersham biosciences hybond ™ - c ) package with 0 . 5 ml of tween 20 to reconstitute 1 liter of pbs - tween0 . 05 % a bottle containing 100 ml of 3 % bsa in pbs ( blocking solution ) a bottle containing 50 ml of 2 ug / ml tau 5 antibody and 1 % bsa in pbs ( antibody solution 1 ) a bottle containing 100 ml of hrp - conjugated anti mouse antibody 0 . 05 ug / ml ( santacruz biotechnology ) and 1 % bsa in pbs ( antibody solution 2 ) chemiluminescense kit supersignal west femto maximum sensitivity substrate ™ ( thermo scientific ) divided in two bottles of 10 ml each container for running acrylamide gels ( eg biorad mini protean iii ™) power source capable of generating at least 100 mv equipment for transfer from gels to nitrocellulose membranes photographic films solutions for developing and fixing photographic films digitizing equipment ( scanner or digital camera ) software for densitometric analysis ( eg imagej http :// rsbweb . nih . gov / ij / index . html ) boxer a l , rabinovici g d , kepe v , goldman j , furst a j , huang s c , baker s l , o &# 39 ; 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