Patent Application: US-85532504-A

Abstract:
the inventive subject matter relates to a competitive method for the diagnosis of latent infectious disease , such as mycobacterium tuberculosis , by estimating , the concentration of cytokine , such as interferon - gamma produced by stimulated immune cells , collected from whole blood , by fluorescence polarization , fluorescence resonance energy transfer or fluorescence lifetime due to antibody - cytokine interaction or by dimerization of the cytokine .

Description:
fluorescent polarization holds great promise as an assay method for frequent , routine , cost - effective means of obtaining quantitation of cytokines and detection of immune cells isolated from blood from potentially infected patients . fluorescent molecules emit polarized fluorescence when they absorb polarized light at a specific wavelength . however , inherent in molecules in solution is their tendency to rotate . when polarized light strikes the molecules in solution , the emitted light does not remain polarized because the molecule is rotating rapidly in solution . the rate of rotation , however , is dependent on the size of the molecule . therefore , an antigen / antibody complex will have an inherently slower rate of rotation causing more of the polarized fluorescence to be emitted in the same plane as the incident light . the polarization - based readouts are less susceptible to environmental interferences , such as ph , cloudiness , and particles in the assay compartment than other light - based assays . fp is fundamentally and theoretically different from most color based photometric techniques and gains its analytical advantage by this intrinsic property of being independent of concentration . the current application utilizes fp technology in a competitive assay to quantitate infection induced cytokines or immune cells in patient blood samples for the diagnosis of infectious diseases such as tuberculosis . it is known that interferon - gamma ( ifn - γ ) plays an important role in the immune response to mycobacterium tuberculosis , as well as other bacterial infectious organisms . therefore , detection of induction ifn - γ in serum or plasma produced by immune cells from infected patients would be an important diagnostic tool . detection can be obtained by either direct measure of interferon - gamma using specific antibody in a competitive fluorescent polarization immunoassay ( cfipa ) or by detection of homodimerization of interferon - gamma by dimerization induced fluorescence polarization ( difp ). in either case , detection and quantitation will be down to less than 11 pg / ml . competitive fp assay for measuring interferon - gamma in serum or plasma by competitive assay using interferon - gamma specific antibody in the current application we disclose a competitive fluorescence polarization immunoassay ( cfpia ) that can measure ifn - γ concentrations in a fraction of the time required for elisa . to measure ifn - γ concentrations , the assay will be conducted as illustrated , generally , in fig1 . reagents include : 1 ) the use of fluorescently labeled ifn - γ which can be made from commercially available reagents , and 2 ) the use of a monoclonal ifn - γ specific antibody . the labeled ifn - γ will produce polarized fluorescence if appropriately aligned when excited by polarized light ( of the appropriate wavelength ). however , only a small portion of the fluorescence will be captured by the polarization detector because ifn - γ is a small molecule that changes its orientation rapidly and becomes depolarized . encumbering the ifn - γ , for example by attaching an antibody , will increase the amount of fluorescence detected . the subsequent addition of unlabeled ifn - γ will decrease the amount of polarized fluorescence detected proportionate to the amount of unlabeled ifn - γ added . the assay is conducted by the following steps : a . collecting whole blood samples ( with anti - clotting agent , e . g . heparin ) from patients suspected of viral , protozoa or bacterial infection , such as m . tuberculosis and containing potential immune ( i . e . primed ) t cells ; b . adding viral , protozoa or bacterial antigen ( such as m . tuberculosis antigen ) to whole blood sample ; c . incubate the whole blood / antigen culture mixture as long as overnight ( the immune ( i . e . primed ) cells are induced to produce cytokine by their recognition of specific antigen in step ( b )); d . collect serum or plasma fractions and prepare dilutions . include in the sample preparation a standard curve of serum with known concentrations of cytokine ( interferon - gamma ) or specific fragments of interferon - gamma ; e . add to the serum or plasma dilutions interferon - gamma fluorescently labeled probe at about 1 nm ; f . add antibody ( polyclonal or monoclonal ) specific to interferon - gamma to each of the dilutions at appropriate antibody dilution ; g . measure the change in fluorescence polarization of the serum dilutions ; h . graphically compare the concentration of test sample dilutions to standard curve to determine concentration of cytokine ( interferon - gamma ). the assay mixture would require 0 . 01 ml plasma ( 20 pg / ml ifn - γ , or about 2 pm ) up to 0 . 1 ml plasma . the intrinsic blood plasma or serum fluorescence can be suppressed by the addition of fluorescent quenchers , if necessary . competitive fp assay for measuring ifn - γ in serum or plasma by competitive assay measuring change in fluorescence polarization due to homodimerization ifn - γ is a 21 to 24 kda protein , synthesized by t cells and natural killer cells , that quickly tends to form homodimers in solution . the concentration of interferon - gamma can be detected , therefore , by the concentration of interferon - gamma can be detected by the change in fluorescence polarization induced by dimerization ( difp ). the assay is conducted by the following steps : a . collecting whole blood samples from patients suspected of viral , protozoa or bacterial infection , such as m . tuberculosis and containing potential immune t cells ; b . adding viral , protozoa or bacterial antigen ( such as m . tuberculosis antigen ) to whole blood sample ; c . incubate the whole blood / antigen culture mixture as long as overnight ; d . collect serum or plasma fractions and prepare dilutions . include in the sample preparation a standard curve of serum with known concentrations ifn - γ ; e . add to the serum or plasma dilutions ifn - γ fluorescently labeled probe at about 1 nm . alternatively , peptide fragments , that are fluorescently labeled can be used as a probe ; f . measure the change in fluorescence polarization of the serum dilutions due to dimerization ; g . graphically compare the concentration of test sample dilutions to standard curve to determine concentration of cytokine ( ifn - γ ). a further aspect of the invention is the detection and quantitation by fluorescence polarization , fluorescence life - time ( flt ) and fluorescence resonance energy transfer ( fret ). if flt is used , then detection is dependent on a change in fluorescence life - time . if fret is used , then detection is by sensitized fluorescence of the acceptor or by quenching of donor fluorescence or by fluorescence depolarization . a still further aspect of the invention is that different fluorochromes , added to the cytokine ( e . g . interferon - gamma or specific m . tuberculosis probes ), can be utilized in order to optimize results , including the incorporation of ph - independent fluorochromes . the fluorochromes that are included as an aspect of the invention include : 7 - aad , acridine orange , alexa 488 , alexa 532 , alexa 546 , alexa 568 , alexa 594 , aminonapthalene , benzoxadiazole , bodipy 493 / 504 , bodipy 505 / 515 , bodipy 576 / 589 , bodipy fl , bodipy tmr , bodipy tr , carboxytetramethylrhodamine , cascade blue , a coumarin , cy2 , cy3 , cy5 , cy9 , dansyl chloride , dapi , eosin , erythrosin , ethidium homodimer ii , ethidium bromide , fluorescamine , fluorescein , ftc , gfp ( yellow shifted mutants t203y , t203f , s65g / s72a ), hoechst 33242 , hoechst 33258 , iaedans , an indopyras dye , a lanthanide chelate , a lanthanide cryptate , lissamine rhodamine , lucifer yellow , maleimide , mant , mqae , nbd , oregon green 488 , oregon green 514 , oregon green 500 , phycoerythrin , a porphyrin , propidium iodide , pyrene , pyrene butyrate , pyrene maleimide , pyridyloxazole , rhodamine 123 , rhodamine 6g , rhodamine green , spq , texas red , tm , toto - 1 , tritc , yoyo - 1 , vitamin b12 , flavin - adenine dinucleotide , and nicotinamide - adenine dinucleotide . 1 . diagnostic standards and classification of tuberculosis in adults and children . this official statement of the american thoracic society and the centers for disease control and prevention was adopted by the ats board of directors , july 1999 . this statement was endorsed by the council of the infectious disease society of america , september 1999 . am j respir crit care med apr 2000 ; 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