Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44704—Details; Accessories
  • G01N27/44743—Introducing samples
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44704—Details; Accessories
  • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
  • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
  • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
  • G01N33/561—Immunoelectrophoresis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

• This invention is in the field of detecting analytes using agents which specifically bind to the analyte to form complexes, such as antigen/antibody complexes.
• U.S. Patent No. 5,006,473, Bouma shows the migration of an alkaline phosphatase labeled antibody in a liposome embedded electrophoresis media. After elec- trophoresis, the liposome is lysed and a staining dye or reactant is released.
• U.S. Patent Nos. 4,205,058 and 4,301,139 describe a chromatography column which separates antigen and antigen/antibody labeled complexes and the reaction is determined by measuring a radio-labeled antigen which migrates on the column.
• T4 I 125 and anti- T4 are reacted and separated on a cross-linked polyvinyl alcohol column.
• the antibody/T4 complex is retained at the bottom of the column and the T4 I 125 migrates.
• These patents represent an example of the direct binding of a labeled hapten T4 I 125 and antibody and separation of these complexes.
• a DNA sequencing system using a multiple lane electrophoresis apparatus. Fluorescent dyes are attached to molecules moving through the lanes. A moving illumina ⁇ tion and detection system scans the multiple lanes. Four color data points are recorded for each of several lanes at a particular time at a fixed distance down the gel. Through a complex analytic procedure, the four colors are related to the concentrations of four dye-labeled DNA components. The object is to identify concentrations of A, C, G, or T which are DNA piece endings where
• A adenosine
• C cytosine
• G guanine
• T thymine. Peak concentrations of a particular dye label are matched with particular bases in DNA sequences.
• a novel type of electrophoresis is described.
• One lane of the gel medium is impregnated with immobilized ligands capable of reacting with a migrating macromolecule, while another lane, a control gel, is untreated.
• a comparison can be made, using electrophoresis, between a macromolecule sample retarded by the affinity gel lane and a similar sample in the control gel lane.
• the gel may incorporate an antibody which interacts with a migrating antigen.
• the two lanes may be calibrated so that different degrees of retardation, for different concentrations of the migrating macromolecule, are known.
• microscopic beads treated with ligands can be entrapped in the gel and similarly serve as a retardant. Beads have the advantage of tight packing in the gel if they are of appropriate size. Activation of the gel involves partial cross- linking so that the gels do not melt on heating. Alter ⁇ native methods of gel preparation are described, all with the result that a macromolecular retardant is immobi ⁇ lized. Electrophoresis proceeds in the usual way. Various types of pulsed electrophoresis are known for use in separating closely related substances (see Kreger EPA 457,748; Slater EPA 395,319, Agawa EPA 320,937; and Allington EPA 396,053).
• This invention relates to methods and test kits for detecting analytes, in a milieu of substances of closely related weights or mobilities, which specifically bind to a labeled binding reagent to form a complex.
• the binding agent is labeled with a detectable marker so that binding between the labeled binding agent and analyte provides a reaction mixture which contains a labeled binding agent and a complex of analyte and labeled binding agent.
• These two labeled substances are placed on a separation medium and the differential rate of migration of the labeled specific binding agent and complex are determined by detecting the label in each as they are being separated on the separation medium. This difference in migration identifies the target analytes.
• a second labeled marker is included which migrates different from the labeled binding agent and complex and most preferably migrates faster than the complex or the labeled binding agent.
• the invention encompasses a method for detecting an analyte in a test sample compris ⁇ ing:
• the analyte may be an antigen and the labeled binding agent a fluorescently labeled anti- body or fragment of an antibody such as an Fab fragment.
• the labeled binding agent and complex are preferably separated by electrophoresis and the label is detected as each migrates past a fixed point in the electrophoresis medium. More particularly, the invention also encom ⁇ passes a method for measuring the concentration of an analyte in a test sample moving in a single lane comprising:
• a preferred assay utilizes a second labeled marker which migrates independent of the labeled binding agent and complex.
• this second labeled marker migrates first in the medium and serves as a quality control check on the system. For example, if the separation media or other reagent in the test kit are not operative, the failure of the second labeled marker to migrate as expected can be readily detected and the reli ⁇ ability of any results from that assay can be discarded. Also, if the monoclonal antibody or immune complex peak fails to appear in the proper relationship to the second labeled marker, this test may be discarded as faulty.
• test kits which contain an analyte standard, a labeled binding agent which specifically binds to the analyte to form a complex, and a separation medium which separates the la ⁇ beled binding agent and complex.
• Such test kits prefera ⁇ bly contain a second labeled marker which migrates inde ⁇ pendent of the labeled binding agent and complex.
• the separation medium is preferably in the form of a modular cartridge having a plurality of electrophoresis lanes as shown in Figure 11a.
• Test kits for detecting an antibody comprise a hapten conjugated labeled carrier, separation medium, and preferably a second labeled marker.
• a test kit for detecting haptens comprises a hapten conjugated labeled carrier, a standard sample of the hapten, and an antibody to the hapten.
• the present invention takes advantage of the specificity of reaction of specific binding mole ⁇ cules, the ability of separation media to separate small amounts of materials, the sensitivity of detecting labels such as fluorescent labels and a quality control agent to provide rapid, sensitive, and reliable quantitative results.
• the methods, test kits, and apparatus of this invention are most advantageously applied to closely related and difficult to separate complexes of closely related molecules such as complexes of isoforms of similar proteins.
• the term "closely related” means not only heavy molecules of close molecular weight, but also molecules whose charge/mass ratio or other characteristic is such that both molecules exhibit similar migration rates, e.g., using electrophoresis, so that previous separation efforts have been difficult.
• analyte refers to a large variety of chemical substances for which there is a specific binding partner.
• the present assay may be applied to the detection of any analyte for which there is a specific binding partner.
• the analyte usually is a peptide, protein, carbohydrate, glycoprotein, ster ⁇ oid, or other organic molecule for which a specific bind ⁇ ing partner exists in biological systems or can be syn ⁇ thesized.
• the analyte in functional terms, is usually selected from the group consisting of antigens and antibodies thereto; haptens and antibodies thereto; and hormones, vitamins, metabolites and pharmacological agents, and their receptors and binding substances.
• analytes immunologically-active polypep- tides and proteins of molecular weights between 1,000 and 4,000,000, such as antibodies and antigenic polypeptides and proteins, as well as haptens of molecular weights between 100 and 1,500.
• antigenic polypeptides are angiotensin I and II, C-peptide, oxyto- cin, vasopressin, neurophysin, gastrin, secretin, and glucagon.
• antigenic proteins are insu- lin, chorionic gonadotropin (e.g., HCG) , carcinoembryonic antigen (CEA) , myoglobin, hemoglobin, follicle stimulat ⁇ ing hormone, human growth hormone, thyroid stimulating hormone (TSH) , human placental lactogen, thyroxine bind ⁇ ing globulin (TBG) , intrinsic factor, transcobalamin, enzymes such as alkaline phosphatase and lactic dehy- drogenase, and hepatitis, HTLV-III, influenza, herpes, and other viral associated antigens.
• HCG chorionic gonadotropin
• CEA carcinoembryonic antigen
• myoglobin e.g., hemoglobin
• follicle stimulat ⁇ ing hormone human growth hormone
• TSH thyroid stimulating hormone
• TSG thyroxine bind ⁇ ing globulin
• TSG thy
• antibody ligands are those antibodies of the IgG, IgE, IgM and IgA classes specific for any of the antigens or haptens, or a class thereof, herein described.
• the class of hapten ligands is exemplified by thyroxine (T 4 ) , triiodothyronine (T 3 ) , the estrogens such as estriol, prostaglandins, vitamins such as biotin, vitamin B 12 , folic acid, vitamin E, vitamin A, and ascorbic acid (vitamin C) , and drugs such as carbamazepine, quinidine, digoxin, digitoxin, theophylline, phenobarbital, primi- done, diphenylhydantoin, morphine, nicotine, and so forth.
• DNA, RNA, and their complementary binding sequences and binding proteins can be determined by method of this invention.
• Cytokines such as interleu- kins, interferons, G-CSF, GM-CSF, M-CSF, tumor necrosis factors (TNF) , erythropoietin and the like are represen ⁇ tative of cytokines that may be determined by methods of this invention.
• labeled binding agent refers to substances which specifically bind to the analyte and which have a detectable label.
• the label may be covalently linked or bound to the binding agent indirectly through another specific binding reaction, for example, a labeled goat antihuman antibody could be used to label a human antibody.
• a labeled goat antihuman antibody could be used to label a human antibody.
• Those skilled in this art will recognize a wide variety of techniques to label proteinaceous, as well as non-protein specific binding substances. For instance, fluorescent dye labeling of proteins in general and antibodies or antigens in particular is well-known.
• a reagent which is a conjugate of the analyte to a labeled carrier. The free analyte in the test sample and analyte on the labeled carrier compete for the specific binding partner prior to separation.
• cytokines and monoclonal antibodies to these cytokines are known, for example, interleukin (l ⁇ , 1/3, 2, 3, 4, 5, 6, 7, 8, 9, 10); interferon ( ⁇ , ⁇ , ⁇ ) ; granulocyte/macrophage colony stimulating factor (CN-CSF) , G-CSF, M-CSF; tumor necrosis factor (TNF ⁇ and ⁇ ) ; and Transforming Growth Factor and their monoclonal antibodies are known.
• interleukin l ⁇ , 1/3, 2, 3, 4, 5, 6, 7, 8, 9, 10
• interferon ⁇ , ⁇ , ⁇
• CN-CSF granulocyte/macrophage colony stimulating factor
• G-CSF G-CSF
• M-CSF granulocyte/macrophage colony stimulating factor
• TNF ⁇ and ⁇ tumor necrosis factor
• Transforming Growth Factor and their monoclonal antibodies are known.
• Antibodies to creatine kinase skeletal, cardiac,
• Lipopolysaccaride an endotoxin
• LPS Lipopolysaccaride
• Monoclonal antibodies to LPS are well-known, see U.S. Patent No. 5,092,235 (Williams et al. and references therein) .
• a specific binding substance may be labeled with any of a variety of dyes, such as fluorescein dyes or rhodamine dyes by conventional chemical techniques.
• Representative fluorescent dyes for making the labeling binding agent are fluorescein isothiocyanate (emission at 520 nm) , 4-chloro-7-nitrobenzo- ⁇ -oxa-l-diazole (emission 550 nm) , tetramethy1rhodamine isothiocyanate (emission 580 nm) and Texas Red (emission 610 nm) . These dyes are available from Molecular Probes, Inc.
• fluorescein 5 or 6 succinimidylcarboxylate fluorescein 5 or 6 iodoaceta- mide
• fluorescein 5 or 6 maleimide are available.
• Similar functional groups are available for tetramethyl- rhodamine dyes.
• separation media refers to size chromatography, affinity and ion exchange chromatography, electrophoresis, such as slab get electrophoresis or capillary electrophoresis.
• Media such as polyacryla- mide, cellulose acetate, agar gel, and agarose gel, are suitable for electrophoresis.
• the separation of the labeled binding agent, complex, and labeled marker can also be achieved by sedimentation techniques where centrifugation causes the component to migrate in the edia. These separation techniques are well-known to those skilled in this art.
• the invention involves the reaction of a labeled binding agent (LB*) and an analyte [A] to form a complex A/LB* and separation of these species on a separation media, measuring the differential rate of migration of A/LB* and LB* on the separation media by detecting the label and then using the difference in migration or migration rate to identify the analyte by comparison with calibration data or other data which establishes expected migration data for the analyte.
• LB* labeled binding agent
• A an analyte
• the reaction can be determined by measuring the decrease in the size of- the LB* peak.
• multiple analytes can be detected by using different labeled binding agents that specifically bind to each analyte and detecting the LB* and A/LB* complex for each label.
• two different labeled binding agents can be bound to one analyte to form a sandwich complex and the migration of the sandwich complex can be compared with either or both of the labeled binding agents.
• two-dimensional electrophoresis can be used to separate multico ponent systems.
• the differential migration data is compared with expected migration information.
• haptens can be detected by binding the hapten to a labeled carrier [C*] such as a polypeptide to form a conjugate in which the hapten or the carrier is labeled (HC*) .
• C* labeled carrier
• the hapten in a test sample is allowed to compete with HC* for antihapten and the mixture is separated on the separation media.
• the species HC* and HC* antibody complex are detected as they separate on the media.
• This reagent is referred to as a "hapten conjugated labeled carrier.”
• a second labeled marker which migrates on the separation medium independent of the labeled binding agent and analyte and preferably the second marker is a fluorescent dye derivative which migrates more rapidly than the labeled binding agent and complex in the separation medium.
• the fluorescent dye can be bound to a protein or other substance which will affect its mobility so that it will migrate as desired in a particular medium.
• human serum albumin is a negatively charged protein which migrates very rapidly toward the positive pole in electrophoresis.
• the labeled marker provides for normalization of channel to channel variability in antibody reaction and detection of signal amplitude. It provides an early warning that the partic ⁇ ular assay is grossly incorrect.
• the labeled marker also provides a reference point for discrimination of both the labeled binding agent peak and complex peak, thus further assuring the quality of the assay.
• the second labeled marker also provides a means for quantitation of the ana ⁇ lyte.
• This invention is most advantageously applied in the diagnosis of molecular variants of proteins.
• creatine kinase (CK) MB isoforms (MB2 and MB1) have been used in the early diagnosis of muscle injury following acute myocardial infarction.
• the determination of the MB2 and MB1 isoforms is also useful in determining the onset of acute cardiac allograft rejection as well as injury following coronary artery bypass grafting.
• the determination of CKMM isoform is important in monitoring atropic skeletal muscle changes.
• mitochondrial creatine kinase in patients with cerebrovascular damages and it is critical that there be a rapid assay to assess such damage so that drug therapy can begin.
• alkaline phosphatase iso ⁇ forms is important in liver, bone, and kidney disease, as well as liver transplant rejection.
• Those skilled in the medical arts will recognize a large number of clinically important proteins which have small differences in struc ⁇ ture which can be determined by the method, test kits, and apparatus of this invention.
• Figure 1 is a plan view of an apparatus for practicing the present invention.
• Figure 2 is a top view of a single gel lane illustrated in Figure 1.
• Figure 3 is a plot of detector signals from unbound and bound fluorescent material.
• Figure 4 is a plot of overlapping detector signals of different wavelength from unbound and bound fluorescent material.
• Figure 5 is a top view of a multiple lane gel arrangement for electrophoresis.
• FIG. 6 shows the electrophoretic migration of Cy5 labeled human serum albumin (HSA) and Cy5 labeled human serum albumin complexed with an antibody.
• HSA human serum albumin
• Figure 7 shows the electrophoretic migration of Cy5 labeled bovine serum albumin (BSA) as a second labeled marker and Cy5 labeled anti-HSA.
• BSA bovine serum albumin
• Figure 8 is a front perspective view of the differential separation assay instrument.
• Figure 9 is a front perspective view of the operating components of the differential separation assay instrument of Figure 8.
• Figure 10 is a schematic drawing of the optics of the differential separation assay instrument of Figure 8.
• Figure 11a is a top plan view of an electropho ⁇ resis cartridge used in the instrument of Figure 8.
• Figure lib is a sectional view of the cartridge of Figure 11a through an electrophoresis channel.
• the electrophoresis apparatus consists of a con ⁇ ventional single lane 18 having a substrate 17, a gel layer 19 and a protective glass cover 21.
• the substrate is usually a self-supporting material which may be glass, Mylar (Trademark) or any well-known gel support.
• the gel itself is usually polyacryla ide or agarose, although other gel materials such as synthetic acrylamide substi ⁇ tutes may also be used. Uniform polymerization and free ⁇ dom from bubbles and irregularities are desirable proper- ties.
• the glass cover is preferably nonreflective glass which merely serves as a protective cover for the gel.
• the well 13 is normally positioned vertically so that it will receive a sample without spillage.
• the well funnels a prepared sample toward the gel.
• the well may combine a stacking and separating gel and creates a slit of sample material on the gel.
• High voltage is then applied to the gel at terminals 15, 16 and charged ions migrate toward the positively charged voltage electrode.
• the end of the gel near well 13 is maintained at negative or ground po- tential so that there is a substantial potential differ ⁇ ence from one end of the gel to the distant end.
• the sample which is placed in well 13 is a fluid, frequently a fractionated blood sample.
• Blood may be pre-processed to remove constituents which will inter- fere with the assay. Removal may be by filtering, ab ⁇ sorption, centrifuging or precipitating either the desired or undesired components so that a desired target analyte may be obtained for electrophoresis.
• the desired target analyte must be one for which there is a specific binding agent.
• Fluorescent tags are commercially avail ⁇ able, such as those manufactured by Molecular Probes Inc. of Oregon which specializes in dyes or dyed beads that can be covalently attached to binding agents to provide a labeled binding agent.
• Mono- clonal antibodies can now be manufactured so that the behavior of this binding agent is uniform and predictable for many assays. Monoclonal antibodies are more expen ⁇ sive than polyclonal antibodies, but the antibodies have greater specificity, are directed toward single epitopes, are easy to produce in large quantities, and are generally more useful and cause precise separation of bound and free material.
• the labeled binding agent is supplied in excess so that the reaction with the analyte will be driven to completion, or nearly to completion, in a reasonable or convenient amount of time.
• the amount of excess labeled binding agent should not be more than twenty times the amount of expected maximum level bound tag, although the number may range between 2 and 50, approximately.
• the labeling binding agent should alter the mass to charge ratio when combined with the analyte and subjected to an electrophoretic field.
• a strongly emitting light source such as light emitting diode or laser 23 is used to generate a beam 25.
• the LED 23 has an output power of about 50 mW in a wave ⁇ length band which will excite fluorescence in the fluo ⁇ rescent tagging material.
• excitation radiation is known as actinic radiation.
• the beam is intercepted by a focusing lens 27 which directs the beam through a slit aperture in barrier 29.
• Light emerging from the slit is divergent and is intercepted by the collimating lens 31.
• the beam is then directed onto a reflecting surface 33 which is part of a prism 35.
• the reflective surface 33 is at a 45 degree angle to the beam so that the reflected beam makes a 90 degree angle with the incident beam.
• the reflected beam is directed toward focusing lens 37 where the beam passes through one half of the focusing lens, while the other half is reserved for light traveling in the opposite direction, reflected from gel layer 19.
• Light passing through the focusing lens carries an image of the slit 29 which is directed onto the gel layer 19.
• Fluorescent light emitted from the complex and some reflected light from the gel layer travels in a retro-beam 39 to the left half of focusing lens 37.
• one half of the focusing lens is used by light travelling in each direction.
• the right half is used by the incoming beam, while the left half is used by the retro-beam. From there, the retro-beam is directed to reflecting surface 41 which is part of prism 35.
• the retro-beam is passed through a filter 43 which rejects any light other than the desired wavelength from the fluorescent target.
• Light transmitted through the filter is directed toward focusing lens 45.
• a light detector such as photomultiplier tube 47 with a slit located at the image plane of the gel.
• the time of arrival of the fluorescent sub- stances is measured relative to the starting time, i.e., the application of high voltage which initiates electro ⁇ phoretic migration. Since the arrival time is not pre ⁇ cise, but rather is a Gaussian curve, the peak time is recorded. The integrated peak area is also used for time discrimination.
• Each analyte and the corresponding la ⁇ beled binding agent are subject to the same procedure in the calibration run. In calibration runs a mean migra ⁇ tion time to the measurement slit or pinhole is deter ⁇ mined. Then, the standard deviation is determined for the time of arrival of the free binding agent, as well as for the bound analyte.
• the mean migration time i.e., the expected arrival times of bound and labeled binding agent for analytes because the times will be used to search for target analyte in a sample where the target substance is possibly present, but not necessarily present.
• the difference in arrival times between the complex and labeled binding agent may be used to establish a time window so that the arrival of one member may be paired with the other member in a search for the other member. If the search reveals that the .her member is present within a standard deviation or v./o, that other material is identified as a member of the pair. If nothing is found within the time window, the first member of the pair is regarded to be an artifact and is discarded.
• the search may be based on the second labeled marker or other labeled component.
• the output of the photomultiplier tube is main ⁇ tained in a buffer memory 49 and a ratio may be formed between the signals representing complex and labeled binding agent.
• a data reader 50 is connected to the buffer memory 49 for receiving recorded signals which represent the fluorescent peaks.
• the data reader is a computer which correlates the various peaks. Each peak is recorded in order to search for complex and unbound labeled binding agent in the recorded data. Normally, the time of appearance of the labeled binding agent could be estimated from prior calibration times. Once the position of the free labeled binding agent is known, a search is conducted for the corresponding complex which should be located a certain time interval away, within a time window defined by statistical limits.
• a peak within this window is identified as a complex that will bind fluorescent substances, i.e., the target analyte.
• the amplitudes of the identified peaks are examined and a ratio is computed in the data reader 50.
• the method whereby labeled binding agent is correlated with complex is explained further below.
• the computer also stores calibrations of known concentrations of target substance so that ratios may be compared in order to obtain an estimate of the unknown concentration.
• the top view of gel 11 shows that the image 29' of slit 29 falls between a positive high voltage terminal 15 and a slit from well 13, coinciding with negative voltage terminal 16.
• the high voltage applied to terminal 15 causes migration of com- plex and labeled binding agents, which are positively or negatively charged molecules which respond to the elec ⁇ tric field from the high voltage supply.
• the labeled binding agent will reach the image 29' of slit 29 which is fixed in position at a time different than the com ⁇ plex.
• the labeled binding agent serves as one marker for a time window which has the bound tagged binding agent as a corresponding marker, the two markers forming a pair of markers which are separated in time within the statisti- cal limit which is defined.
• a plot of the detector signal is shown where the horizontal axis is time and the vertical axis is amplitude of the detected signal.
• t 2 a relatively large peak 51 is observed, representing free fluorescent labeled marker of a first color.
• Another signal 54 discussed below, is detected after peak 51.
• t 3 a weaker signal 53 of the same color is observed.
• the peak 53 exists in the mid-region of a window, Wl, between XI and X2. The existence of window Wl is established by the labeled binding agent signal 51.
• Peak 53 is within window Wl and is recognized as a signal from the complex. Peak 54 is not within window Wl and is treated as a false positive or artifact, after being checked to determine whether the signal is not mistaken for the labeled binding agent signal 51.
• a search of all signals is made to determine the most logical positions for free and bound fluorescent substances. If no signal is found in time window Wl, the absence of target analyte is inferred.
• Each window W acts as a time domain filter, allowing discrimination of spurious fluorescent signals and noise. Note that all signals are recorded and signal discrimination occurs after recording by analyzing recorded data. Even though gel to gel characteristics may vary, the present inven ⁇ tion has immunity to most variations because the complex and labeled binding agent traverse the same path.
• the ratio of the two signals represented by the area under the peaks 51 and 53 represents an estimate of the ratio of a complex to labeled binding agent after normalizing data relative to calibrations, assuming good binding efficiency. A further time later, another large peak 55 is observed. This represents another fluorescent binding agent. This defines another time window W2 at a subsequent time and a lesser peak 57 is measured in the window. This is taken to represent a complex. Again, the ratio of complexes to free dye is computed and once again the target analyte associated with the second dye may be estimated in concentration.
• the peaks may overlap e->.ch other as shown in Figure 4.
• the labeled binding agent substance peak 61 having a relatively large - amplitude, overlaps the second peak 65 of similar amplitude in a test where two different fluorescent substances were used.
• the second peak 65 is the second free fluorescent substance signal.
• Peak 61 establishes the time window W3 where a peak 63, representing a bound fluorescently labeled binding agent of a color which is the same as that associated with the unbound peak 61, occurs totally within the second peak
• peak 63 may be spatially and optically differentiated from peak 65.
• the ratio of bound to unbound signal amplitudes appears to be about 2:1.
• the corresponding molecular amounts of complex and labeled binding agent are estimated to be in the same ratio.
• a time window W4 is established, but no fluorescent signal is found within the window and so the absence of target analyte is inferred.
• a multiple lane electrophoresis sheet gel is shown.
• the sheet 71 is provided with two lanes 73 and 75. Each of the lanes has a respective well 83 and 85 and a respective slit image 87 and 89.
• the two lanes are constructed similarly, with the spot image locations in the same position.
• Lane 73 is used to run a calibrated amount of target analyte, a known amount of free fluorescently labeled binding agent, and a fixed amount of a second labeled marker.
• an unknown amount of target analyte is run with free fluorescent labeled binding agent and the same fixed amount of the second labeled marker.
• the two lanes may be compared after normalization of peak area between lanes using the peak area of the second labeled marker to determine the amount of unknown analyte in lane 75.
• multiple runs may be made in lane 73 of various amounts of target analytes so that many ratios may be stored in a memory.
• a ratio from a run of an unknown amount of target analyte may then be looked up and compared with known ratios, with the best match indicating the amount of target analyte.
• FIG. 8 Preferred apparatus for practicing the invention is set out in Figures 8-lla and b.
• the instrument 101 which has two main modules, a computer module 103, serving to log and display data, and a measurement module 105, serving to receive samples and subject the samples to testing and measurement in accord with the procedures disclosed herein.
• Computer module 103 is a standard PC of the 386 or 486 MS-DOS kind, running familiar software suitable for manipulating numbers, such as Excel or Lotus spreadsheets.
• a display device 104, a keyboard 106 and a disk drive 108 are normal input/output devices associated with the computer.
• Keyboard 106 is used for signalling commands to the measurement module, such as start, stop, repeat, and so on.
• the measurement module 15 includes a U-shaped frame 107 for receiving an electrophoresis cartridge 109 and internal optics and electronics covered b the shroud 201. Once a cartridge 109 is placed on frame 107, the frame is pulled under the shroud 201 for the measurements disclosed herein using a signal from keyboard 106. Principal features of the measurement modules are shown in Figure 9. Frame 107, shown without a pro ⁇ tective lip apparent in Figure 8, supporting electro ⁇ phoresis cartridge 109, is moved by a pinion gear 203, in the direction indicated by arrows A, to a position wherein the cartridge rests over a Peltier device 205, including a fan within housing 207.
• the Peltier device is a commercially available refrigeration apparatus which chills a metallic mass 209 of good thermal conductivity, such as an aluminum block, to a temperature suitable for electrophoresis measurements of a particular media.
• the mass 209 is kept in thermal contact with cartridge 109 by means of a movable pressure plate 301 which is supported from a slotted plate 303 to apply downward pressure on opposed edges of cartridge 109.
• the pressure plate 301 and the slotted plate 303 are moved downwardly by means of a spring biased actuator, thereby forcing the car ⁇ tridge 109 into thermal contact with metallic mass 209 with downward force transferred to the cartridge at edges of the cartridge which are supported by frame 107.
• a first slot 305 in slotted plate 303 admits a scanning beam 307 which is directed onto a locus of spots on the cartridge, described below.
• Light scattered, reflected or fluorescing from the impinging light is directed back into optical fibers 309, one fiber corre ⁇ sponding to each beam spot in the locus of spots.
• Each of the fibers leads to a single optical detector which is always on. The detector output is synchronized with the beam position so that a single fiber is identified as the one providing the optical signal at the detector at any particular time.
• incoming light is seen to originate at laser 401, which is a 5 mw helium-neon laser, or a laser of any appropriate wavelength.
• the beam 307 has its path folded by mirrors 403 and 405, while the beam is collimated by optics not shown and focussed by a lens 407 to form a small spot on a scanning mirror 409.
• the mirror 409 is located sufficiently close to cartridge 109 that beam spots on the cartridge will not be significantly out of focus.
• Mirror 409 is rotated about a scanning axis in steps, defined by pivots 501 and 503 for mirror motion indicated by arrows B.
• a motor, 500 steps the mirror in discrete angular amounts of a few degrees per step starting from a home position at an edge of the support frame where an optical detector is located.
• a beam scan always starts from the home position and the beam is stepped by known angular amounts to create a limited number of spots at desired locations on a cartridge.
• the number of steps is counted by an electrical counter, which is a beam synchronizer 502, so that the beam position is known at all times.
• the beam position can be synchronized with the detector 505, a photomultiplier or PMT tube which receives light from the optical fibers 309 which are gathered in a bundle.
• a lens or lenses and a filter, not shown, may be used to optimize coupling between the fiber bundle and the PMT 505. The filter reduces spurious light.
• a holder block 507 is used to secure each optical fiber in a desired location and to space each fiber very close to the locus of spots where the incoming beam will impinge.
• a cartridge 109 is seen to include opposed edges 601 and 603 having apertures 701 and 703 partially extending into the edges. These apertures are known as wells where fluid samples are placed. Additional apertures 705 and 707 may be used as detents to index the location of the cartridge in the support frame. Between opposed cartridge edges, slightly above a heat conducting plate 605, are very thin capillary tubes 607 having open ends which extend into the wells 701 and 703 for communicating with any fluid in the wells.
• the capillary tubes are clear, low reflection material or treated with a coating for low reflection.
• the tubes have a diameter suitable for electrophoretic migration of a sample in view of the viscosity of the sample.
• one beam spot is provided for each capillary tube so that the number of angular steps of the beam needs to at least equal the number of capillary tubes and must be such that the beam lands squarely on a capillary tube for possibly exciting a response from a fluid in the capillary.
• electrodes 609 and 709 are seen to extend into apertures 701 and 703 in order to make contact with fluid in the well and migrating between the opposed elec ⁇ trodes under the influence of an electric field. In op- eration, the electrodes come into contact with the wells at the same time a pressure plate clamps the cartridge in place as described above.
• Electrodes 609 and 709 cause electro ⁇ phoretic migration in the capillary tubes.
• the beam 307 repetitively scans a locus of spots transverse to the length of the capillaries, with a spot falling on each capillary and passing into its center where it illumi ⁇ nates fluid sample material under electrophoresis migration. Fluorescent light emitted from the sample falls upon an end of a nearby optical fiber and the light is guided back to the PMT.
• One of the advantages of the present invention is that analysis of peaks representing bound and free dye can be computed before electrophoresis is complete, i.e., before the migrating substances reach the distant high voltage electrode.
• Another advantage is that the present system uses only a single lane of an electrophoresis apparatus so that gel to gel non-uniformities are nulled. It is possible to use a second lane in an electrophoresis device as a reference or calibration, but such calibra ⁇ tions may be done beforehand and results stored in a memory. It is also possible to use a second or third or fourth lane for additional analytes of interest creating panels of relevant analytes.
• analysis of target analytes usually requires completion of the electrophoresis and subsequent analysis by a plurality of stains, colored or fluorescent substances, etc.
• the analysis may be done in real time as soon as sufficient separation exists between the bound and free fluorescent material. Such a separation can be at a point which is only twenty-five percent or thirty-three percent of the length of a lane. Once a point is found where adequate separation exists, the image of the slit or pinhole is positioned at that loca ⁇ tion and then all measurements are made from there.
• this is an open-ended electro ⁇ phoresis system, i.e., there is no need to stop the electrophoresis at a defined point to get all materials "on scale.” Materials that migrate slowly can be detected just as well as fast moving target analytes. Amplitude thresholds may be used as further discrimina ⁇ tion against noise and artificial signals. To discriminate between two or more target ana ⁇ lytes in the same gel lane, different fluorescent wave ⁇ lengths can be used, so long as filter 43 in Figure 1 can adequately resolve the different wavelengths. Multiple tests can be run simultaneously, each test associated with a particular wavelength.
• Creatine kinase is an enzyme present in various mammalian tissue. It occurs in three different forms known as isoenzymes: CK-MM (skeletal) , CK-MB (cardiac) and CK-BB (brain) . After release from tissue and on circulation in blood the MM and MB forms themselves break down to smaller fragments known as isoforms or subfor s. In the event of myocardial infarction, the MB isoenzyme, present in cardiac muscle, is released into plasma. Hence, it serves as a specific diagnostic molecular marker for cardiac ischemia or necrosis.
• the labeled intact immune complex will migrate differently than the labeled antibody.
• the fluorescence associated with bound and free markers will be detected and arrival times measured and recorded.
• Free marker is identified by a large peak. Any substance within the expected time of the free substance is regarded to be target analyte. Anything else is an artifact.
• Example 2 Detection of the presence of sexually transmitted diseases
• Many sexually transmitted pathogens such as chlamydia, herpes, etc., form lesions in the urogenital area.
• samples are taken with a swab directly from the lesion and a number of different types of tests are performed on this extract. These tests include culture and/or immunochemical tests.
• the swab is treated with a solubilization reagent to liberate micro-organism present. This process will also solubilize target analytes originating from the micro ⁇ organisms.
• HSA is the target substance which is detected by tagging with fluorescent HSA.
• HSA, Fraction V was obtained from Sigma Chemical Company (St. Louis, Missouri) .
• Monoclonal anti-HSA was obtained from Biospacific Inc., California.
• Cy5-labeled HSA was synthesized by the coupling of Cy5 fluorescent dye to HSA (Biological Detection Systems, Inc. , Pittsburgh, Pennsylvania) . This fluorescent sub ⁇ stance is the binding agent.
• DSA Differential separation assay
• the real time detection of fluorescent proteins during electrophoresis was performed using a He-Ne laser beam focussed at a point 1.3 cm below the wells of the gel.
• the emitted fluorescence was collected using a photo ultiplier (PMT) tube.
• PMT photo ultiplier
• Data was collected using a Lab-PC from National Instruments (Trademark) (Austin, Texas) data acquisition board on the IBM-PC and imported into a Microsoft Windows (Trademark) Excel (Trademark) file for analysis and graphics.
• This example demon ⁇ strates that the relevant time window for this pair of binding agent (Anti-HSA) and fluorescent tag (Cy5-HSA) is 17.5 minutes.
• the 8 minute peak 91 defines the reference position in the data acquisition window for finding the peak of the immune complex Cy5-HSA-Anti-HSA. Peak 95 is the residual uncomplexed labeled Cy5-HSA.
• Septic shock is the most common cause of death in a medical-surgical intensive-care unit. Mortality rates range from 40% for early phase sepsis to more than 70% for refractory septic shock. Septic shock develops in a cascade fashion. Bacterial antigens (including endotoxin) activate local tissue macrophages, blood monocytes, and serum complement. Local complement activation induces (directly and indirectly) migration and activation of blood neutrophils—as do the macrophage and monocyte activation products (which include interleukin-1 and tumor necrosis factor) .
• activated macro ⁇ phages and lymphocytes produce (again directly or indi ⁇ rectly) molecules that stimulate the endothelial cells to produce more neutrophil chemotactic factors.
• cytokines increase endothelial permeability. This in turn promotes blood-neutrophil adhesion and migration.
• the neutrophil releases cyto- lytic enzymes and reactive oxidants.
• Chronic exposure destroys the local endothelial vasculature. Such persistent local damage impairs vascular integrity to such an extent that hemodynamic homeostasis cannot be restored. Death is the result.
• Unstimulated neutrophils constitutively express lectin adhesion molecules (LECAM-1) .
• ICAM-1 intercellular adhesion molecule-1
• ELAM-1 endothelial-leukocyte adhesion molecule-1
• Platelet activating factor PMN leukocyte products (toxic oxygen radicals, proteolytic enzymes) Serotonin Transforming growth factor beta
• Tumor necrosis factor A Vascular permeability factor
• LPS tumor necrosis factor
• interleukin-1 interleukin-1
• interleukin-8 gamma interferon
• LPS Lipopolysaccaride
• Monoclonal antibodies to TNF interleukin 1 and 8 and y interferon are described in Current Protocols in Immunology, supra. Following the procedures in Example 3, except the antibody is labeled and the cytokine is the analyte, LPS, interleukin-1, interleukin-2, TNF , and gamma interferon are determined.
• Bovine serine albumin is dissolved at about 1 mg/ml in 50 mM phosphate buffered saline PBS. This bovine serum albumin solution is reacted with a solution of carboxy ethylindocyanine succinimidyl ester in accordance with the procedure in Cytometry 11: 418-430 (1990) .
• a kit for such labeling is sold by Biological Detection Systems, Inc., 4617 Winthrop Street, Pittsburgh, Pennsylvania 15213.
• Figure 7 shows the differential migration of Cy5-BSA peak 97 and Cy5-labeled monoclonal anti-human serum albumin peak 98.
• Cy5-BSA is a suitable second labeled marker.

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