JPH028744A - Use of magnetic particle for isolation, collection and assaying of ligature for diagnosis - Google Patents

Abstract

PURPOSE: To realize a simple and economical assay by forming a required complex containing a first ligand using magnetically reactive particles, collecting the complex to produce a complex cluster and then quantitatively determining the amount of cluster based on the measurement of ligate concentration in the liquid. CONSTITUTION: Magnetically reactive particles are added to a humor and a first specific ligant is bonded to the ligate in humor thus forming a complex of magnetic particle/ligand/ligate. The complex is then collected from the humor by magnetic field and aggregated to produce a complex cluster. When the ligate concentration in humor is measured under that state, the amount of cluster can be determined based on the measurements. Consequently, the ligate concentration in humor can be determined simply and economically by separating the ligate from humor and taking out the separated ligate.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for isolating, extracting and quantifying unique components present in a liquid. More specifically, the present invention provides methods for in vivo isolation and recovery of unique components present in body fluids, and methods for indicating disease states. The present invention further provides a novel assay method for quantifying the composition. Also included are pertinent materials and equipment useful in the practice of the invention.

[Conventional technology]

The body fluids of healthy individuals (humans and other animals) contain various biochemical components. However, during illness the chemistry of normal body fluids may change. In some diseases, specific components in specific body fluids increase, while in other disease states, the unique components normally present in body fluids decrease by 1 ton.
'destruction' or a decrease in concentration occurs. In either case, the concentration of specific body fluid components indicates signs of disease.

Body fluid components that are indicative of, or characteristic of, a disease state can be classified as ligates. The lack of suitable methods for accurately quantifying ligate concentrations in the body has prevented the use of ligates in disease diagnosis. Quantitation requires complete recovery of the ligate from the body fluid in question. This problem is particularly important when body fluids are present in body cavities that contain only small amounts of fluid, e.g.
Diagnostic methods based on ligates contained in gingival fluid require the removal of material containing no more than 1 to 2 microliters of fluid from the body cavity.

[Problem to be solved by the invention]

Even if ligates can be successfully isolated and removed from the body, it is known that in many cases it is extremely difficult to accurately quantify their component concentrations. Conventionally, tlA
It has been necessary to use expensive, cumbersome and time consuming assay methods such as lt immunoassays and high performance liquid chromatography.

It is therefore an object of the present invention to provide a method for successfully isolating and extracting ligates from body fluids.

It is also an object of the present invention to provide an hour wheel, inexpensive assay for quantifying ligate concentration in body fluids.

[Means for solving the problem]

In order to achieve the above-mentioned object, the present invention provides a method for quantifying Liga Il concentration in body fluids in vivo. This method consists of a step knob that attaches a ligate-specific biological or other molecule (ligand) in a body fluid to a magnetically responsive particle.

In this case, magnetically responsive particles are introduced into body fluids with attached ligands. The magnetic particles and attached ligands are left in contact with body fluids for a period of time during which the ligands and ligates combine to form magnetic particle/ligand/ligate complexes. ! Once the particle/ligand/ligate complex is formed, the complex is recovered from the body fluid by applying a magnetic field. After the complexes are collected, they are aggregated to form clusters, and the ligation H in body fluids is
Quantify Q degree.

Further, to accomplish the above objectives, the present invention provides a magnetic collection or sampling device designed to be anatomically compatible with each body cavity containing the fluid of interest.

[Action and effect]

Magnetically responsive particles useful in the present invention can be of any type as long as they are magnetic and can be attracted by a magnetic field. Paramagnetic particles are preferred (ie particles that respond to 3n fields without becoming permanent magnets).

However, the method of the present invention does not require such particles. Commercially available particles are used in many applications of the invention. However, this requires specially designed particles.

Suitable magnetically responsive particles have a core of a metal oxide, usually iron oxide, surrounded by a coating of organic material to which a suitable ligand can be bound. The composition of the coating varies depending on the nature of the substance to which the ligand is bound and the number of binding sites present in the coating. The size of magnetically responsive particles varies and depends on the specificity of the application. Two important issues to consider that influence the selection of particle size are the magnitude of the magnetic field required to retrieve the particles from the specific body cavity containing the fluid in question, and the size of the coating. It is the number of attachment sites that has. Generally, the size range of magnetically responsive particles is 1 to 20 microns.

Magnetic particles or beads commonly known to those skilled in the art and useful in the practice of the present invention are described in US Pat.
These are Dynal J beads available from Dyna II Inc.

"Dynal" beads are paramagnetic, spherical polystyrene particles with a ferrous oxide core. The particles have a uniform diameter of 2.85 or 4.5 microns. The functional group on the bead surface, ie, the component that binds to the ligand on the bead surface, is OHi, which is hydrophilic. Before the beads can bind ligand, they must be "activated", which is done by reacting the hydroxyl groups with tosyl chloride.

Other commercially available magnetic particles suitable for the practice of this invention include P.C., Indianapolis, Indiana;
O, Box 1210. Serady
n Inc.) [SEr?ADYN]
"MAGN Sort" manufactured by DuPont Co.
I5ORT)J. "Ceradein" beads are latex paramagnetic particles with an iron oxide core.

The active functional group on the surface is C0OH (carboxyl group). The size of these beads is 0.7 to 1.7 microns,
The beads, whose shape is non-spherical and non-uniform, do not need to be activated before binding the ligand. "Magnisort" particles are paramagnetic chromium dioxide particles coated with silane. The particles are amorphous in shape and have a size of 0.5 to 5 microns. Silane is the functional group and the particles are activated when the silane is reacted with glutaraldehyde.

Activation times for the commercially available particles listed above range from a few hours to two days. For example, activation of "Ceradein" beads requires overnight and "Dynal" beads require two days. However, activated forms of beads can be purchased from both Dynal and DuPont.

As mentioned above, the ligands capable of binding to the specific ligates present in the body fluid in question are attached to a coating that surrounds the metal oxide core of the magnetically responsive particle.

(Hereinafter, the ligand bound to the magnetic particle is referred to as MAb)
, this binding interaction is commonly referred to as ligand/ligate binding and includes, but is not limited to, enzyme/substrate, antibody/antigen, receptor/hormone, and protein/substrate binding. do not have.

The specific ligands bound to the magnetic particles are dependent on specific diagnostic ligates present in body fluids. Oll
Only after it has been confirmed that the ligate is indicative of a specific disease state will the ligand that binds to the ligate be identified and selected. For example, two characteristic ligates associated with periodontal ligament disease and present in gingival exudates are interleukin I and cachectin. The second of these, cachectin (also known as Uffi111 necrosis factor), has been linked to periodontal ligament disease.
This was revealed through research by the present inventor.

Developing methods for disease diagnosis requires knowledge of the disease process. As with many bacterial infections,
Periodontitis occurs in a series of ways or stages. The first step is activation of macrophages by ribopolysaccharide, a component of bacterial cell walls. Activated macrophages then secrete a number of compounds called cytokines that act as signaling molecules to fibroblasts, which are connective tissue cells. In response to this signal, fibroblasts produce and secrete a special degrading enzyme, collagenase, which is the collagenase that causes the decomposition of &II tissue seen in periodontal ligament disease.

Cachectin (Cx) is one of the cytokines secreted by macrophages.8 The present inventor studied the effect of this polypeptide on dental fibroblasts and discovered that CX causes the production of collagenase. This molecule is
It is an ideal molecule that serves as an indicator of gingival membrane disease as a precursor that initiates collagenase secretion. Preliminary studies conducted in human subjects showed that subjects with gingival disease had detectable levels of Cx, whereas Cx was not detectable in subjects without any clinical signs of the disease. It turns out. Furthermore, since Cx levels are expected to increase before collagenase production, detecting Cx can predict the onset of disease.

By using the ELISA (enzyme-linked immunosorbent assay) assay, the inventors established a correlation between dental health and the presence of Cx in the gingival sulcus. The ELISA test method used is a modification and improvement of methods known in the literature. It was developed using a murine anti-human Cx monoclonal antibody. The 96-hole (Guinate Quimron 1) plate is
9119.6 in 0.1 M sodium carbonate/bicarbonate buffer coated with a 1000-fold dilution of the monoclonal antibody (MAb). Plates were incubated overnight at room temperature, emptied, carbonate buffer pH 9.6 containing 2% fetal bovine serum (Fe2) was added, and incubated for 4 hours at room temperature or at 4°C until use. Then, use Biotique's automatic microplate washer to remove 0.
0.01 with 05% Tween 20 (PBST)
Washed 4 times with M phosphate buffer (PBS), 0.7
A cachiectin stock solution at a concentration of 5 mg/m 14 was diluted in PBST to a concentration of 1500 picomolar and
Serial dilutions were performed on the plate to create a standard curve from 0 pmol to 2 pmol. Veriopaper (Halco, Tustin, Calif.), which was block-locked with 1 volume of 0.1% bovine serum alpha in PBS, was used to collect gingival samples from patients.

Verio paper 100. c+7! PBST-2%F
The plate was placed in a well with C3 and incubated for 1 hour at 37°C.Then, the berriopaper was removed and the plate was washed four times with PBST. Furthermore, rabbit anti-tiectin serum diluted 100 times with PBS-2% FC3 was added to each well.
The cells were cultured at 37°C for 1 hour and washed 4 times with PBST. Horseradish peroxidase-conjugated goat anti-rabbit (Organon Technica, Westchester City, Pennsylvania)
A 35,000-fold dilution of PBST 2% FC was added to each well, incubated at 37°C for 1 hour, and washed 4 times with PBST during each wash cycle. Then, 150 μl of an aqueous solution of 2.2′-adenobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) with a concentration of 44 mg/m 12 was added to the PH
Add 4,0 ml of 10 mM citric acid/sodium citrate buffer; add 40 ttl of 3% hydrogen peroxide to A
Added to Tl'3S-citrate buffer. 10 of this solution
0 μβ was added to each well to develop color. The plate was read for absorption at 414 nm using a Titertic Multiscan MCC/340, and the data was analyzed using Linpro's Basic Time BT/XT scan software. The assay is reproducible and the 2-op
Sensitivity up to M or 340 pg/ml, 1500
The curve from pM to 209M is linear.

Patients with mature periodontal ligament disease were selected from outpatient visits to the University of Connecticut School of Dentistry for treatment. Mature (medium ) The patient was diagnosed with dental disease. The design of periodontal status for all periodontal tissues followed the criteria described by Raney et al. For the insertion test, patients were required to have no history of diabetes, acute ulcerative gingivitis under anesthesia, and any gingival membrane treatment in the last 6 months. Clinical signs and/or symptoms of systemic disease and pregnancy were also excluded. Volunteers from among the dental school students constituted the periodontitis-free control population.

Each complainant underwent an initial dental examination. The test was conducted at the University of Connecticut Clinical Laboratory Center. All of these examinations are internal and external examinations for cavities. In addition, the dental plaque index (P) and the periodontal disease index (Gl) were determined.

In addition, measurements of periodontal ligament pocket depth (PD) were performed using corrected (to the nearest whole number of millimeters) gingival probes. The diameter of the probe was 0.6 mm, and a probe "power" of about 20 g was used.

Test data were entered and stored in a computer database. Each patient's file has a patient's! ,D.

number, number of teeth tested, Cx value in gingival crevicular fluid (GCF), gingivitis index (pocket depth, floss and periodontal disease index) of the tested teeth and current medical condition, i.e. cancer, etc. It was a record. All relevant aspects of assay and analysis conditions were also recorded.

The correlation between cachiectin and periodontal disease is shown in Figure 9. In this figure, the abscissa is the periodontal disease index (Gl), the plaque index (PI), and the pocket depth (Gl) obtained at each site.
PD) indicates the total value: (bad) is the value obtained from 6 samples,
(Tuesday) indicates the value obtained from 5 samples, (x) indicates the value obtained from 4 samples, and (-) indicates the value obtained from 1 sample. The ordinate indicates the amount of cachectin in each site. show. A total of 23 specimens were collected from 6 patients. - These data show that when the global index reaches 5 or more, the value of cacheckin increases. Surprisingly high values of cachectin were obtained in relatively healthy gingiva [see Figure 9, data shown in (1)], as this tooth had a subgingival amalgam filling, so the value may be due to stimulation by the filling. The result is possible.

As mentioned above, both cytokines, interleukins and cachectins are useful as diagnostic ligates for periodontal disease. Two molecules of the immunoglobulin G (1gG) class of antibodies, interleukin-1 monoclonal antibodies and cachectin antibodies, are each suitable ligands for binding to these ligates.

The adhesion between the ligand and the magnetic particle must be strong enough to retain the ligand on the particle while both are introduced into the body fluid and the binding reaction between the ligand and the ligate takes place. . Furthermore, the adhesion between the particle and the ligand must be strong enough to remove the particle/ligand/ligate complex from the body as a unit; the nature of the adhesion depends primarily on the chemical structure of the ligand. For example, it is conceivable that the above-mentioned monoclonal antibodies are directly covalently bonded to magnetic particles in a displacement reaction. Alternatively, a binding medium such as a protein A linker could be used to link IgG to magnetic beads.

The time required for the binding reaction, ie, the attachment of the ligand to the activated magnetic particles, ranges from several hours to several days. The type of active surface functional groups a particle has influences the binding reaction. For example, IgG binds to carboxyl groups more easily than to hydroxyl groups.Assays of binding efficiency using IgG labeled with S1 revealed that for the various magnetic particles listed above, Dynal beads, Sera-Din beads, and Magnisort beads were 26.7% of the IgG used, respectively.
It was found that the binding was 50.7% and 67%. It should be noted that the results of this assay are due, at least in part, to differences in the total surface area of each sample of beads.

After completion of the binding reaction, the magnetically responsive particles, together with the ligands attached to them, are added to the body fluid containing the diagnostic ligate. In the case of periodontal disease, a suspension of MAb particles is prepared and introduced into the sulcus using a microdispenser, using either cachectin or interleukin-1 as a diagnostic ligate.

After the magnetic particles are introduced into the body fluid with their attached ligands and the binding reaction between the ligand and the diagnostic ligate is completed, the magnetic particles/ligand/ligate complex must be removed from the body. This is accomplished with a magnetic gathering or harvesting device that attracts and holds the complexes by applying a localized magnetic field. The magnetic collection device must be anatomically compatible with the body cavity containing the fluid into which the magnetic particles are introduced, and the collection device must be capable of applying a magnetic field that corresponds to the size of the magnetic particles used. Therefore, when applying the present invention, each unique magnetic sampling device is required. However, these devices are generally hand held and consist of an electromagnet and a tip suitable for insertion into a body cavity.

Figure 1 shows an electromagnetic collector (lO) designed to extract magnetic particle/ligand/ligate complexes from the gingival sulcus.
exemplify. The sampling device is powered by two 1.25 volt batteries (1
It has a handle (ll) that houses a power supply device consisting of (2, 12). Furthermore, the collector is made of 351 24 gauge copper wire.
It has an electromagnetic coil (14) and a chip (16). The tip illustrated in FIG. 1 is shaped similar to a standard gingival plug. However, a variety of other chip designs are possible, such as a Schahel-type chip.

According to standardized tests, the magnetic sampler weighs 0.63 g
produces a magnetic field that can move an object by 6.7 cm. A magnetic field one-third this strength is approximately 25% efficient in displacing magnetic beads from the test solution. A magnetic field 8 times the strength produced by this collector is 100% efficient in displacing beads from the same solution. It is readily apparent to those skilled in the art that the strength of the magnetic field of the collector can be easily adjusted to accommodate variables such as the type of magnetic particles used and the particular body cavity from which the complex must be removed. be understood.

For the diagnosis of periodontal disease, the inventors needed to design and fabricate a special tray to collect the magnetic particles/ligand/ligate complexes from the tip of the collector.

FIG. 2 shows a collection tray (18
) is exemplified. A large number of depressions (22, 22) are formed on the sheet (2
0). There is a recess representing each tooth, the recesses being arranged in two arcuate parts (24 and 26), the arch (24) corresponding to the lower side of the tooth and the arch (26) corresponding to the upper side of the tooth. There is. As illustrated in FIG. 3, a permanent magnet (2 days) is placed at the bottom of each well to facilitate collection of the complexes.

Each well is filled with a buffer such as 0.05M Tris-HCe (
A removable plastic cup containing 2.5 ml of pH 7) is placed. Place the collector tip containing the attached complex into a buffer solution. The complex is removed from the chip with a nylon needle brush rotating at low speed and suspended in buffer. A standard dental engine (low speed handpiece) can be used to rotate the brush. however,
Excessive swirling, foaming and splashing must be avoided.

Once the magnetic particle/ligand/ligate complex is removed from the body, ligate concentration in body fluids can be quantified.

The present invention is a simple and straightforward assay in which the aggregation of complexes into clusters is at least large enough to be seen in the field of a microscope at low magnification. As described above, the particle/ligand/ligate complexes extracted from body fluids are suspended in a buffer. A second suspension of aggregated particles in this suspension?
IFa (add &.

Agglomerating particles are created by binding magnetically responsive particles with a ligand specific for the diagnostic ligate, but different from the ligand used for in vivo binding. (Hereinafter, the ligands used for magnetic particles and agglomerating particles are referred to as PAb particles). For example, as already mentioned, if cachectin is the ligate in question,
Monoclonal antibodies are used as ligates to bind in vivo, and polyclonal antibodies are used to form aggregated particles. Since ligates typically have at least two sites where ligands can bind, the ligands of the aggregated particles attach to the ligates at one or more sites where the ligand is absent. Clusters of magnetic particles/ligand/ligate-aggregated particles are thus formed.

FIG. 4 schematically illustrates a magnetic particle/ligand/ligate complex. As previously described, the complex consists of a ligand bound to a magnetic particle and a ligate bound to the ligand. In Figure 4, the magnetic particle and bound ligand are numbered 32.32, and the unbound ligate is numbered 33.
．． The complete complex is indicated by the number 3434, with the number 33.

FIG. 5 schematically shows a cluster of magnetic particles/ligand/ligate-aggregated particles. The cluster (36) contains at least one complex (34) and at least one aggregated particle (3 days). As shown in FIG. 5, the clusters can take a linear, rosette or branched form.

The overall properties of a cluster assay are influenced by many factors. One such factor is the formation of non-specific raster. Functional groups on the magnetic particles that are not blocked by IgG bind body fluid components that do not bind at all or bind non-specifically to the diagnostic ligate. In this case, clusters that do not contain the diagnostic ligate will be generated, thus obscuring the assay results. It is known that adding non-reactive proteins to the suspension suppresses non-specific and rasterization.

A suitable protein for this type of spring is bovine serum albumin (BS
A). Alternatively, surfactants such as Tween 20 or Triton X may be added to the suspension. However, surfactants are less effective at reducing non-specific rasterization than non-reactive proteins; the conclusion is that surfactants are less effective at reducing non-specific rasterization than buffering The ionic strength and chemical composition of the fluid was varied. NaCl, KCI, TrisHC1' and 1st/
All dibasic potassium phosphates were effective.

However, adding 0.01% of BSA to pH 7.0
A 0.5% solution of Tris HC/ was the most suitable formulation to suppress the formation of non-specific clusters.

Attempts have been made to use various magnetic particles to attach monoclonal and polyclonal antibodies in the same assay. However, even using the above-mentioned method of inhibition, beads with different functional groups on their surfaces were found to form clusters based on surface functional groups rather than antibody/ligate interactions.

The size and shape of the magnetic particles also affect the properties of the cluster assay. For example, in the case of Ceradine beads, which are 0.7-1.7 microns in size and non-spherical, it is difficult to see individual beads at 30x magnification, and random aggregation occurs in the blank sample. Makes cluster quantification difficult. The diameter of "Magnisort" particles is 0.5 to 5 microns. The 0.5 micron beads are not visible at 30x magnification and the amorphous particles do not cluster.

Ultimately, it was determined that Guinar beads were the most suitable particles for the assay. Guinar beads, which are spherical and have a size of 2.85 microns, were found to be most effective in promoting cluster formation. For example, place the beads at a neutral pH.
22.5 f when diluted in 0.05 M Tris buffer with
MAb forms a rosette with mol of cachectin.
The PAbO to beads ratio is 20:1.

As mentioned above, the clusters formed should be of a size that can be seen in a low magnification microscopic field of view. Clusters of this size allow direct or raster measurements, or microscopic images of the clusters are recorded on a suitable storage medium and counted by computer. Alternatively, clusters are counted by a computer of the type used to count cells. Alternatively, clusters are counted by a device of the type used for cell counting. This device is tailored for particles of various sizes.

When observing with a microscope and counting without using a computer, clustering is defined as groups of four or more beads being observed. The net micrometer disk in the microscopic field is the area to be counted.

FIG. 6 is a photograph of clustering of MAb and PAb beads in the presence of 22.5 f mol of cachectin. FIG. 7 is a photograph of low concentrations of non-specific rasterization in control samples. The photograph was taken with a Hoffmann phase contrast optic at 300x magnification.

The number of clusters was found to increase with cachiectin concentration. The detection limit was 2.5 fmol. However, it is likely that 10- to 100-fold lower amounts could be detected with further optimization of assay conditions. A correction curve for the number of bead clusters as a function of hemtomoles of cachiectin is shown in FIG. Figure 8 shows s! The number of clusters of 4 or more in the 10 corrected fields of view of the JI microscope is
Expressed against a known amount of cachiectin.

〔Example〕

The method of the invention is illustrated by the following examples.

Example 1 is an illustration of a method for attaching a cachectin/TNF (CIC) monoclonal antibody to magnetic particles containing a protein linker.

1. Pipette 1 mJ of protein A-coated magnetically responsive particles (approximately 5 mg/m 1 ) into a vial.

2. Collect the particles at the bottom of the vial with a magnet and remove the liquid 3.
,0. I M Nag HPO, (pH 8,0) 1
Add m2.

4. 140 pl Cx-T at a rate of 0.5 mg/ml
- Add the noclonal antibody to the vial.

5. Incubate with shaking at room temperature for 2 hours.

6.Similar to step 2, 0-I M N a z
Wash three times with HP Oa (pH 8,0).

7. Tris buffer after slanting the third wash? &(T
Resuspend in 4mff1 of BS; pH 7,5).

Particles with attached ligands become liquid for binding with cachectin.

Example 2 illustrates the preparation of aggregated particles by attaching polyclonal antibodies to magnetic particles for use in cluster assays.

1. Pipette the protein A-covered particles (approximately 5 mg/ml) into a vial.

2. Collect the particles at the bottom of the vial with a magnet and remove the liquid.

3, 0. I M Nag HPOa (pH 8,0
).

4. Add 140 μl of Cx polyclonal antibody (rabbit anti-Cx) to the vial at a ratio of 0.5 mg 7 ml.

5. Incubate with shaking at room temperature for 2 hours.

6.Same as step 2, 0. I M Nag ) (p
Wash 3 times with o, (pH 8,0).

7. After decanting the third wash, resuspend in 4 ml of Tris buffer (TBS; pH 7,5).

The aggregated particles are used for cluster assay.

Example 3 illustrates a method for confirming cachectin (Cx) binding to Cx MAb particles by clustering the particles.

1.5. uIl/mj! Prepare a Cx solution containing TBS.

2. Pipette 10 μl of Cx MAb particles into the well.

3. Cx solution (0 to 50 μm) was poured into the cavity from O to 1
Dilute to 0 μl.

4. Adjust the solution in the well to 50 μl with TBS.

5. Incubate at room temperature for 2 hours with rocking.

6. Add 10 μl of aggregated particles to each enrichment.

7. Pipette 90 μl of TBS into the well corresponding to the first well.

8. Pipette 10μ2 from each first well and add to the second corresponding well (110 dilution) at 9° room temperature.
Incubate for an hour with gentle rocking.

10. Observe the enrichment under the bright field of a microscope with low magnification.

11. Evaluate the aggregation of the depressions using an evaluation score of 1+ to 4+ for the aggregation pattern.

Toru Ohki ↓ Example 4 illustrates that magnetically responsive particles can be placed into the gingival sulcus of a monkey and retrieved with an electromagnet.

Fill a 1.1 mff syringe with 1 ml of particle suspension.

2. Using an anesthetized monkey, remove the quadrant of the jaw with a cotton swab.

3. Attach a flexible tube with a narrow hole to the tip of the syringe,
A tube is placed at the entrance to the gingival sulcus and at the lower end of the gingival tissue.

4. Administer the solution from the syringe into the groove.

5. An electromagnet with a sterile tip measuring 8 ml in length and approximately 4 mm in diameter, which can be held in the hand (capable of generating a magnetic field of 3750 Gauss), is inserted into the gingival sulcus. The tip of the magnet must be in contact with the tissue. Particles are observed to gather at the tips of the magnets.

6. The number of aggregated particles can be quantified by direct counting under a microscope using a counter graduated with "emission lines".

Example 5 exemplifies manual cluster measurement. Clusters are visualized using an Olympus inverted microscope equipped with a Hoffmann phase contrast apparatus and an attached Polaroid camera. 1.5 ml Enopendorf polypropylene tubes are used as culture vessels. final i'aW
1 to a mounting glass with a coverslip or use a Linpro 96-well plate as a reaction vessel. M
Before use, Ab beads were diluted to concentrations of the order of 7 ml of l x 10' beads, 1500 pM, 500 pM and 16 pM.
React with Cachectin solution of M. These mixtures are incubated at room temperature for 3 minutes, then polyclonal beads with a 20-fold concentration of MAb are added and allowed to react for 10 minutes. Linpro 9
The 6-well plate was found to be most suitable for the assay with minimal distortion of the flat bottom of the wells compared to those from other manufacturers. Due to the affinity of cachectin for glass, plastic plates are optimally used. The photograph in Figure 6 shows the clusters obtained by this method.

Example 6 illustrates a method for attaching a cachectin/TNF (Cx) monoclonal antibody to magnetic particles.

1. Pre-activated magnetically responsive particles (approximately 30 μg
Pipette 200 μe of /mJ) into a vial.

2. Collect the particles at the bottom of the vial with a magnet and remove the liquid. Resuspend the beads in 1 ml of water.

3. Wash by stirring once with water and repeat step 2.

4. Prepare a monoclonal antibody (MAb) container with a final concentration of 150 μg/ml by dissolving the antibody in 0.2 M Il phosphate buffer (pH 9,5).

5. Add 1 ml of MAb solution (from step 2) to 1 ml of beads (from step 2).

6. Incubate at room temperature for 24 hours with gentle rotation.

7. Collect the beads with a magnet and discard the supernatant.

8. Wash for 10 minutes with 1 mff1 of 0.1 M phosphate buffer.

Wash for 2 hours with 1 ml of 1 M ethanolamine hydrochloride (pH 9,5) containing 9, O, 1% Tou Bean 20.

10, 0. I MN a 0.05 M in C1 solution
) Squirrel, 0.1% Serum Albumin (BSA)
, 0.01% merthiolate, 20 1 mj of Oll % Tween! (pH 7.5) for about 12 hours.

11.) Wash for 2 hours with 1 ml of the mild i4i solution described in step IO, excluding Vienna 20.

12. Collect particles, discard supernatant liquid, 0. IMNaC#
200 μl of buffer containing 0.05 M l-Lys and 0.1% USA and 0.01% merthiolate in solution
Resuspend in When stored at 4°C, the particles are stable for at least 6 months.

Example 7 illustrates the preparation of aperture particles for use in cluster assays by attaching polyclonal antibodies (PAbs) to magnetic particles.

1-3. Same as Example 6.

4. Same as above except using PAb.

5-12. Same as Example 6.

Example 8 illustrates that Cx binding to MAb particles can be identified by clustering of the particles.

1, 0.05M) Liss hydrochloric acid (pH 7,5) and 0
．． 3000n of Cx in buffer containing 0.01% BSA
Prepare a Cx preservation solution containing M.

2. Use the MAb particles made in Example 6. Collect the beads from the storage solution with a magnet. Separate the buffer stock.

The beads were treated with Tris-HCl mfljiffl (pH 7,5
). This suspension is diluted 25 times with Tris buffer and further diluted 10 times with the same buffer. This MA
′M of b particle, ? r: Transfer 5 μl of J solution to the well with a pipette.

3. Perform serial dilutions of the stock solution of Cx (made in step 1) to create two new solutions containing 11000 nM and 333 nM of Cx. Make another solution containing only the buffer.

4. Add 10 μl from each of the three solutions containing Cx and 10 μl of dilution buffer from step 1 to the MAb-containing well made in step 2.

5. Incubate for 5 minutes at room temperature.

6. Use the PAb particles made in Example 7. Collect the beads from the stock solution, separate the buffer stock and resuspend the beads in Tris-HCl (pH 7.5). 6.0μ of this solution
Dilute 2 to 500 μl of Tris buffer and further dilute 30 μl of this solution to 300 μl of buffer. Add 30 μl of 41 g particles (PAb) to each well.

7. Incubate for 5 minutes at room temperature.

8. Observe using Hoffman phase contrast microscopy at low magnification.

9. Measure clusters of four or more beads within a limited field of view with a "net" micrometer. Repeat the measurements 10 times and average.

Figure 6 is a micrograph with clusters. FIG. 7 is a micrograph without clusters.

The data obtained from step 9 is illustrated by plotting the number of clusters observed at the three concentrations used in step 4 (ordinate) as a function of the concentration of 3111111 (abscissa). Subtract the number of clusters observed in the blank solution from each experimental value.

Example 9 illustrates that magnetically responsive particles can be placed into a human gingival sulcus and retrieved with an electromagnet.

Fill a 1.25 μl syringe (Durmond's “ordered” Pihenot) with approximately 15 μl of the MAb slurry made in Example 6.

2. Remove the quadrant from the human subject with a cotton swab.

3. Place the flexible plastic tip of the syringe at the entrance of the gingival sulcus and the lower end of the gingiva & 11 tissue.

4. Do you have a hard time? Administer & into the groove from a syringe.

5. An example of an electromagnetic sampling device is shown in Figure 1. ) insert the tip into the gingival sulcus. The excited magnet and particles are seen to collect on the tip of the magnet.

6. Remove the sampler from the subject's mouth and transfer it to the tray shown in Figure 2. The chip is placed in the well shown in Figure 3, the magnetic field is interrupted and the bristle brush is slowly rotated to remove particles from the chip.

7. Remove excess buffer.

8. Add PAb particles made in Example 7. Clusters are generated as described in Example 8.

9. The amount of CxO recovered from the gingival sulcus can be measured by directly counting the clusters under the microscope using low magnification Hoffmann phase contrast microscopy as described in Example 8.

The invention is illustrated by examples, but is not limited thereto. Many modifications and changes may be made to the invention without departing from the scope of the appended claims. For example, although the invention described above is directed to in-vivo methods of diagnosing disease states, it is also suitable for separating and recovering special compounds present in industrial processes and environments. The present invention can be applied to veterinary medicine and is also effective for drug administration. Such applications are particularly useful when liquid industrial waste contains toxic or other environmentally harmful compounds.

[Brief explanation of the drawing]

Figure 1 shows magnetic particles/ligands/ligates from the gingival sulcus.
Figure 2 shows a magnetic collection device for collecting x coalescence. FIG. 2 shows a tray for collecting complexes. FIG. 3 shows a sectional view taken along line 3-3 of the tray shown in FIG. FIG. 4 shows a schematic of the magnetic particle/ligand/ligate complex. FIG. 5 shows a schematic of a cluster of magnetic particles/ligand/ligate-a assembled particles. FIG. 6 is a photograph showing a cluster of magnetic particles/ligand/ligate-aggregated particles. FIG. 7 is a photograph showing non-specific rasterization in the control sample. FIG. 8 shows a calibration line plotting the number of clusters as a function of the cachectin concentration. FIG. 9 is a graph showing the correlation between cachectin and periodontal ligament disease. 10...Electromagnetic collector, 11...Handle, 12...
・Battery, 14... Electromagnetic coil, 16... Chip, 18... Collection tray 20... Sheet,
22・ 24.26... Arcuate part, 28... Permanent magnet, 32 33... Rigate, 34 36... Cluster 38... a particle collection.・Indentations, Ligands, ・Complexes, Rearranged Applicant's Representative The University of Connecticut

Claims (1)

[Claims] (1) A first ligand specific to the ligate in a liquid is attached to magnetically responsive particles, and the magnetically responsive particles are introduced into the liquid together with the attached first ligand. , the first ligand is bound to the ligate such that a magnetic particle/ligand/ligate complex is formed with the ligand attached to a magnetically responsive particle, the complex is retrieved from the liquid by a magnetic field, and the complex is removed from the liquid. 1. A method for measuring ligate concentration in a liquid, which comprises aggregating the complexes to form clusters of the complex, and measuring the ligate concentration in the liquid to quantify the number of clusters. (2) aggregating the complexes to form clusters, the step of aggregating the complexes to form clusters, comprises combining the agglomerated particles comprising the magnetically responsive particles and a second attached ligand capable of binding to the ligate with the magnetic particles/ligands/ligates; A method according to claim 1, characterized in that it is added to a complex. (3) the liquid is a body fluid, and the step of introducing the particles into the liquid together with the attached ligand, combining the ligand with the ligate to form a particle/ligand/ligate complex, and recovering the complex from the liquid, 2. The method according to claim 1, wherein the method is performed in vivo. 4. The method of claim 1, wherein the step of attaching a ligand further comprises the step of attaching a ligand, wherein the ligand is a monoclonal antibody. 5. The method of claim 1, wherein the step of attaching a ligand further comprises attaching the ligand to the magnetically responsive particle by a protein A linker. (6) The method according to claim 1, wherein the step of aggregating the complexes to form clusters further comprises the step of aggregating the complexes to form clusters such that the clusters have a size that is observable in the field of view of a low-magnification microscope. (7) The step of quantifying the number of clusters in order to measure the ligate concentration in the liquid further includes recording the microscope image on a suitable recording device and measuring the number of clusters present in the image using a computer counting method. 7. The method according to claim 6, characterized in that: (8) The step of quantifying the number of clusters to measure the ligate concentration in the liquid further comprises measuring the number of clusters with a counter adjustable for particles of different sizes. the method of. (9) Attach the first ligand, which is specific to the ligates present in the gingival exudate and is an indicator of periodontal ligament disease, to the magnetically responsive particles, and attach the magnetically responsive particles to the gingival tissue together with the first attached ligand. The magnetic particle/ligand/ligate complex is then introduced into the exudate fluid and bound to the first ligand such that a magnetic particle/ligand/ligate complex is formed with the ligand attached to the magnetically responsive particle, and the magnetic field causes the complex to invade the gingiva. Collected from the exudate,
A method for diagnosing periodontal ligament disease, which comprises aggregating the complex to form clusters of the particles, and quantifying the number of clusters in order to measure the ligate concentration, which is an indicator of periodontal ligament disease. (10) The method according to claim 9, wherein the step of attaching a first ligand specific to the ligate in the gingival exudate, which is an indicator of periodontal ligament disease, further comprises that the ligate is a cytokine. . (11) The step of attaching a first ligand specific to the ligate present in the gingival exudate and serving as an indicator of periodontal ligament disease is further characterized in that the ligate is selected from the group consisting of interleukin-1 and cachectin. Claim 10
The method described in. (12) Retrieving the particle/ligand/ligate complex further comprises collecting the complex with a magnetic collector comprising a hand-held electromagnet and having an attached tip anatomically adapted to the oral cavity. 10. A method according to claim 9, characterized in that it is collected. (13) The step of attaching the first ligand, which is specific to the ligate present in the gingival exudate and is an indicator of periodontal ligament disease, to the magnetically reactive particles further includes the step of attaching the first ligand, which is an immunoglobulin G monoclonal antibody, to the magnetically reactive particles. 10. A method according to claim 9, characterized in that. (14) The step of aggregating the complexes to form a cluster further includes magnetically reactive particles and aggregated particles containing an immunoglobulin G polyclonal antibody capable of binding to a ligate that is an indicator of periodontal ligament. 10. A method according to claim 9, characterized in that it is added to a ligand/ligate complex. (15) The method according to claim 9, wherein the step of aggregating the complex to form a cluster further comprises suspending the complex in a buffer solution. (16) The method according to claim 15, further characterized in that the buffer solution is 0.5M Tris HCl. (17) The method according to claim 15, further characterized in that the suspended substance is a non-reactive protein. (18) The method according to claim 17, further characterized in that the non-reactive protein is bovine serum albumin. (19) The method according to claim 15, further characterized in that the suspended substance is a surfactant. 20. The method of claim 19, further characterized in that the surfactant is selected from the group consisting of Tween 20 and Triton X. (21) A magnetic collector consisting of a hand-held electromagnet with an attached tip anatomically adapted to the oral cavity to retrieve magnetic particles/ligand/ligate complexes from the gingival sulcus. (22) A generally rectangular sheet with many depressions in which the complex is formed, each depression corresponding to a unique part of the tooth, and suspending the complex within each depression. A magnetic particle/ligand/ligate complex collection tray consisting of a cup containing the medium and a permanent magnet placed at the bottom of each well to facilitate collection of the complex.