Patent Application
20080124813
A method of small volume bead-based radioimmunoassay which includes utilization of an immunoassay particle having a solid phase substrate linked to a target particle capture moiety which dissociably captures target particles in an immunoassay sample. Specifically, a bead linked to antibody which competitively dissociably captures target particles and labeled particles of an immunoassay sample in a small volume bead-based radioimmunoassay to allow determination of the concentration of target particles, the level of protease activity, or the level of protease inhibitor activity in such immunoassay samples.
Radioactive elements, commonly referred to as radionuclides, are detectable because they emit energy in the form of alpha, beta or gamma rays as they decay. The use of radio-labeled proteins or peptides to trace biological activity may be one of the easiest and specific methods. The advantage of capturing radiolabeled antigen with antibody (hereinafter “radioimmunoassay”) can be that the signal of the radiolabeled antigen (whether a peptide or protein) is not affected, or only affected to a limited extent, by the physical state or chemical combination of the antibody with the radiolabeled antigen during the radioimmunoassay. This allows for the detection of a radiolabeled target particle such as a radiolabeled target antigen in complex mixture of biological particles from the radiation profile emitted.
A conventional radioimmunoassay (hereinafter “RIA”)(referring also to Example 1 below and
Conventional enzyme immunoassay (hereinafter “EIA”) or enzyme linked immunosorbent assays (hereinafter “ELISA”) can for example utilize biotin-labeled peptide to compete with unlabeled peptide for a limited quantity of antibodies and subsequently detects bound biotin complex by reaction with streptavidin horseradish peroxidase and sequent quantitative reaction of the horseradish peroxidase with colormetric or fluorescence substrates is quite similar to RIA. However, biotinylation of peptide or protein may not be as specific as iodination of the same peptide and can affect the binding of peptide to the antibody. Also, detection of fluorescence, chemoluminescence, or colormetric regents may be impaired by a high background. Analysis using fluorescence and chemiluminescence may also require detection and analytical instrumentation which may not be available in a medical environment or if available may not readily be utilized for high throughput of immunoassay samples. In contrast, a radioactive detector useful in the detection of I125-peptide, such as a gamma-counter, will likely be available and can be utilized for high throughput of immunoassay samples. Therefore, it remains a conventional practice to label peptides and proteins with radioactive atoms such as I125 and employ RIA for their biological measurement. Even though conventional RIA, EIA, and ELISA have been widely practiced for many years, there remain long felt but unresolved problems with these conventional procedures.
Another significant problem with conventional RIA can be that the formation of antigen antibody complex requires a long period of time to achieve equilibrium or that the primary antibody having the required specificity or affinity to the target particle cannot be solubilized, cannot be solubilized in suitable concentration, or can only be solubilized in an amount of liquid having a sufficiently large volume to render conventional RIA unsuitable or less suitable for detection of the corresponding target particle(s).
Another significant problem with conventional RIA can that precipitation of target protein or peptide for analysis may require the use of a solubilization material which may degrade the protein or peptide (such as trichloroacetic acid) making differentiation between degradation due to proteolysis and degradation due to solubilization (such as cleavage of disulfide bonds) difficult or impossible to assess.
Another significant problem with conventional RIA may be a lack of sensitivity. Conventional RIA methods as to certain immunoassay samples cannot increase concentration of antigen sufficiently to allow capture by antibody for detection. While it is known that abnormal levels of certain low abundance proteins and peptides (such as plasma orexin A and troponin) can be an indicator or biomarker of disease, the concentration or change in concentration of these low abundance proteins and peptides in immunoassay samples cannot be measured by conventional RIA or ELISA.
Another significant problem with conventional RIA can be that certain immunoassay samples have a volume too small to be analyzed by conventional RIA methods. Certain target particles may only be obtainable in small volume immunoassay samples because the animal only produces a limited volume of the liquid containing the target particle of interest whether due to the size of the animal or because the target particle is only present in fluids collectable in small volumes from larger animals.
Another significant problem with conventional RIA can be that the determination of concentration of a target molecule takes too long a period of time. This may be due to the necessity to obtain immunoassay samples of sufficient size, the duration of time to perform the conventional assay analysis which can be several days as to certain types of RIA methods, or that a plurality of target particles may have to be quantified which may require analysis of a corresponding plurality of immunoassay samples in serial or in parallel.
Another significant problem with convention RIA can be that degradation of only one target particle can be analyzed in a single assay procedure because the detection signal is generated from a common reporting system. However, in biological fluids a plurality of target particles such as ANP, amylin, and glucagon may be coincident and degradation analysis of each coincident target particle may be desirable from the same immunoassay sample.
Additionally, there are significant problems associated with other conventional non-radioisotopic methods of assessing target particles in solution such as EIA and ELISA. Assays such as EIA and ELISA which utilize fluorescent substrates may be subject to interference from environmental factors such as pH, ionic strength, temperature, or the like. Interference from environmental factors such as these can limit the detection sensitivity and the dynamic range of these conventional assays or be misinterpreted as an inhibition of an enzyme, thus requiring extensive follow-up assays to distinguish true positives from the false positives.
Also, as to certain fluorescent assays, cleavable substrates may only be available for specific proteases. For example, in the case of measuring the activity of matrix metalloproteinases (MMPs) in solution using fluorescence resonance energy transfer assay, the fluorescent substrate is labeled with a fluorescent rodamine group on one end of a peptide and a fluorescence resonance energy donor on the other. In the absence of MMP, the quatum dot (the “QD”) emission is red light (590 nm). When the peptides are cleaved by MMP, the rhodamine groups are released, and the QD emission changes to green light (545 nm). However, fluorescent substrates for many other proteases may not yet be available.
As to each of these significant problems with conventional RIA, EIA, ELISA, or the like, the invention affords a practical solution.
Accordingly, a broad object of the invention can be to provide an immunoassay particle which affords a solid substrate linked to at least one of a variety of target particle capture moieties which can be suspended in a immunoassay sample for the dissociable capture of target particles and subsequently isolated from the immunoassay sample without the conventional use of a secondary antibody and precipitation.
A second broad object of the invention can be to provide a method of immunoassay which can be performed with an amount of immunoassay sample containing target particle(s) which can be of lesser volume than utilized in performing conventional RIA, or a volume which limits or precludes the use of conventional RIA for the detection of target particles.
A third broad object of the invention can be to provide a method of immunoassay which allows detection of lesser concentration of target particles in an amount of sample solution as compared to conventional RIA, or allows detection of target particles at concentrations at which conventional RIA does not allow, or which limits or precludes the use of conventional RIA.
A fourth broad object of the invention can be to provide a method of immunoassay which allows detection of target particles in an amount of sample solution in less elapsed time as compared to conventional RIA, or allows detection of target particles in an amount of elapsed time which does not allow the use of conventional RIA.
A fifth broad object of the invention can be to provide a method of immunoassay which allows for coincident analysis of the rate of degradation of a plurality of target particles in the same sample solution. The coincident analysis of the degradation of a plurality of target particles in a single sample solution can be useful in the evaluation of the regulatory ability of a first target particle on a second target particle in the same physiological or pathophysiological condition, or the competition of the first target particle with the second target particle, or the inhibitory effect of the first target particle on the second target particle, or the like.
Naturally, further objects of the invention are disclosed throughout other areas of the specification, drawings, photographs, and claims.
A method of small volume bead-based radioimmunoassay which includes utilization of an immunoassay particle having a solid phase substrate linked to a target particle capture moiety which dissociably captures target particles in an immunoassay sample. Specifically, a bead linked to antibody which competitively dissociably captures target particles and labeled target particles of an immunoassay sample in a small volume bead-based radioimmunoassay to allow determination of the concentration of target particles, the level of protease activity, or the level of protease inhibitor activity in such immunoassay samples.
The term “immunoassay particle” means a solid phase substrate which includes at least one type of link element such as a molecule, functional group, ion, or atom which retains a target particle capture moiety having specificity for at least one target particle. The link element typically provides a functional group(s) such as amino groups or carboxyl groups available for linking to the target particle capture moiety in the presence of a crosslinking material, for example, glutaraldehyde, carbodiimide, diazoto compounds, or other suitable crosslinking material which generates a linking bond between the solid phase substrate and the target particle capture moiety which can be a covalent bond such as an amide, ester, ether, sulfonalmide, disulfide, azo, or the like, or as to other link elements a non-covalent bond such as physical adsorption, and specifically includes, without limitation of the forgoing, silanized iron oxide magnetic particles such as BIOMAG particle available from Polyscience, Inc., 400 Valley Road, Warrington, Pa. 18976 and non-magnetic particles available from Spherotech Inc. 1840 Industrial Dr., Suite 270, Libertyville, Ill. 60048. [catalog number].
The term “solid phase substrate” means a material such as latex, polystyrene, polypropylene, polyethylene, iron oxide, magnetic iron oxide which can further include a substrate matrix comprising a plurality of pores, interstices, or the like.
The term “immunoassay sample” means an amount of sera, tissue culture medium, plasma, or biological fluid such as blood, lymph, urine, sputum, joint fluid, spinal fluid, saliva, or an amount of a liquid which contains an amount of the target particle, and with respect to the various embodiments of the inventive immunoassay can be combined or diluted with an amount of a diluent to establish a concentration of the target particle in the inventive assay detection range, or a solution containing soluble target particle, molecular analogs of the target particle, or fragments thereof.
The term “target particle” means the molecule or molecules such as protein(s), protein fragment(s), peptide(s), antibody(ies), nucleic acid(s), or regions or fragments thereof, capable of dissociable capture by the target particle capture moeity.
The term “labeled particle” means a particle such as protein(s), protein fragment(s), peptide(s), antibody(ies), nucleic acid(s), or regions or fragments thereof, capable of dissociable capture by the target particle capture moeity which further includes a non-dissociably bound radioactive material such as 125 Iodine (“I125”).
The term “target particle capture moiety” means a molecule or molecules capable of dissociable capture of at least one target particle including, but not limited to a protein or polypeptide such as an antibody, or an antibody fragment, specific to a particular target particle which can be generated by various procedures for the production of polyclonal antibodies or monoclonal antibodies in rabbits, mice, rats, sheep, goats, horses, or the like, or produced by recombinant biological techniques.
Now referring primarily to
Now referring primarily to
When utilizing the immunoassay particle (2) in accordance with the invention, or as part of the inventive immunoassay methods, or the inventive immunoassay methods specifically described in Examples 3-12, the immunoassay particle (2) can be utilized in a lesser volume of immunoassay sample (6) as compared to conventional RIA to dissociably bind target particles (7) such as ghrelin, obestatin, amylin, or glucagons with similar or better results as compared to conventional RIA for a variety of applications. The immunoassay particle (2) can then be incubated with the immunoassay sample (6) for a period of time typically between 2 hours and 12 hours to allow dissociable capture (equilibrium between rate at which the target particle (7) is bound to the target particle capture moiety (4) and the rate at which the target particle (7) is released from the target particle capture moiety (4)) of the target particle (7) by the target particle capture moiety (4). In a subsequent step, an amount of labeled particle (9) can be combined with the immunoassay sample (6) to allow dissociable capture of the labeled particle (6) with the target particle capture moiety (4) in competition with the amount of target particle (7) in the immunoassay sample (6). As can be understood the greater the concentration of target particle (7) in the immunoassay sample (6) the less the amount of labeled target particle (9) that will be dissociably captured by the target particle capture moiety (4).
Because the inventive immunoassay particle (2) can then be removed or separated from the immunoassay sample (6) (whether by magnetic force, centrifugal force, or filtration) without the conventional use of a secondary antibody or the use of precipitation, and washed with an amount of wash solution (such as an amount 150 mM NaCl, 100 mM sodium phosphate, 1% BSA and 1% Triton X-100) as further described below, nonspecific binding of other particles (8) in the immunoassay sample (6) to the immunoassay particle (2) can be decreased, decreased as compared to conventional RIA, or substantially eliminated. As such, the background signal generated using the immunoassay particle (2) in accordance with the inventive immunoassay methods can be correspondingly decreased, decreased as compared to conventional RIA, or substantially eliminated. The corresponding increase in the signal to noise ratio obtained using the inventive immunoassay particle (2) or inventive immunoassay methods, or both, affords a decreased period of time to complete the inventive immunoassay method. As shown for example by Examples 4, 6 and 9, the inventive immunoassay method can be completed for a sample containing nesfatin in a one day period of time and for obestatin or ghrelin in a two day period as compared to the conventional method of radioimmunoassay for ghrelin as described by Example 1 which requires 3 days to achieve substantially the same results. As such, the inventive immunoassay method can afford a decreased sample volume and a decreased immunoassay time period with the same or similar sensitivity as compared to the conventional immunoassay methods.
Now referring primarily to Table 1 and
As can be understood from the results summarized in Table 1, the inventive immunoassay particle can be utilized in accordance with the methods of Examples 4 and 5 respectively to achieve results which are the substantially the same or better than utilizing the conventional RIA method with the further advantages of using a lesser volume of human plasma and performance of the assay in lesser period of time.
Now referring to Table 2, which summarizes the results using the inventive immunoassay system (1) in accordance with the procedures described in Examples 1, 4 and 5 to compare the inventive immunoassay method which utilizes an immunoassay sample (6) having a total volume of about 60 μL or about 150 μL to the conventional RIA method which utilizes a total volume of about 300 μL for the assay of the mouse peptide obestatin to produce a standard curve of mouse peptide obestatin concentrations of about 1 picogram per mililiter (“pg/mL”), about 2 pg/mL, about 4 pg/mL, about 8 pg/mL, about 16 pg/mL, about 32 pg/mL, about 64 pg/mL, and about 128 pg/mL to determine the concentration pg/mL or amount of mouse peptide obestatin in samples of mouse plasma. The numerical values represent a mean with S.E. from three discrete lots of silanized magnetic iron oxide beads each measurement taken in duplicate.
Again as can be understood from the results summarized in Table 2, the immunoassay particle (2) can be utilized in accordance with the methods of Examples 4 and 5 respectively to achieve results which are the substantially the same or better than conventional RIA methods with the further advantages of using a lesser volume of immunoassay sample (6) and to perform the immunoassay method in less time.
Now referring primarily
Now referring to Table 3 which provides a summary of the results of Example 7, it can be understood that the inventive immunoassay method can achieve consistent results even when the immunoassay sample (6) contains very low concentration of target particle (7). For example, when an original 20 μL immunoassay sample (6) of human saliva having a known concentration of ghrelin peptide of about 80.8 pg/mL is diluted to one half the original concentration of ghrelin peptide or one-twenty fifth the original concentration of ghrelin peptide by dilution (the original 20 μL sample brought to 40 μL or 500 μL respectively) for incubation with the immunoassay particle (2) and the subsequent incubation with the labeled particle (9), accurate determination can be made of the concentration of ghrelin peptide in the human saliva immunoassay sample (6).
Similarly, and now referring to Table 4, the concentration of a target peptide (7) such as the ghrelin peptide in an immunoassay sample (6) such as human plasma can be accurately determined over a wide range of concentrations in the immunoassay sample (6). As shown by Example 8, immunoassay samples (6) having a volume of 20 μL of human plasma of known ghrelin peptide concentration of 126 pg/mL can be diluted to reduce the original immunoassay sample (6) concentration by about 3 times, about 12 times, and about 25 times respectively prior to dissociable capture of the target particle (7) and subsequent dissociable capture of the labeled particle (9) by the inventive immunoassay method. Even at very dilute concentration, determination can be made of the concentration of ghrelin peptide in the original immunoassay sample (6).
Now referring to
Now referring to Table 5 and Example 10, an embodiment of the inventive immunoassay system (1) can be utilized to provide determination of a plurality of different target particles (7) in the same immunoassay sample (6)(also referred to as a “Mulitplex Assay”). For example, the embodiment of the inventive immunoassay system (1) utilized in Example 10 allows determination of concentration of orexin A, ghrelin, and obestatin in the same sample of human plasma. To achieve determination of concentration of a plurality of different target particles (7) in the same immunoassay sample (6), two different immunoassay particles (2)(see
As further shown by the results summarized by Table 5, the Multiplex Assay can be utilized to determine the concentration of at least two target particles (7)(7A) in single volume of immunoassay sample (6) of less than 100 μl and in those embodiments of the invention utilized to further determine a concentration of a target particle (7) which includes use of a conventional RIA methods than an immunoassay sample (6) of greater volume can be used such as 150 μl or more.
Now referring primarily
Because the inventive immunoassay particle (2) does not non-specifically bind fragments of degraded labeled particles (9) but continues to specifically bind fragments with intact epitopes, the inventive immunoassay particle (2) can be used to screen protein digests to identify the epitopes which bind particular antibodies. Generally speaking, the invention allows for the identification of antibodies which bind specific protein fragments or the identification of specific protein fragments which bind specific antibodies utilized as the target particle capture moiety (4) of the immunoassay particle (2). The protein fragments can be generated by protease cleavage of a peptide or protein using various proteolytic enzymes. Typically, the smallest synthetic peptide that will consistently elicit the production of antibodies that bind the original peptide or protein are about 6 residues in length. Certain protease cleavage sites can be predicted using a computer program, such as Peptidecutter, to analyze the peptide or protein to be treated with one or more proteases.
For example, ghrelin peptide in the naturally occurring form has 28 residues. Using a computer program such as Peptidecutter for analysis of cleavage, trypsin can cleave at positions 10,14,15,18, 23 and 27. Thermolysin can cleave at positions 2, 3, 10, 21, and 23. The anti-ghrelin antibody utilized to produce the results summarized by
Now referring primarily to Example 11 and
Now referring primarily to
As shown in
Conventional RIA methods can include the following steps:
3. Add 125I-labeled particle (typically an amount of 125I-labeled protein or peptide generating about 15,000 counts per minute by gamma counter) prepared by reaction of Na125I with the target particle (about 1 mCi per 100 μg of purified peptide or protein) and purification of the 125I-labeled particle by application to column chromatography gel (Bio-Gel P30 Bio-Rad #150-4154). Mix and incubate overnight (16-24 hrs.) at 4° C.
Total assay time between about 36 hours to about 48 hours.
Now referring to
A particular embodiment of the immunoassay particle (2) can be prepared as follows:
BIOMAG beads responsive to a magnetic field having a mean diameter of about 1.5 μm were obtained from Polysciences, Inc., 400 Valley Road, Warrington, Pa. 18976. The BIOMAG beads were activated by glutaraldehyde in 0.01 M pyridine immediately prior to conjugation of the antibody to the BIOMAG beads. Incubation of the BIOMAG beads in the presence of an antibody in 0.01 M pyridine overnight at 21° C. (1 to 500 μg antibody per mL supernatant depending upon the antibody conjugated and specifically with respect to anti-ghrelin polyclonal antibody about 500 μg/mL). The antibody-conjugated BIOMAG beads were separated from the supernatant by application of magnetic force. The separated BIOMAG bead conjugate was then incubated in 1 M glycine (pH 8.0) and 0.1% w/v BSA for 1 hr. on an orbital shaker. The antibody-conjugated bead was then washed five times with a wash buffer (0.01M Tris. 0.15M NaCl, 0.1% w/v BSA and 0.001 M EDTA) and then suspended in a solution containing 0.15M NaCl, 0.01M Tris, 0.1% BSA and 0.05% NaN3, pH 7.4, for storage.
The use of the BIOMAG bead as described is not intended to be limiting with respect to the numerous and varied solid phase substrates which can be utilized in the inventive immunoassay system (1) but rather provides an example sufficient for the person of ordinary skill in the art to make and use numerous embodiments of the inventive immunoassay system (1).
A particular embodiment of the immunoassay particle (2) can be prepared as follows:
Amino polystyrene beads having a mean diameter of about 11.2 μm were obtained from Spherotech, Inc., 1840 Industrial Dr. Suite 270, Libertyville, Ill. 60048. The amino polystyrene beads were activated by glutaraldehyde in 0.01 M pyridine immediately prior to conjugation of the antibody to the non-magnetic bead. Incubation of the non-magnetic beads in the presence of an antibody (such as 500 μg/mL anti-ghrelin polyclonal antibody) in 0.01 M pH 7.0 phosphate buffer overnight at 21° C. The antibody-conjugated non-magnetic beads was separated from the supernatant by centrifugation at 3000× g. The separated non-magnetic bead conjugate was then incubated in 200 mM ethanolamine (pH 7.0) and 0.1% w/v BSA for 1 hr. on an orbital shaker. The antibody-conjugated bead was then washed five times with a wash buffer (0.01M Tris. 0.15M NaCl, 0.1% w/v BSA and 0.001 M EDTA) and then suspended in a solution containing 0.15M NaCl, 0.01M Tris, 0.1% BSA and 0.05% NaN3, pH 7.4, for storage.
The use of the amino polystyrene bead as described is not intended to be limiting with respect to the numerous and varied solid phase substrates which can be utilized in the inventive immunoassay system (1) but rather provides an example sufficient for the person of ordinary skill in the art to make and use numerous embodiments of the inventive immunoassay system (1).
A particular embodiment of the immunoassay method for dissociable capture of radiolabeled ghrelin peptide by solid phase linked antibody can be performed as follows:
1. Combine 100 ul of I125-ghrelin (specific activity 3.15 p mole/ml) prepared in accordance with Example 1.3 (Example 1 step 3) to each of eight aliquots of 50 μL antibody-conjugated beads and to each of two aliquots of 50 μL glycine-conjugated beads prepared in accordance with Example 2B suspended in stock solution (0.01 M Tris (pH 7.4), 0.1% NaN3, 0.1% w/v bovine serum albumin (BSA), 0.15M NaCl, and 0.001M EDTA.
3. To each incubated mixture of antibody-conjugated beads and I125-ghrelin add 1.5 ml RIA buffer (150 mM NaCl, 100 mM sodium phosphate, 1% BSA and 1% Triton X-100), mix, and centrifuge for 5 minutes at 17,000× g. Aspirate off the supernatant.
4. Wash once with 1 ml RIA buffer. Aspirate off the supernatant
Now referring primarily to
A particular embodiment of the immunoassay method for competitive dissociable capture of radiolabeled ghrelin peptide by solid phase linked antibody to determine ghrelin peptide concentration in a 150 μl small volume immunoassay sample can be performed as follows:
5. Add 1.5 ml RIA buffer to incubated mixture and centrifuge for 5 minutes at 17,000× g. Aspirate off the supernatant (except total counts tubes).
6. Wash once with 1 ml RIA buffer. Aspirate off the supernatant (except total counts tubes).
Now referring primarily to
A particular embodiment of the immunoassay method for competitive dissociable capture of radiolabeled ghrelin peptide by solid phase linked antibody to determine ghrelin peptide concentration in a 60 μl small volume immunoassay sample can be performed as follows:
3. Add 10 μl 125I-peptide. Mix and incubate overnight (16-18 hrs.) at 4° C.
4. Add RIA buffer. Mix and centrifuge for 5 minutes at 1,700 g. Aspirate off the supernatant (except total count tubes).
5. Wash once with 1 ml RIA buffer. Aspirate off the supernatant (except total count tubes).
A particular embodiment of a one day small volume inventive immunoassay method for competitive dissociable capture of radiolabeled nesfatin peptide by solid phase linked antibody to determine nesfatin peptide concentration in rat plasma can achieve results comparable to conventional RIA methods.
An aliquot of 10 μL of magnetic beads prepared in accordance with Example 2A suspended in stock solution was combined with 10 μL of RIA buffer in 5 mL assay tubes. The resulting 20 μL mixture was combined with 20 μL of 100 μL or 200 μL, or 400 μL of rat plasma or with nesfatin standard and incubated at room temperature on an orbital shaker 250 rpm for 2 hr. The assay tubes were then placed in a magnetic field of sufficient strength to generate a pellet of the magnetic beads. The volume of supernatant was adjusted by aspiration to 40 μl and brought to 60 μl by addition of 20 μL of I125-labeled nesfatin. After the incubation with I125-nesfatin 2 hr, the dissociable capture of nesfatin were terminated by addition of 1.5 ml of RIA buffer. The magnetic beads were centrifuged for 20 min. at 1,700× g. The resulting pellet of magnetic beads was re-suspended with 1.5 ml of RIA buffer and centrifuged for 20 min. at 1,700×g. The supernatant was removed and the resulting pellets of magnetic beads were place in a Gamma counter to obtain counts per minute. The conventional RIA method of Example 1 was performed with 200 μL and 400 μL aliquots of rat plasma for comparison.
Using the conventional RIA of Example 1, the plasma concentration of nesfatin was interpolated from standard curves as about 1624±32 pg/mL. The immunoassay method utilizing the small volume immunoassay samples to determine the concentration of nesfatin in rat plasma yielded results of 1879±81, 1406±325, and 1937±66 pg/ml with respect to the corresponding 100 μL, 200 μL, and 400 μL immunoassay samples. The results of this embodiment of the inventive bead-based immunoassay are comparable to those obtained by the conventional RIA method of Example 1. However, there are substantial advantages in using the inventive immunoassay system because the assay time can be reduced from about three days to about one day.
A particular embodiment of a one day small volume inventive immunoassay method for competitive dissociable capture of radiolabeled ghrelin peptide by solid phase linked antibody was utilized to determine effects of immunoassay sample dilution on determination of ghrelin peptide concentration in human saliva.
Human saliva was collected and heated to 100° C. for 5 min and subsequently centrifuged for 10 min. at 15000 rpm. 0.5 mL of saliva sample or physiologic saline control were incubated with 20 μL of magnetic beads conjugated with anti-ghrelin antibody as described in Example 2A on an orbital shaker for 3 hr at room temperature. After incubation, the magnetic beads were separated by application of a magnetic field of sufficient strength to aggregate the magnetic beads in the saliva sample. An aliquot of 480 μL of the saliva sample was removed resulting in a 40 μL volume. An aliquot of 20 μL of 125I labeled ghrelin was added and incubated at 4° C. for 12 hr. The dissociable capture of 125I labeled ghrelin was terminated by addition of 1.5 ml of RIA buffer. The magnetic beads were centrifuged for 20 min. at 1,700×g. The resulting pellet of magnetic beads was resuspended with 1.5 ml of RIA buffer and centrifuged for 20 min. at 1,700×g. The supernatant was removed and the resulting pellets of magnetic beads were place in a Gamma counter to obtain counts per minute. The determined ghrelin concentration in human saliva samples by interpolation to a standard curve was 81.5 pg/ml ghrelin.
To compare the effect of different incubation volumes in determining ghrelin concentration in the inventive immunoassay system, samples of human saliva of 20 μL, 40 μL, and “20 μL diluted to 0.5 mL” were each assayed. Each human saliva sample was assayed as above described in this Example 7. The results in Table 3 indicate that a broad range of the dilution values can be utilized in the inventive immunoassay method in determining concentration of target particles in samples using the inventive immunoassay methods.
A particular embodiment of a small volume inventive immunoassay method for competitive dissociable capture of radiolabeled ghrelin peptide by solid phase linked antibody was utilized to determine effects of immunoassay sample dilution on determination of ghrelin peptide concentration in human plasma.
Human plasma was collected and a 20 μL aliquot, a 20 μL aliquot (brought to 60 μL with RIA buffer), and a 20 μL aliquot (brought to 500 μL with RIA buffer) were transferred to immunoassay tubes. A volume of 20 μL of immunoassay beads prepared in accordance with Example 2B suspended in stock solution was added to each assay tube. Standard samples and a 0.5 mL of peptide free plasma was spiked with four picograms ghrelin as a positive control for recovery rate calculation were prepared according to the same protocol. The resulting mixture in each assay tube was incubated on an orbital shaker for 3 hr at 300 rpm at room temperature. The incubated mixtures were centrifuged for 3 min at 15000 rpm for a brief duration to establish a pellet of immunoassay beads. From the 20 μL aliquot of human plasma brought to 60 μL with RIA buffer, 40 μL of the supernatant was removed, and from the 20 μL aliquot of human plasma brought to 500 μL with RIA buffer 460 μL was removed. Then 20 μL of I125-labeled ghrelin was added to bring the volume of each immunoassay sample to a total volume of 60 μL accordingly. The mixture in each immunoassay tube was incubated for a duration of 18 hrs on an orbital shaker at 250 rpm at 4° C. The incubated mixtures were centrifuged at 15000 rpm for 15 min to pellet the immunoassay beads and the supernatant was removed. The pellet was washed with 1.5 ml of RIA buffer and centrifuged at 15000 rpm for 15 min pellet the immunoassay beads. The wash solution was aspirated and the CPM was counted by gamma counter.
The results are set out in Table 4, a volume of 20 μL, 60 μL, and 500 μL and the final assay results of plasma ghrelin were 126, 118, and 135 pg/mL from 20 μL, 60 μL, and 500 μL, respectively which indicate that consistent results can be generated by the inventive immunoassay method regardless of variation in sample volume. Additionally, very dilute samples can be enriched by use of embodiments of the inventive immunoassay method to achieve results consistent with the assay in small volume of sample.
A particular embodiment of a two day small volume inventive immunoassay method for competitive dissociable capture of radiolabeled obestatin peptide by solid phase linked antibody was utilized to determine the range of linear detection.
Aliquots of human plasma of 5 μL (diluted to 20 μL with RIA buffer 1:4), 10 μL (diluted to 20 μL with RIA buffer 1:2), 20 μL or 100 μL were combined with 20 μL of antibody conjugated beads suspended in stock solution prepared in accordance with Example 2B. The resulting mixtures were incubated at 4° C. on a orbital shaker 250 rpm for 12-16 hrs and centrifuged at 15000 rpm 15 min. to pellet the antibody conjugated beads. A 20 μL aliquot of the supernatant was removed from each assay tube and replaced with a 20 μL aliquot of radio-labeled obestain. The resulting mixtures were incubated at 4° C. on an orbital shaker 250 rpm for 12-16 hrs. and then centrifuged to pellet the antibody conjugated beads. The supernatant was removed and the pellets of antibody conjugated beads were washed with 1.5 mL of wash solution followed by centrifugation at 15000 rpm 15 min. to pellet the antibody conjugated beads. The wash solution was removed and the pelleted antibody conjugated beads and the CPM counted by gamma counter. The counts were interpolated by comparison to a standard curve and the results were summarized in
A particular embodiment of a small volume inventive immunoassay method for competitive dissociable capture of radiolabeled ghrelin peptide by solid phase linked antibody was utilized to determine the concentration of three target particles in a single sample.
Aliquots of 200 uL or 60 uL of human plasma or physiologic saline control were incubated with 20 μL antibody-conjugated magnetic bead prepared in accordance with Example 2A in stock solution for dissociable capture of orexin A and antibody-conjugated non-magnetic beads prepared in accordance with Example 2B in stock solution for dissociable capture of ghrelin on an orbital shaker for 3 hr in room temperature. The antibody-conjugated magnetic beads were separated from the supernatant by application of magnetic field and introduced into a “first target particle assay” to determine concentration of orexin A. The remaining non-magnetic beads were separated from the supernatant by centrifugation at 2000×g for 10 min and introduced into a “second target particle assay”. The supernatant was saved and a portion transferred to each of the first target particle assay, the second target particle assay, and into the conventional RIA method of Example 1 as a “third target particle assay” as further described below.
To each sample of magnetic beads separated as above described, add 20 μL supernatant correspondingly collected from that sample. Subsequently, add 20 μL 125-I labeled orexin peptide prepared in accordance with Example 1.3 to each sample of magnetic beads to generate a total volume of about 60 μL. Incubate the resulting mixture for 8-12 hr. Pellet the magnetic beads by application of magnetic field or by centrifugation at 15000 rpm 15 min. and remove the supernatant by aspiration. After removal of the supernatant, the magnetic bead pellets are washed with 1.5 ml of RIA buffer and centrifuged at 15000 rpm 15 min pellet the magnetic beads and the RIA buffer removed. The counts per minute for each magnetic bead pellet was obtained by gamma counter.
To each sample of non-magnetic beads separated as above described, add 20 μL supernatant correspondingly collected from that sample. Subsequently, add 20 μL 125-I labeled ghrelin peptide prepared in accordance with Example 1.3 to each sample of non-magnetic beads to generate a total volume of about 60 μL. Incubate the resulting mixture for 8-12 hr. Pellet the non-magnetic beads by centrifugation at 15000 rpm 15 min. and remove the supernatant by aspiration. After removal of the supernatant, the non-magnetic bead pellets are washed with 1.5 ml of RIA buffer and centrifuged at 15000 rpm 15 min pellet the magnetic beads and the RIA buffer removed. The counts per minute for each non-magnetic bead pellet was obtained by gamma counter.
For each sample transfer 100 μL of the supernatant to an assay tube. Add 100 μL rabbit antibody solution and incubate 8-12 hrs at 4° C. Add 100 μL 125-I labeled third target particle to each assay tubes and incubate 8-12 hrs at 4° C. Then add 100 μL goat anti-rabbit antibody and 100 μL of normal rabbit serum to each assay tube and incubate at room temperature for 2 hr. Add 0.5 ml RIA buffer to each sample assay tube. Centrifuge for 20 min and aspirate off the supernatant from sample assay tubes and standard tubes but not total counts tubes. Determine CPM by gamma counter and interpolate counts to the standard curve to determine concentration of the third target particle in the original sample.
Three different digestion conditions were established to measure the inhibition of degradation of the ghrelin peptide by the inventive immunoassay system. The first digestion condition established a plurality of ghrelin samples digested with increasing concentration of trypsin enzyme without any inhibitor for 1 hr at 37° C.
The second condition established a plurality of ghrelin samples digested with increasing concentration of trypsin enzyme in the presence of peptide free heat inactivated human serum (“HS”) purchased from Scantianbodies Laboratory Inc. in Santee, Calif. The HS prior mixed with active charcoal (7% w/w) for 16 hours in room temperature and then centrifuged at 16000× g followed by filtration (0.22 um filter) to remove particulates and peptides. 20 μL of peptide free HS was then added to each digestion sample having different concentration of trypsin and then incubated for 1 hr at 37° C.
The third condition established a plurality of ghrelin samples digested with increasing concentration of trypsin enzyme in the presence of the protease inhibitor Aprotinin (0.3 TIU/ml), a bovine lung kallikrein inactivator purchased from Calbiochem in San Diego, Calif.
As to each digestion condition, the initial time was recorded and a 1 hr digestion period was allowed to elapse before adding of 10 μl of PMSF and 250 μl of 0.02M ammonium chloride (pH 5.0) to stop the digestion reaction. Then, each ghrelin sample digest was heated for 15 min at 85° C. and then frozen immediately on dry ice. The inventive RIA was carried by combining for each of the plurality of ghrelin digested samples 50 μL immunoassay bead presenting anti-ghrelin antibody as the capture moiety with 150 μL of radio-labeled ghrelin mixture for a total volume of 300 μL for each sample. After incubation of 1 hour, those tubes were centrifuged at 1700× g for 10 min to generate pellets. The pellets were washed with 1.5 ml of radioimmunoassay buffer once and the counts taken by gamma counter.
Specifically, with regard to the results shown in
Prior to performing the multiplex assay single peptide degradation plots were prepared for each of glucagon shown in
As shown by
As shown by
2. n each tube, 200 μL of above digestion solution add with I125-labeled peptide to incubate at 37° C. at the indicated time of 15, 10, 5, 3, 1 min to stop reaction and a tube of no incubation as the time zero.
As can be easily understood from the foregoing, the basic concepts of the present invention may be embodied in a variety of ways. The invention involves numerous and varied embodiments of an inventive bead-based radioimmunoassay and methods of using such embodiments of the inventive bead-based radioimmunoassy.
As such, the particular embodiments or elements of the invention disclosed by the description or shown in the figures or tables accompanying this application are not intended to be limiting, but rather exemplary of the numerous and varied embodiments generically encompassed by the invention or equivalents encompassed with respect to any particular element thereof. In addition, the specific description of a single embodiment or element of the invention may not explicitly describe all embodiments or elements possible; many alternatives are implicitly disclosed by the description and figures.
It should be understood that each element of an apparatus or each step of a method may be described by an apparatus term or method term. Such terms can be substituted where desired to make explicit the implicitly broad coverage to which this invention is entitled. As but one example, it should be understood that all steps of a method may be disclosed as an action, a means for taking that action, or as an element which causes that action. Similarly, each element of an apparatus may be disclosed as the physical element or the action which that physical element facilitates. As but one example, the disclosure of an “assay” should be understood to encompass disclosure of the act of “assaying”—whether explicitly discussed or not—and, conversely, were there effectively disclosure of the act of “assaying”, such a disclosure should be understood to encompass disclosure of an “assay” and even a “means for assaying.” Such alternative terms for each element or step are to be understood to be explicitly included in the description.
In addition, as to each term used it should be understood that unless its utilization in this application is inconsistent with such interpretation, common dictionary definitions should be understood to included in the description for each term as contained in the Random House Webster's Unabridged Dictionary, second edition, each definition hereby incorporated by reference.
Thus, the applicant(s) should be understood to claim at least: i) each of the bead-based radioimmunoassy devices or systems herein disclosed and described, ii) the related methods disclosed and described, iii) similar, equivalent, and even implicit variations of each of these devices and methods including but not limited to ELISA and EIA, iv) those alternative embodiments which accomplish each of the functions shown, disclosed, or described, v) those alternative designs and methods which accomplish each of the functions shown as are implicit to accomplish that which is disclosed and described, vi) each feature, component, and step shown as separate and independent inventions, vii) the applications enhanced by the various systems or components disclosed, viii) the resulting products produced by such systems or components, ix) methods and apparatuses substantially as described hereinbefore and with reference to any of the accompanying examples, x) the various combinations and permutations of each of the previous elements disclosed.
The background section of this patent application provides a statement of the field of endeavor to which the invention pertains. This section may also incorporate or contain paraphrasing of certain United States patents, patent applications, publications, or subject matter of the claimed invention useful in relating information, problems, or concerns about the state of technology to which the invention is drawn toward. It is not intended that any United States patent, patent application, publication, statement or other information cited or incorporated herein be interpreted, construed or deemed to be admitted as prior art with respect to the invention.
The claims set forth in this specification, if any, are hereby incorporated by reference as part of this description of the invention, and the applicant expressly reserves the right to use all of or a portion of such incorporated content of such claims as additional description to support any of or all of the claims or any element or component thereof, and the applicant further expressly reserves the right to move any portion of or all of the incorporated content of such claims or any element or component thereof from the description into the claims or vice-versa as necessary to define the matter for which protection is sought by this application or by any subsequent application or continuation, division, or continuation-in-part application thereof, or to obtain any benefit of, reduction in fees pursuant to, or to comply with the patent laws, rules, or regulations of any country or treaty, and such content incorporated by reference shall survive during the entire pendency of this application including any subsequent continuation, division, or continuation-in-part application thereof or any reissue or extension thereon.
The claims set forth in this specification, if any, are further intended to describe the metes and bounds of a limited number of the preferred embodiments of the invention and are not to be construed as the broadest embodiment of the invention or a complete listing of embodiments of the invention that may be claimed. The applicant does not waive any right to develop further claims based upon the description set forth above as a part of any continuation, division, or continuation-in-part, or similar application.
This United States Non-provisional patent application claims the benefit of U.S. Provisional Patent Application No. 60/859,762, filed Nov. 17, 2006, hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60859762 | Nov 2006 | US |
