阅读说明：本技术 一种定量检测肿瘤坏死因子α的试剂卡及其制备方法 (Reagent card for quantitatively detecting tumor necrosis factor alpha and preparation method thereof ) 是由 陈庆全 李伟甲 于 2019-10-28 设计创作，主要内容包括：本发明公开了一种定量检测肿瘤坏死因子α的试剂卡,包括盒体和设置在盒体内的试剂条,试剂条包括衬片,衬片上设有免疫硝酸纤维素膜,免疫硝酸纤维素膜的一端衔接有免疫荧光材料释放垫、另一端衔接有吸水纸；免疫荧光材料释放垫包被有TNFα单克隆抗体；免疫硝酸纤维素膜上设有TNFα检测线和质控线,TNFα检测线位于质控线与免疫荧光材料释放垫之间；盒体上开设有加样孔和观察窗。本发明还公开了定量检测肿瘤坏死因子α的试剂盒的制备方法。本发明采用干式荧光免疫层析技术及双抗体夹心法原理检测样本中肿瘤坏死因子α的浓度,可以有效的排除非特异性荧光的干扰,提高分析灵敏度和定量测定的准确性,适用于肿瘤坏死因子α的检测技术领域。(The invention discloses a reagent card for quantitatively detecting tumor necrosis factor alpha, which comprises a box body and a reagent strip arranged in the box body, wherein the reagent strip comprises a lining, an immune nitrocellulose membrane is arranged on the lining, one end of the immune nitrocellulose membrane is linked with an immunofluorescence material release pad, and the other end of the immune nitrocellulose membrane is linked with absorbent paper; the immunofluorescence material release pad is coated with a TNF alpha monoclonal antibody; the immune nitrocellulose membrane is provided with a TNF alpha detection line and a quality control line, and the TNF alpha detection line is positioned between the quality control line and the immunofluorescence material release pad; the box body is provided with a sample adding hole and an observation window. The invention also discloses a preparation method of the kit for quantitatively detecting the tumor necrosis factor alpha. The invention adopts dry-type fluorescence immunochromatography and double-antibody sandwich method to detect the concentration of the tumor necrosis factor alpha in the sample, can effectively eliminate the interference of nonspecific fluorescence, improves the analysis sensitivity and the accuracy of quantitative determination, and is suitable for the technical field of the detection of the tumor necrosis factor alpha.)

1. The utility model provides a quantitative determination tumor necrosis factor alpha's reagent card, includes the box body and sets up the reagent strip in the box body, its characterized in that: the reagent strip comprises a lining, wherein an immune nitrocellulose membrane is arranged on the lining, one end of the immune nitrocellulose membrane is connected with an immunofluorescence material release pad, and the other end of the immune nitrocellulose membrane is connected with absorbent paper;

the immunofluorescence material release pad is coated with a fluorescently-labeled TNF alpha monoclonal antibody;

the immune nitrocellulose membrane is provided with a TNF alpha detection line coated with a TNF alpha monoclonal antibody and a quality control line coated with a goat anti-mouse IgG polyclonal antibody, and the TNF alpha detection line is positioned between the quality control line and the immunofluorescence material release pad;

and a sample adding hole is formed in the box body corresponding to the immunofluorescence material release pad, and an observation window is formed in the box body corresponding to the TNF alpha detection line and the quality control line.

2. The reagent card for quantitatively detecting tumor necrosis factor alpha according to claim 1, wherein: the anti-TNF monoclonal antibody is a murine or rabbit antibody;

the polyclonal antibody is rabbit-derived, mouse-derived, chicken-derived and horse-derived.

3. The reagent card for quantitatively detecting tumor necrosis factor alpha according to claim 1 or 2, wherein: the TNF alpha monoclonal antibody coated by the immunofluorescence material release pad is marked by Eu fluorescent microspheres.

4. A method for preparing the reagent card for quantitatively detecting tumor necrosis factor alpha according to any one of claims 1 to 3, wherein the method comprises the following steps: comprises the following steps which are carried out in sequence,

firstly, preparing the immunofluorescence material release pad

Labeling an anti-TNF alpha monoclonal antibody-1 with a fluorescent material, preparing an anti-TNF alpha monoclonal antibody-1 solution labeled with an immunofluorescence material, adjusting the solution to a specified concentration range, spraying the solution on a release pad, and drying;

secondly, preparing the immune nitrocellulose membrane

Preparing a TNF alpha detection line by scribing an anti-TNF alpha monoclonal antibody-2 solution on a nitrocellulose membrane, preparing a quality control line by scribing a goat anti-mouse IgG solution on the nitrocellulose membrane, and drying;

third, sticking film

Sequentially sticking an immunofluorescence material release pad, an immunonitrocellulose membrane and absorbent paper on a lining to form a reagent strip;

fourthly, packaging

And (3) packing the dried reagent strip into a prepared box body to obtain the reagent card for quantitatively detecting the tumor necrosis factor alpha.

5. The method for preparing the reagent card for quantitatively detecting tumor necrosis factor alpha according to claim 4, wherein the method comprises the following steps: and after the fourth step is finished, filling the reagent card for quantitatively detecting the tumor necrosis factor alpha and the drying agent into an aluminum foil bag, sealing and packaging.

6. The method for preparing the reagent card for quantitatively detecting tumor necrosis factor alpha according to claim 4 or 5, wherein the reagent card comprises: the first step is carried out according to the following step sequence,

firstly), adding a marking buffer solution into a fluorescent material, and diluting the fluorescent material to 0.2-0.3% to obtain a solution A;

wherein the amount of fluorescent material = batch volume × target fluorescent material concentration 0.25% ÷ original fluorescent material concentration 1%, the amount of added labeling buffer = batch volume-fluorescent material volume;

secondly), centrifuging the solution A for 10-20 minutes under the conditions of 12000-13000rpm, discarding the supernatant, redissolving the precipitate by using a marking buffer solution, performing ultrasonic treatment, centrifuging for 10-20 minutes under the conditions of 12000-13000rpm, discarding the supernatant, adding the marking buffer solution, and performing ultrasonic treatment to obtain a solution B;

thirdly) adding the EDC solution with the concentration of 0.01-0.03mL/mL and the NHS solution with the concentration of 0.08-0.2mL/mL into the solution B, and uniformly mixing for 0.5-1.5 hours to obtain a solution C;

wherein, the dosage of EDC solution = batch volume (0.01-0.03) mL/mL, and the dosage of NHS solution = batch volume (0.08-0.2) mL/mL;

centrifuging the solution C for 10-20 minutes under the conditions of 12000-13000rpm, discarding the supernatant, redissolving the precipitate by using a marking buffer solution, and performing ultrasonic treatment to obtain a solution D;

adding the antibody into the solution D, and stirring for 2-4 hours to obtain a solution E;

wherein the antibody dosage = batch volume × target antibody concentration 0.1-0.3mg/mL ÷ antibody concentration

Centrifuging the solution E for 10-20 minutes under the conditions of 12000-13000rpm, removing the supernatant, adding the TBS solution with batch volume into the precipitate, ultrasonically mixing the solution uniformly, and stirring the solution for 3-5 hours to obtain a solution F;

centrifuging the solution F for 10-20 minutes under the conditions of 12000-13000rpm, re-dissolving the precipitated immune material into a batch volume by using TBS, and performing ultrasonic treatment at 160-220W to obtain an immunofluorescence material diluted solution;

fourthly) spraying the immunofluorescence material diluted solution to a release pad according to the parameter of 2.0-3.0 mu L/cm, and then drying for 4-24 hours at the temperature of 40-45 ℃ to obtain the immunofluorescence material release pad;

batch volume refers to the volume of the immunofluorescent material dilution solution.

7. The method for preparing the reagent card for quantitatively detecting tumor necrosis factor alpha according to claim 6, wherein the reagent card comprises: the second step is carried out according to the following step sequence,

① quality control line solution preparation

Measuring a goat anti-mouse IgG solution, diluting the goat anti-mouse IgG solution with a CT-S buffer solution until the final concentration is 0.8-1.5mg/mL, and storing the diluted goat anti-mouse IgG solution at the temperature of 2-8 ℃ for later use;

② TNF alpha detection line solution preparation

Measuring an anti-TNF alpha monoclonal antibody-2, diluting the anti-TNF alpha monoclonal antibody-2 by using a CT-S buffer solution until the final concentration is 0.8-1.5mg/mL, and storing the anti-TNF alpha monoclonal antibody-2 at the temperature of 2-8 ℃ for later use;

③ spraying the prepared quality control line solution onto the nitrocellulose membrane according to the parameter of 2.0-3.0 μ L/cm to prepare a quality control line, spraying the prepared TNF alpha detection line solution onto the nitrocellulose membrane to prepare a TNF alpha detection line, and drying the nitrocellulose membrane at 40-45 deg.C for 4-24 hr to obtain the immune nitrocellulose membrane.

8. The method for preparing the reagent card for quantitatively detecting tumor necrosis factor alpha according to claim 7, wherein the reagent card comprises: the third step is carried out according to the following step sequence,

i, sticking the immune nitrocellulose membrane at the middle position of the gasket;

II, attaching the water absorbing paper to the water absorbing paper end of the lining, covering the upper end of the attached immune nitrocellulose membrane downwards, covering the immune nitrocellulose membrane by 1.5-2 mm, and pressing;

III, attaching the immunofluorescence material release pad to the lower end of the immunonitrocellulose membrane, covering the immunonitrocellulose membrane by 1.5-2 mm, and pressing.

Technical Field

The invention belongs to the technical field of biology, and relates to a reagent card, in particular to a reagent card for quantitatively detecting tumor necrosis factor alpha and a preparation method thereof.

Background

Tumor necrosis factor is a cytokine with a wide range of biological activities. Shalalby named tumor necrosis factor alpha from macrophage in 1985, and research shows that the tumor necrosis factor alpha is closely related to apoptosis, and some cell surfaces have death receptors which sense cell cycle environment death signals and stimulate the apoptosis mechanism in cells through the binding of ligand and receptor. Tumor necrosis factor is a 17kDa nonglycosylated protein whose biologically active composition is in the form of a trimer. In addition to the soluble component alpha TNF, a membrane-bound component with a molecular weight of 17kDa is present on the surface of the TNF-producing cells, which acts as a tumor necrosis factor sink and is proteolytically separated from the cell surface.

The human TNF-. alpha.precursor consists of 233 amino acids (26 kDa) and contains a signal peptide consisting of 76 amino acid residues, which is cleaved off by the TNF converting enzyme TACE to form the mature 157 amino acid residues TNF-. alpha. (17 kDa). Since there is no methionine residue, there is no glycosylation site, with the two cysteines at positions 69 and 101 forming an intramolecular disulfide bond. Human TNF-alpha has 79 percent of amino acid composition homology with mouse TNF-alpha, and the biological effect of TNF-alpha has no obvious species specificity. Recently, it has been reported that 155 amino acid human TNF-alpha with less 2 amino acids (Val, Arg) at the N-terminal is expressed by genetic engineering technology, and has better biological activity and anti-tumor effect. In addition, 7 amino acid residues at the amino terminal of the TNF-alpha molecule are deleted by a genetic engineering method, 8Pro, 9Ser and 10Asp are changed into 8Arg, 9Lys and 10Arg, or 157Leu is changed into 157Phe at the same time, the activity of the modified TNF-alpha for killing L929 cells in vitro is increased by about 1000 times compared with the natural TNF, and the in vivo tumor hemorrhagic necrosis effect is also obviously increased. The natural form in which TNF- α and β exert their biological effects is a homotrimer.

Alpha tumor necrosis factor can be produced by a variety of cells, for example macrophages, CD4+ T cells and natural killer cells can produce alpha tumor necrosis factor following lipopolysaccharide stimulation. In addition, smooth muscle cells, segmented nucleus neutrophils, astrocytes and a large number of tumor cells can produce alpha tumor necrosis factor. The action of alpha tumor necrosis factor is produced by two distinct receptors on the cell surface, which are called alpha tumor necrosis factor receptor i (p55) and alpha tumor necrosis factor receptor ii (p75), respectively. These receptors are recognized on the surface of almost all cell types, except red blood cells. In addition to the cell binding component, the lytic component of the alpha tumor necrosis factor receptor is generally believed to have the ability to bind alpha tumor necrosis factor.

TNF alpha participates in the generation and development process of various immune inflammations and is a main medium of autoimmune diseases, systemic inflammatory response syndrome and the like; mainly produced by mononuclear macrophages, neutrophils, NK cells, activated T lymphocytes and the like; the biological activities of TNF α are very complex, including the regulation of hematopoiesis, immunity and inflammation, the effects on blood vessels and coagulation and on various organs (liver, heart, bone, cartilage, muscle and other tissues), the ability to enhance the effects of cytotoxic T cells, increase the expression of MHC antigens, causing leukocytosis and enhanced endothelial cell adsorption; in addition, it can inhibit various tumor cells and virus-infected cells. Normally, low levels of TNF α are present in plasma and have the effect of enhancing antiviral, antitumor, and anti-infective properties. TNF α plays an important role in inflammatory response, development of immune system, apoptosis and lipid metabolism, and is closely related to many diseases including asthma, crohn's disease, rheumatoid arthritis, neuropathic pain, obesity, diabetes, autoimmune diseases and tumors. However, since abnormality of TNF α has no disease specificity, detection of TNF α concentration in serum or body fluid cannot be a specific index for differential diagnosis of diseases, but can be an evaluation index for disease state change, therapeutic effect, and prognosis.

The tumor necrosis factor alpha is an important proinflammatory cytokine, the level of the tumor necrosis factor alpha is obviously increased within 24 hours in cerebrospinal fluid and serum of an acute stroke patient, a large amount of the tumor necrosis factor alpha can aggravate brain tissue damage and is positively correlated with the severity of the disease, and the tumor necrosis factor alpha plays an important role in inflammation caused by early stroke. Transient cerebral ischemia mouse model shows that tumor necrosis factor alpha can cause blood brain barrier permeability increase, and the anti-tumor necrosis factor antibody can obviously reduce blood brain barrier damage. Tumor necrosis factor alpha has an effect of damaging ischemic tissues and can induce damage to transplanted stem cells. The tumor necrosis factor alpha can also promote the continuous activation of NF-kappa B and induce apoptosis.

TNF alpha is one of important biological indexes of immunological research, and relates to occurrence mechanism, prognosis judgment and clinical treatment of various diseases such as infectious diseases, autoimmune diseases, tumors and the like, so that a simple and easy quantitative method is established, and accurate determination of TNF alpha levels in different samples has important significance for clinical research.

The POCT reagent has the advantages of simple operation, quick interpretation, low cost and the like, and is widely applied to the quick detection of diseases such as reproductive diseases, cardiovascular and cerebrovascular diseases, blood diseases, infection diseases, endocrine diseases and the like. Immunochromatography has been unable to perform accurate quantitative determination due to many interfering factors. The fluorescence immunochromatography method is characterized in that the antigen or the antibody is labeled by using a material, so that the interference of nonspecific fluorescence can be effectively eliminated, the analysis sensitivity is greatly improved, the accuracy of quantitative determination is improved, the detection method is convenient and easy to implement, the detection sensitivity and accuracy are high, the specificity is strong, a large number of samples can be simultaneously and rapidly detected, the time consumption of the method is low, and the precious time is saved for clinical patients.

If the fluorescence immunochromatography method can be adopted to detect the tumor necrosis factor alpha, the fluorescence immunochromatography method plays an important role in TNF-alpha related basic and clinical research.

Disclosure of Invention

The invention aims to provide a reagent card for quantitatively detecting tumor necrosis factor alpha, which adopts the fluorescence immunochromatography technology and the double-antibody sandwich method principle to measure the content of TNF alpha in human serum, plasma and whole blood samples.

The invention also aims to provide a preparation method of the reagent card for quantitatively detecting the tumor necrosis factor alpha.

In order to achieve the purpose, the invention adopts the following technical scheme:

a reagent kit card for quantitatively detecting tumor necrosis factor alpha comprises a lining, wherein an immune nitrocellulose membrane is arranged on the lining, one end of the immune nitrocellulose membrane is connected with an immunofluorescence material release pad, and the other end of the immune nitrocellulose membrane is connected with absorbent paper;

the immunofluorescence material release pad is coated with a fluorescently-labeled TNF alpha monoclonal antibody;

the immune nitrocellulose membrane is provided with a TNF alpha detection line coated with a TNF alpha monoclonal antibody and a quality control line coated with a goat anti-mouse IgG polyclonal antibody, and the TNF alpha detection line is positioned between the quality control line and the immunofluorescence material release pad;

and a sample adding hole is formed in the box body corresponding to the immunofluorescence material release pad, and an observation window is formed in the box body corresponding to the TNF alpha detection line and the quality control line.

As a limitation: the anti-TNF monoclonal antibody is a murine or rabbit antibody;

the polyclonal antibody is rabbit-derived, mouse-derived, chicken-derived and horse-derived.

As a second limitation: the TNF alpha monoclonal antibody coated by the immunofluorescence material release pad is marked by Eu fluorescent microspheres.

The preparation method of the reagent card for quantitatively detecting the tumor necrosis factor alpha comprises the following steps of sequentially carrying out,

firstly, preparing the immunofluorescence material release pad

Labeling an anti-TNF alpha monoclonal antibody-1 with a fluorescent material, preparing an anti-TNF alpha monoclonal antibody-1 solution labeled with an immunofluorescence material, adjusting the solution to a specified concentration range, spraying the solution on a release pad, and drying;

secondly, preparing the immune nitrocellulose membrane

Preparing a TNF alpha detection line by scribing an anti-TNF alpha monoclonal antibody-2 solution on a nitrocellulose membrane, preparing a quality control line by scribing a goat anti-mouse IgG solution on the nitrocellulose membrane, and drying;

third, sticking film

Sequentially sticking an immunofluorescence material release pad, an immunonitrocellulose membrane and absorbent paper on a lining to form a reagent strip;

fourthly, packaging

And (3) packing the dried reagent strip into a prepared box body to obtain the reagent card for quantitatively detecting the tumor necrosis factor alpha.

As a limitation: and after the fourth step is finished, the reagent card and the drying agent are filled into an aluminum foil bag, sealed and packaged.

As a second limitation: the first step is carried out according to the following step sequence,

firstly), adding a marking buffer solution into a fluorescent material, and diluting the fluorescent material to 0.2-0.3% to obtain a solution A;

wherein the amount of fluorescent material = batch volume × target fluorescent material concentration 0.25% ÷ original fluorescent material concentration 1%, the amount of added labeling buffer = batch volume-fluorescent material volume;

secondly), centrifuging the solution A for 10-20 minutes under the conditions of 12000-13000rpm, discarding the supernatant, redissolving the precipitate by using a marking buffer solution, performing ultrasonic treatment, centrifuging for 10-20 minutes under the conditions of 12000-13000rpm, discarding the supernatant, adding the marking buffer solution, and performing ultrasonic treatment to obtain a solution B;

thirdly) adding the EDC solution with the concentration of 0.01-0.03mL/mL and the NHS solution with the concentration of 0.08-0.2mL/mL into the solution B, and uniformly mixing for 0.5-1.5 hours to obtain a solution C;

wherein, the dosage of EDC solution = batch volume (0.01-0.03) mL/mL, and the dosage of NHS solution = batch volume (0.08-0.2) mL/mL;

centrifuging the solution C for 10-20 minutes under the conditions of 12000-13000rpm, discarding the supernatant, redissolving the precipitate by using a marking buffer solution, and performing ultrasonic treatment to obtain a solution D;

adding the antibody into the solution D, and stirring for 2-4 hours to obtain a solution E;

wherein the antibody dosage = batch volume × target antibody concentration 0.1-0.3mg/mL ÷ antibody concentration

Centrifuging the solution E for 10-20 minutes under the conditions of 12000-13000rpm, removing the supernatant, adding the TBS solution with batch volume into the precipitate, ultrasonically mixing the solution uniformly, and stirring the solution for 3-5 hours to obtain a solution F;

centrifuging the solution F for 10-20 minutes under the conditions of 12000-13000rpm, re-dissolving the precipitated immune material into a batch volume by using TBS, and performing ultrasonic treatment at 160-220W to obtain an immunofluorescence material diluted solution;

fourthly) spraying the immunofluorescence material diluted solution to a release pad according to the parameter of 2.0-3.0 mu L/cm, and then drying for 4-24 hours at the temperature of 40-45 ℃ to obtain the immunofluorescence material release pad;

batch volume refers to the volume of the immunofluorescent material dilution solution.

As a further limitation: the second step is carried out according to the following step sequence,

① quality control line solution preparation

Measuring a goat anti-mouse IgG solution, diluting the goat anti-mouse IgG solution with a CT-S buffer solution until the final concentration is 0.8-1.5mg/mL, and storing the diluted goat anti-mouse IgG solution at the temperature of 2-8 ℃ for later use;

② TNF alpha detection line solution preparation

Measuring an anti-TNF alpha monoclonal antibody-2, diluting the anti-TNF alpha monoclonal antibody-2 by using a CT-S buffer solution until the final concentration is 0.8-1.5mg/mL, and storing the anti-TNF alpha monoclonal antibody-2 at the temperature of 2-8 ℃ for later use;

③ spraying the prepared quality control line solution onto the nitrocellulose membrane according to the parameters of 2.0-3.0 muL/cm to prepare a quality control line, spraying the prepared TNF alpha detection line solution parameters onto the nitrocellulose membrane to prepare a TNF alpha detection line, and drying the nitrocellulose membrane at 40-45 ℃ for 4-24 hours to obtain the immune nitrocellulose membrane.

As a further limitation: the third step is carried out according to the following step sequence,

i, sticking the immune nitrocellulose membrane at the middle position of the gasket;

and II, attaching the water absorption paper to the water absorption paper end of the lining, covering the upper end of the attached immune nitrocellulose membrane downwards, covering the immune nitrocellulose membrane by 1.5-2 mm, and pressing.

III, attaching the immunofluorescence material release pad to the lower end of the immunonitrocellulose membrane, covering the immunonitrocellulose membrane by 1.5-2 mm, and pressing.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:

(1) the reagent card provided by the invention has the advantages that the detection method is convenient and easy to implement when in use, the detection sensitivity and accuracy are high, the specificity is strong, the recovery rate is between 85% and 115% in the linear range of concentration, and the linearity is as follows: within a linear range of (10-1000) pg/mL, the correlation coefficient r is more than or equal to 0.990, and meanwhile, a large number of samples can be detected rapidly, so that the time consumption is low, and precious time is saved for clinical patients;

(2) the blank limit of the reagent card provided by the invention is not higher than 5 pg/mL;

(3) the reagent card provided by the invention has the following repeatability: in the linear interval of the reagent card, low-concentration and high-concentration TNF alpha samples are repeatedly detected for 10 times respectively, and the coefficient of variation CV is less than or equal to 15 percent;

(4) the material used for preparing the fluorescent material is a mixture of substances containing any one or more of europium (Eu), samarium (Sm), erbium (Er) and neodymium (Nd) in rare earth lanthanide elements; the lanthanide ion chelate has a long fluorescence decay time which is 106 times that of the conventional fluorescence, such as: when the fluorescence is measured by a fluorescence analyzer, after the excitation of the pulse light source, the measurement can be carried out after a certain time delay, at the moment, the short half-life fluorescence of other components is already decayed, and only Eu exists³⁺Specific fluorescence of the marker, by measuring Eu on test strip after immunoreaction³⁺The content of the chelate can be quantitatively obtained by referring to a standard concentration curve to obtain the concentration of the corresponding antigen in the sample to be detected;

(5) the reagent card provided by the invention has high detection speed, only needs 10 minutes in the whole process, and can realize the detection of a single sample or a large number of samples; the sensitivity is high, and the lowest detection unit is pg/mL; strong specificity and no cross interference with other markers of inflammation or immune diseases; the operation is simple and convenient, the operator can finish the operation according to the instruction without professional training, and the popularization and the use are easy; the detection sample range is wide, whole blood, serum and/or plasma can be used as test samples, the required reagent and sample amount are small, and the sample amount can be as low as 50-100 mu L.

The invention is applicable to the technical field of detection of tumor necrosis factor alpha.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

In the drawings:

FIG. 1 is a schematic structural view of example 1 of the present invention;

FIG. 2 is a partial structural view of a reagent strip according to embodiment 1 of the present invention.

In the figure: 1. a box body; 2. an observation window; 3. a reagent strip; 31. a lining; 32. an immunonitrocellulose membrane; 33. an immunofluorescent material release pad; 34. absorbent paper; 4. a quality control line; 5. a TNF alpha detection line; 6. and (4) sample adding holes.

Detailed Description

Preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the present invention.