CN113514634A - Preparation of double-index immunochromatography kit for detecting Adipo/SHBG - Google Patents

Abstract

The invention belongs to the field of immunodetection, and particularly relates to a double-index immunochromatography kit for detecting adiponectin (Adipo) and Sex Hormone Binding Globulin (SHBG). The detection line 1(T1 line) is formed by fixing the Adipo antibody on a nitrocellulose membrane, the detection line 2(T2 line) is formed by fixing the SHBG antibody, and the quality control line (C line) is formed by fixing the anti-IgG antibody. The invention also discloses a coupling microsphere process of the Adipo antibody and the SHBG antibody. The invention uses the immunochromatography kit for detecting the Adipo/SHBG to detect the concentrations of Adipo and SHBG in blood or other body fluids, and is used for early predicting insulin resistance, type 2 diabetes and the risk of gestational diabetes.

Description

Preparation of double-index immunochromatography kit for detecting Adipo/SHBG

Technical Field

The invention belongs to the field of immunology, and particularly relates to a process for preparing an Adipo antibody-fluorescent microsphere and an SHBG antibody-fluorescent microsphere and establishing a fluorescence immunochromatography kit for detecting Adipo/SHBG.

Background

By 2030, the worldwide population of diabetics is expected to reach 3.6 billion, with the majority of these cases being Type 2 Diabetes mellitis, T2 DM. Diabetes mellitus is often associated with two metabolic defects, insulin resistance and impaired pancreatic beta cell function. However, factors such as genetic predisposition, obesity, pregnancy, female hormonal increase, etc. contribute to the development of diabetes. The traditional diabetes diagnosis adopts fasting blood glucose, postprandial blood glucose and oral glucose tolerance tests to detect glycosylated hemoglobin and glycosylated albumin indexes, but does not have biological indexes for predicting the occurrence of diabetes, and needs to develop the biological indexes for predicting the occurrence of diabetes.

It has been found that Adiponectin (adipeonectin, Adipo) and Sex hormone-binding globulin (SHBG) levels in blood can predict the risk of diabetes.

Adipo, also known as 30KDa adipocyte complement-associated protein (Acrp30), is an adipocyte-derived hormone that is produced by other cell types, including skeletal cells, cardiac myocytes, and endothelial cells. Adipo consists of 244 amino acids, structurally similar to collagen and C1q complement factor, and possesses a short N-terminal variable region, connecting several collagen repeats, a large C-terminal globular domain. Adipo forms multiple higher-order structures, Adipo monomers aggregate into homotrimers, three globular domains are adjacent, three collagen-like regions form collagen triple helices, and trimers reassemble into homohexamers and High Molecular Weight (HMW) multimers.

Adipo is a protein abundant in blood circulation and involved in homeostatic control of circulating glucose and lipid levels. The various types of Adipo multimers act differently on hepatocytes and skeletal muscle cells. The Adipo hexamer or HMW multimer inhibits glucose production by activating AMP-protein kinase (AMPK) in hepatocytes, and can stimulate glucose uptake by acting on myocyte AMPK; adipo also increases fatty acid oxidation and energy consumption in peripheral tissues, promotes ATP production, and thereby reduces intracellular triglyceride levels and decreases insulin resistance.

Adipo has anti-atherosclerotic, inflammatory response inhibiting and insulin sensitivity increasing effects. Levels of Adipo in the blood are negatively associated with obesity, hypertension, blood lipids and coronary heart disease, and decreased levels of Adipo are associated with increased risk of developing obesity, insulin resistance and T2 DM. The level of Adipo in blood was tested for the assessment of T2DM development, particularly in the population with a history of T2DM parents, Adipo being a strong pre-diabetic risk assessment marker. The Adipo level of the people in the early stage of diabetes is lower than that of the healthy people; increased levels of Adipo are associated with a decreased risk of T2DM and subsequently reduce the incidence of cardiovascular disease.

Another study showed that, at the early pregnancy stage of less than 13 weeks, plasma Adipo concentrations were less than 6.4ug/ml, the risk of developing Gestational Diabetes Mellitus (GDM) was increased 4.6 times, and the reduction in Adipo levels was strongly correlated with GDM development after removal of known GDM risk factors. The Adipo may play a role in the pathophysiological process of GDM, levels of Adipo in serum are down-regulated several months before the GDM is clinically diagnosed, and detection of Adipo levels in blood can be used for predicting the risk of GDM occurrence.

The Adipo indexes are not conventional detection items, and when the Adipo indexes are detected more and more widely, the Adipo indexes are used for predicting the risks of T2DM and GDM, and an immunological method is required to be established for detecting the Adipo kit.

SHBG is a glycoprotein produced by the liver, also known as testosterone-estradiol binding globulin, which is a carrier for sex hormone transport, but during the action of sex hormones, it exerts its influence in a variety of physiological and pathological conditions. In bound testosterone, 44% -60% binds SHBG; estrogen binds to SHBG specifically about 95% of its circulation, allowing sex hormones to be transported in the blood in an inactive form. SHBG levels in circulation are affected by age and sex, testosterone and estrogen levels, as well as certain diseases and conditions, such as liver disease, hyperthyroidism or hypothyroidism, and obesity.

Insulin and insulin-like growth factors result in the inhibition of SHBG secretion, and in the insulin resistant state, a decrease in SHBG levels is detected, which has been considered a marker of insulin resistance. Epidemiological studies have found that there is a link between decreased testosterone in the male serum and subsequent central obesity, increased insulin levels, metabolic syndrome and diabetes, while elevated testosterone in the female blood is closely associated with glucose intolerance and insulin resistance, moving towards diabetes. In obese, insulin resistant, metabolic syndrome and type 2 diabetic populations, SHBG levels are reduced and circulating SHBG concentrations become a potential predictor of T2DM development.

The research shows that insulin resistance is a danger signal of GDM, the SHBG level of GDM and insulin treatment women is low, in addition, the SHBG concentration in the first trimester of pregnancy is reduced, and the GDM suffering risk is obviously increased in the later stage of pregnancy. A prospective study showed that evaluating 269 pregnant women at 15 weeks for several biomarkers showed that low levels of SHBG were associated with increased risk of GDM, independent of other risk factors. Using a 211.5mM/L cut-off value, detection of SHBG levels predicted a sensitivity of 85% for GDM generation and a specificity of 37%. Detecting SHBG levels in blood can help predict GDM occurrence.

Meanwhile, the kit for detecting the Adipo and the SHBG has the advantages that the specificity and sensitivity of T2MD and GDM are improved, and the kit for quantitatively detecting the Adipo and the SHBG in blood is simple and quick to develop.

The invention establishes an immunochromatography kit for detecting Adipo/SHBG, measures the levels of Adipo and SHBG in blood, is used for judging insulin resistance and predicting the occurrence risk of T2MD and GDM.

Disclosure of Invention

The invention discloses an immunochromatography kit for detecting Adipo and SHBG in blood, which mainly comprises an immunochromatography test strip, wherein the immunochromatography test strip is formed by sequentially connecting and fixing a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper on a PVC (polyvinyl chloride) bottom plate, and the figure 1 is a schematic diagram of the immunochromatography test strip. Adsorbing an Adipo antibody-microsphere and an SHBG antibody-microsphere on the binding pad; the nitrocellulose membrane is provided with a fixed Adipo antibody forming detection line 1(T1 line), a fixed SHBG antibody forming detection line 2(T2 line) and a fixed anti-IgG antibody forming quality control line (C line).

Chromatography liquid: 20-200mM Tris, pH7.0-8.0, 0.1-1.0% BSA, 100-400mM NaCl, 0.1-1.0% Tween-20.

The microsphere can be one of a fluorescent microsphere, a colored microsphere, a gold-labeled microsphere, a magnetic microsphere and an up-conversion luminescent microsphere, and the europium chelate fluorescent microsphere is selected, wherein the exciting light wavelength is 360-650 nm, and the emitting light wavelength is 600-650 nm.

The surface modification functional group of the microsphere is one of carboxyl, hydroxyl or epoxy, and preferably the carboxyl modified fluorescent microsphere. The diameter range of the fluorescent microspheres is 100-300nm, and the fluorescent microspheres with the diameters of 100nm and 200nm are preferred.

The invention discloses a preparation process of an Adipo antibody and SHBG antibody coupled fluorescent microsphere, which comprises the following steps:

(1)5-20mM carbodiimide (EDC) and 10-50mM N-hydroxyl thiosuccinimide (Sulfo-NHS) activated carboxyl modified fluorescent microspheres; (2) adding an Adipo antibody or an SHBG antibody for coupling reaction; (3) adding 100-400mM Tris solution to terminate the coupling reaction; (4) and (3) after washing, re-dissolving the Adipo antibody-fluorescent microspheres and the SHBG antibody-fluorescent microspheres by using a Tris buffer solution, and storing at the temperature of 2-8 ℃ for later use.

The invention discloses a preparation process of a conjugate pad adsorbed with an Adipo antibody-fluorescent microsphere and an SHBG antibody-fluorescent microsphere, which comprises the following steps:

and (3) diluting the concentration of the Adipo antibody-fluorescent microspheres to 0.1-1.0mg/ml and the concentration of the SHBG antibody-fluorescent microspheres to 0.05-0.5mg/ml by using Tris buffer solution, spraying the fluorescent microspheres on a bonding pad at the speed of 2-10ul/cm, drying at the temperature of 25-37 ℃ for 4-12 hours, drying and sealing for later use.

The invention discloses a preparation process of a T line and a C line on a nitrocellulose membrane, which comprises the following steps:

diluting the Adipo antibody to 1.0-4.0mg/ml, SHBG antibody to 0.5-2.5mg/ml and anti-IgG antibody to 0.5-2.5mg/ml with phosphate buffer solution, spraying the antibody solution onto nitrocellulose membrane at a speed of 0.4-1.0ul/cm to form T1 line, T2 line and C line at an interval of 4-6mm at 25-37 deg.C, drying for 4-12 hr, drying, and sealing.

The invention discloses an immunochromatographic test strip assembly process:

sticking a nitrocellulose membrane in the middle of the PVC bottom plate, sticking absorbent paper at one end, locating above the nitrocellulose membrane, and overlapping for 2-3 mm; the other end is stuck with a bonding pad, is positioned above the nitrocellulose membrane and is overlapped for 2-3 mm; and adhering a sample pad on the other end of the combination pad, overlapping the sample pad by 2-5mm to form a test paper board, and cutting into test paper strips with the width of 3-5 mm.

The invention discloses an immunochromatography test paper card assembly process which comprises the following steps:

the invention comprises a shell which is composed of a shell surface and a shell base. And (3) placing the test strip on a shell base, and closing the shell surface to form the test strip card. A detection window is arranged on the shell surface corresponding to the position of the nitrocellulose membrane, and a sample hole is arranged on the shell surface corresponding to the position of the sample pad.

The invention adopts the principle of double-antibody sandwich immunochromatography: the method comprises the following steps that Adipo and SHBG in a sample move forwards through a chromatography effect and are combined with a combination pad to adsorb an Adipo antibody-fluorescent microsphere or an SHBG antibody-fluorescent microsphere to form an Adipo-Adipo antibody-fluorescent microsphere compound or an SHBG-SHBG antibody-fluorescent microsphere compound; under the action of capillary power, the diameter of a fluorescent microsphere of the SHBG-SHBG antibody-fluorescent microsphere compound is 100nm, the fluorescent microsphere enters a nitrocellulose membrane firstly, and is combined with a fixed SHBG antibody at a T2 line of the nitrocellulose membrane through a T1 line to form a fluorescent microsphere-SHBG antibody-SHBG-SHBG antibody compound which is gathered at a T2 line; the diameter of a fluorescent microsphere of the fluorescent microsphere composite is 200nm, the fluorescent microsphere enters a nitrocellulose membrane, and is combined with an Adipo antibody fixed on a nitrocellulose membrane T1 line to form the fluorescent microsphere-Adipo antibody-Adipo antibody composite which is gathered on a T1 line; the free Adipo antibody-fluorescent microsphere or SHBG antibody-fluorescent microsphere does not bind at the T1 line or T2 line, continues to move forward, and binds with the C line immobilized anti-IgG antibody; the unbound components continue to move to the absorbent paper position. The quantity of fluorescent microspheres captured by the T1 line is positively correlated with the concentration of Adipo in the sample, the quantity of fluorescent microspheres captured by the T2 line is positively correlated with the concentration of SHBG in the sample, and the quantity of fluorescent microspheres captured by the C line indicates that the immunochromatography process is completed. Scanning signals of a T1 line, a T2 line and a C line by an immunofluorescence analyzer, and obtaining the concentration of the Adipo in the sample according to the positive correlation between the signal intensity of the T1 line and the concentration of the Adipo in the sample; the SHBG concentration in the sample is obtained by positively correlating the intensity of the T2 line signal with the SHBG concentration in the sample.

Immunofluorescence analysis appearance, its characterized in that: the excitation wavelength is 350-430nm, and the receiving wavelength is 600-650 nm. The analyzer is used for interpreting the test paper card result, can realize automation, and provides a simple, quick and reliable detection result.

Adding an Adipo/SHBG calibrator into a sample hole of an Adipo/SHBG test paper card, reacting for 5-20 minutes, measuring the fluorescence intensity of a T1 line, a T2 line and a C line on the test paper card by using an immunofluorescence analyzer, preparing an Adipo and SHBG concentration standard curve, obtaining parameters related to Adipo and SHBG concentration and fluorescence intensity units, setting an immunofluorescence analyzer system for automatically detecting the Adipo and SHBG concentrations, and further detecting the Adipo and SHBG concentrations in a sample.

The immunochromatography kit for detecting the Adipo/SHBG is simple to operate, is rapid and specific, quantitatively determines the concentrations of the Adipo and the SHBG in blood, and can meet the requirements of clinical examination.

Drawings

FIG. 1 is a schematic diagram of an immunochromatographic test strip

FIG. 2 Immunochromatographic test card front

FIG. 3 Adipo concentration versus RFU correlation

FIG. 4 correlation curve of SHBG concentration with RFU

Detailed Description

The principles and results of the present invention are further illustrated in conjunction with specific experiments, which set forth the multi-step experimental procedures of the present invention.

Preparation and detection of Adipo/SHBG immunochromatographic test strip

Coupling Adipo antibody to europium chelate fluorescent microsphere

In the present example, Europium Chelate (Europium Chemate) fluorescence-labeled carboxyl-modified microparticles (Europium Chemate PS-COOH) with a diameter of 100-300nm were purchased from Bangs Laboratories Inc.

In the present example, an Adipo monoclonal antibody, purchased from Medix Biochemical, was conjugated to Europium Chemate PS-COOH using EDC and Sulfo-NHS in the following procedure:

1. 0.1ml of Europium Chemate PS-COOH, 10mg/ml in concentration and 200nm in diameter, was put into a 1.5ml centrifuge tube, centrifuged at 15000rpm for 15 minutes, and the supernatant was removed.

2. Then 1.2ml of 20mM MES buffer, pH6.0, was added to the centrifuge tube and the microspheres were suspended.

3. Centrifugation at 15000rpm for 15 minutes removed the supernatant.

4. Then, 0.2ml of the MES buffer containing 5mM EDC and 10mM Sulfo-NHS was added to the centrifuge tube, mixed, and reacted at 25 ℃ for 30 minutes.

5. Add 1.2ml of MES buffer to the centrifuge tube.

6. Centrifugation at 15000rpm for 15 minutes removed the supernatant.

7. 0.2ml of PBS containing 60ug of the Adipo monoclonal antibody at pH7.4 was added to the above centrifuge tube and reacted at 25 ℃ for 2 hours.

8. 20ul of 200mM Tris, pH7.4 was added to the centrifuge tube and reacted at 25 ℃ for 30 minutes.

9. Add 1.2ml Tris buffer, 20mM Tris, pH7.40.2% BSA, 0.01% Tween-20 to the centrifuge tube.

10. Centrifugation at 15000rpm for 15 minutes removed the supernatant.

11. The Adipo antibody-fluorescent microspheres were suspended in the above Tris buffer and stored at 4 ℃ for further use.

Coupling SHBG antibody to europium chelate fluorescent microsphere

In the examples of the present invention, SHBG monoclonal antibody, purchased from Medix Biochemical, was conjugated to Europium Chemate PS-COOH by the following steps:

1. 0.1ml of Europium Chemate PS-COOH, 10mg/ml in concentration and 100nm in diameter, was put into a 1.5ml centrifuge tube, centrifuged at 20000rpm for 20 minutes, and the supernatant was removed.

2. Then 1.2ml of 20mM MES buffer, pH6.0, was added to the centrifuge tube and the microspheres were suspended.

3. Centrifugation at 20000rpm for 20 minutes removed the supernatant.

4. 0.2ml of MES buffer containing 5mM EDC and 10mM Sulfo-NHS was added to the centrifuge tube, mixed, and reacted at 25 ℃ for 30 minutes

5. Add 1.2ml of MES buffer to the centrifuge tube.

6. Centrifugation at 20000rpm for 20 minutes removed the supernatant.

7. 0.2ml of PBS containing 80ug of SHBG monoclonal antibody at pH7.4 was added to the above centrifuge tube and reacted at 25 ℃ for 2 hours.

8. 20ul of 200mM Tris, pH7.4 was added to the centrifuge tube and reacted at 25 ℃ for 30 minutes.

9. Add 1.2ml Tris buffer 20mM Tris, Ph7.4, 0.2% BSA, 0.01% Tween-20 to the centrifuge tube.

10. Centrifugation at 20000rpm for 20 minutes removed the supernatant.

11. And suspending the SHBG antibody-fluorescent microspheres in the Tris buffer solution, and storing at 4 ℃ for later use.

(III) treatment of sample pad and conjugate pad

The invention selects a glass fiber MF1 sample pad, purchased from GE Healthcare, and soaked in a sample pad treatment solution, 20mM Tris, pH7.4, 0.5% BSA, 0.2% Tween-20, 0.01NaN3, dried for 1 hour at room temperature, dried for 6 hours at 30 ℃, dried and sealed for later use.

The invention selects a glass fiber GFDX binding pad which is purchased from EMD Millipore, soaked in a binding pad treatment fluid, 20mM Tris, pH7.4, 0.5% BSA, 0.1% PVP, 5% Sucrose, 0.2% Tween-20, 0.01% NaN3, dried for 6 hours at room temperature for 1 hour and 30 ℃, dried and sealed for standby.

(IV) spray coating of conjugate pad and nitrocellulose Membrane

1. The Adipo antibody-europium chelate fluorescent microspheres were diluted to 0.25mg/ml and 0.15mg/ml with Tris buffer, 20mM Tris, pH7.0, 10mM NaCl, 1.0% BSA, 10% sucralose, 0.5% PVP, 0.5% Tween20, and the Adipo antibody-europium chelate fluorescent microspheres were sprayed onto GFDX binding pads at 30 ℃, for 8 hours, dried and sealed for use at a speed of 4ul/cm using an XYZ three-dimensional cross-cut gold spraying apparatus (HM3035) available from shanghai jinbiao scientific and scientific biology ltd.

2. The 3 antibodies were sprayed on a nitrocellulose membrane using phosphate buffer, 10mM NaPO4, pH7.0, 1% Trehalo, 0.1% PVP to dilute the Adipo antibody to 2.0mg/ml, SHBG antibody to 1.5mg/ml, anti-mouse IgG antibody to 1.5mg/ml, using XYZ three-dimensional striping gold spraying machine (HM3035), micro-quantitative striping, speed 1.0ul/cm to form T1 line, T2 line and C line, spacing between lines 5mM, 30 ℃, dried for 8 hours, dried and sealed for use.

(V) assembling immunochromatographic test paper card

The test strip is assembled in a room with the humidity of less than 30 percent and the temperature of 25-37 ℃. An immunochromatographic test strip is shown in figure 1, and the immunochromatographic test strip is a schematic diagram, and comprises a PVC base plate, a 22mm sample pad, a 10mm binding pad, a 25mm nitrocellulose membrane and 30mm absorbent paper, wherein the 22mm sample pad, the 10mm binding pad, the 25mm nitrocellulose membrane and the 30mm absorbent paper are sequentially adhered to the base plate along the length direction of the base plate; the nitrocellulose membrane is adhered to the middle part of the bottom plate, and is provided with an interval Adipo antibody coating to form a T1 line, an SHBG antibody coating to form a T2 line and an anti-mouse IgG antibody coating to form a C line; the binding pad is sprayed with Adipo antibody-europium chelate fluorescent microspheres and SHBG antibody-europium chelate fluorescent microspheres.

In the embodiment of the invention, the T1 line and the T2 line are arranged in parallel, the distance between the T1 line and the T2 line is 5mm, the T2 line and the C line are arranged in parallel, and the distance between the T2 line and the C line is 5 mm.

The absorbent paper is positioned at one end of the nitrocellulose membrane close to the line C, is partially overlapped with the nitrocellulose membrane, and has the overlapping length of 2 mm; the absorbent paper was overlaid on top of the nitrocellulose membrane.

The combination pad is positioned at one end of the nitrocellulose membrane close to the T1 line; partially overlapping the nitrocellulose membrane, wherein the overlapping length is 2 mm; the conjugate pad was overlaid on top of the nitrocellulose membrane.

The sample pad is positioned at the outer side of the combination pad and is partially overlapped with the combination pad, and the overlapping length is 3 mm; the sample pad overlaps the conjugate pad.

And (3) assembling each group of parts into a test paper board, and cutting the test paper board into test paper strips with the width of 4 mm.

Putting the test strip into a shell base, and closing the shell surface to form a test strip card, wherein the shell surface is provided with a sample adding hole (9) and a detection window (10) to expose a local area of the test strip as a front view of the immunochromatography test strip card shown in figure 2; the sample adding hole is opened at the upper part of the sample pad (2) to expose part of the sample pad area; the detection window is opened on the upper part of the nitrocellulose membrane (4), and all the T1 line (5), the T2 line (6) and the C line (7) are exposed.

Second, the process of detecting the concentration of Adipo and SHBG by an immunofluorescence analyzer

Operation process of Adipo/SHBG immunochromatography kit

When the Adipo/SHBG immunochromatographic test paper card is used for quantitatively detecting Adipo and SHBG in blood, 5ul of Adipo/SHBG calibrator solution or 5ul of serum/slurry is added into a sample hole, 100ul of chromatography solution, 20mM Tris, pH7.4, 100mM NaCl, 0.5% BSA and 0.5% Tween-20 are added into the sample hole for reaction for 10-15 minutes, an immunofluorescence analyzer is used for selecting an excitation wavelength of 365nm and a detection wavelength of 610nm, and the fluorescence intensity on a T1 line, a T2 line and a C line is detected.

(II) establishing an immunofluorescence analyzer system for detecting concentration of Adipo and SHBG

1. Establishing an Adipo and SHBG concentration standard curve

The Adipo/SHBG calibrator was prepared by diluting the Adipo and SHBG proteins with PBS, pH7.4, 1.0% BSA at Adipo concentrations of 0, 1.25, 2.5, 5, 10, 20ug/ml and SHBG concentrations of 0, 12.5, 25, 50, 100, 200 mM/L. Adding 5ul of an Adipo/SHBG calibrator into sample wells of an Adipo/SHBG immunochromatographic test card, adding 100ul of a chromatography solution into the sample wells, performing a membrane chromatography reaction, after 10-15 minutes, selecting an excitation wavelength of 365nm and a detection wavelength of 610nm by using an immunofluorescence analyzer, and measuring Relative fluorescence intensity units (RFU) of a test strip card T1 line, a T2 line and a C line. Taking the concentration of the Adipo calibrator as an ordinate and a T1 line RFU as an abscissa, preparing an Adipo calibrator concentration standard curve to obtain an equation, wherein y is 0.0003x-0.5272, and R is²Referring to fig. 3, a curve relating Adipo concentration to RFU is shown as 0.9982, and an Adipo concentration standard card is obtained from the standard curve as a basis for quantitative analysis of the Adipo concentration contained in the sample.

Taking the concentration of the SHBG calibrator as an ordinate and the RFU line T2 as an abscissa, preparing a standard concentration curve of the SHBG calibrator to obtain an equation, wherein y is 0.0028 x-8.4439, and R is²Referring to fig. 4, a SHBG concentration-RFU correlation curve is shown at 0.9892, and a SHBG concentration standard card is obtained from the standard curve as a basis for quantitative analysis of the SHBG concentration contained in the sample.

SHBG	0	12.5	25	50	100	200mM/L
							RFU	389	6634	13653	22579	43415	72147
	436	6217	12785	24170	40412	68756
								517	5862	12014	21874	44578	73106
Average	447.3333	6237.667	12817.33	22874.33	42801.67	71336.33

And inputting the standard card into an immunofluorescence analyzer, and establishing a system for automatically displaying the concentrations of the Adipo and the SHBG in the sample by the immunofluorescence analyzer.

2. Repeatability and accuracy of immunochromatography kit for determining Adipo/SHBG

The samples were prepared as 5ug/ml Adipo and 50mM/L SHBG calibrators, and the Adipo/SHBG samples were repeatedly measured 20 times using the immunochromatographic kit described above, and the mean value (M) and Standard Deviation (SD) were calculated, respectively, and the SD/M x100 (100%: Coefficient of Variation (CV), (1-M/sample concentration) x 100%: relative deviation (Bias) was calculated, and the results are shown in the following table.

Sample	Adipo		SHBG
					4.87		48.75
	5.12		49.24
					5.43		47.54
	4.69		49.28
					4.85		51.12
	4.754		50.35
					4.97		48.65
	4.86		47.37
					4.682		50.28
	4.97		48.63
					5.26		50.91
	4.83		52.46
					4.79		48.82
	4.91		47.69
					4.78		48.77
	4.92		48.56
					5.18		47.32
	5.22		48.86
					4.86		47.17
	4.68		47.64
				M	4.9313		48.9705
SD	0.21		1.42
				CV	4.30％		2.90％
Bias	1.40％		2.10％

The CV of Adipo is 4.3%, the Bias is 1.4%, the CV of SHBG is 2.9%, and the CV of Bias is 2.1%, which indicates that the Adipo/SHBG immunochromatography kit has good repeatability and accuracy.

3. Detection of Adipo and SHBG concentrations with Adipo/SHBG immunochromatographic kit

Adding 5ul of serum into an Adipo/SHBG immunochromatography test paper card sample adding hole, adding 100ul of chromatography liquid into the sample adding hole, performing membrane chromatography reaction, and after 10-15 minutes, automatically detecting the concentrations of Adipo and SHBG by using an immunofluorescence analyzer, wherein the results are as follows:

serum sample	1	2	3	4	5	6	7	8	9	10
											RFU	35574	42187	18863	25126	29675	24076	46053	37402	16894	18205
Adipo	10.1ug/ml	12.2ug/ml	5.1ug/ml	7ug/ml	8.4ug/ml	6.7ug/ml	13.3ug/ml	10.7ug/ml	4.5ug/ml	4.9ug/ml
											RFU	24265	37592	13496	28764	17563	43821	24538	51225	10142	26785
SHBG	60mM/L	97mM/L	29mM/L	72mM/L	41mM/L	114mM/L	60mM/L	135mM/L	20mM/L	66mM/L

Claims (7)

1. An immunochromatography kit for rapid quantitative detection of Adipo/SHBG, which mainly comprises an immunochromatography test strip and a chromatographic solution; the immunochromatographic test strip comprises a PVC base plate, and a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper are sequentially connected and fixed on the PVC base plate. The binding pad adsorbs an Adipo antibody-microsphere and an SHBG antibody-microsphere; an Adipo antibody is fixed on the nitrocellulose membrane to form a detection line 1(T1 line), an SHBG antibody is fixed to form a detection line 2(T2 line), and an anti-IgG antibody is fixed to form a quality control line (C line).

2. The chromatography liquid as claimed in claim 1, wherein the concentration of Tris is 20-200mM, pH7.0-8.0, 0.1-1.0% BSA, 100-400mM NaCl, 0.1-1.0% Tween-20.

3. The microsphere of claim 1 can be fluorescent microsphere, colored microsphere, gold-labeled microsphere, magnetic microsphere, upconversion luminescent microsphere, etc., and europium chelate fluorescent microsphere is selected.

4. The microsphere as claimed in claim 1, wherein the surface modification functional group is one of carboxyl, hydroxyl or epoxy, the diameter of the microsphere is 100-300nm, the microsphere is selected from 100nm and 200nm, and the microsphere is modified by carboxyl.

5. The process for preparing fluorescent microspheres labeled by the Adipo antibody and the SHBG antibody according to claim 1 comprises the following steps:

(1) activating carboxyl groups to modify the fluorescent microspheres by using carbodiimide (EDC) and N-hydroxyl thiosuccinimide (Sulfo-NHS); (2) adding an Adipo antibody and an SHBG antibody for coupling reaction; (3) adding Tris buffer solution to terminate the coupling reaction; (4) and (3) after washing, re-dissolving the Adipo antibody-fluorescent microspheres and the SHBG antibody-fluorescent microspheres by using a Tris buffer solution, and storing at the temperature of 2-8 ℃ for later use.

6. The process for preparing the immunochromatographic test paper card for detecting the Adipo/SHBG comprises the following steps:

(1) preparing an Adipo antibody-fluorescent microsphere solution and an SHBG antibody-fluorescent microsphere solution, spraying the solutions on a bonding pad, and drying at 25-37 ℃ for 4-12 hours; (2) preparing a detection line and a quality control line: spraying an Adipo antibody solution, an SHBG antibody solution and an anti-IgG antibody solution on a nitrocellulose membrane to form a T1 line, a T2 line and a C line, and drying at 25-37 ℃ for 4-12 hours; (3) assembling an immunochromatography test strip: sequentially adhering a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper on a PVC (polyvinyl chloride) base plate to form a test paper plate, and cutting the test paper plate into test paper strips with the thickness of 3-5 mm; (4) and putting the test strip into a shell to form the immunochromatographic test strip card.

7. The Adipo/SHBG immunochromatographic kit according to claim 1, which detects Adipo and SHBG concentrations in blood or other body fluids for early prediction of insulin resistance, type 2 diabetes and risk of onset of gestational diabetes.

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