CN115728491A - Kit for multiple detection of immune factors and application thereof - Google Patents

Abstract

The invention provides a kit for multiple detection of immune factors and application thereof, belonging to the technical field of clinical immunoassay. The kit provided by the application comprises a reagent R1 of a first ligand coated magnetic rare earth material coding microsphere and a reagent R2 containing a detection group marked second ligand. The kit can be used for simultaneously detecting the concentrations of multiple immune factors of clinical samples, and has the advantages of high sensitivity, high specificity, high accuracy, cost saving, less required sample amount, simultaneous and efficient detection of multiple indexes and the like.

Description

Kit for multiple detection of immune factors and application thereof

Technical Field

The invention belongs to the technical field of clinical immunoassay, and particularly relates to a kit for multiple detection of immune factors and application thereof.

Background

The detection kit widely used for in vitro diagnosis in clinic mainly comprises biochemical diagnosis detection, immunodiagnosis detection and molecular diagnosis detection. The biochemical diagnosis and detection are mainly based on the Lambert-beer law, the immunodiagnosis and detection are mainly based on antigen-antibody specific reaction, and the molecular diagnosis and detection are mainly based on the nucleic acid complementary pairing principle.

In terms of market distribution, biochemical diagnosis tends to saturate, and domestic reagents almost completely replace imports. The key is to realize the lean and refinement of core products and actively develop the independent intellectual property and technical barrier of foreign markets.

The molecular diagnosis technology is still in the beginning stage at present, mainly based on qPCR and second-generation sequencing technology, and mainly distributed in the detection of tumor markets and the detection of genetic diseases. Because the detection analyte is based on molecular level and combines biological big data information and abundant gene information, the detection result is easier to become the gold standard.

The reverse immunodiagnosis technology is in a high-speed development stage through precipitation for many years, especially chemiluminescence and immunofluorescence gradually become the main technical stream, especially the technology with ultrahigh sensitivity, specificity and wide linear range gradually replaces enzyme-linked immunosorbent assay and colloidal gold technology, and slowly permeates large hospitals such as the third hospital for import replacement. Under the policy of graded diagnosis and treatment, primary hospitals bring wider development space for immunodiagnosis.

Clinically, although relatively reliable results can be detected by immunodiagnostic reagents, technologies such as enzyme immunoassay, chemiluminescence, chromatography and the like are mainly single-index detection. With the continuous increase of the sample size or the detection number of patients and the requirements of doctors and patients on the accuracy of detection results, the detection time is shortened, the detection accuracy is improved, and the problem can be well solved by carrying out multiple detections under the limited resources.

At present, the multiplex detection appearing in the market is mainly based on luminex 200, luminex xMAP, barcoded Magnetic Beads (BMB), scientific research products developed by membrane strip technology or IVD products. However, due to high patent costs and strict procurement pressure of the same owner, the imported luminex technology and BMB technology have not achieved the expected cooperative effect with most of the enterprises in China. The membrane strip detection technology has a limited clinical application range due to low sensitivity and specificity.

Disclosure of Invention

In view of this, the present invention aims to provide a kit for multiplex detection of immune factors and applications thereof, which have the advantages of high detection sensitivity and high efficiency.

The invention provides a kit for multiple detection of immune factors, which comprises a reagent R1 and a reagent R2;

the reagent R1 is a mixture of a plurality of first ligands coated magnetic rare earth material coding microspheres;

in the reagent R1, magnetic rare earth material coded microspheres of one color are coated with a first ligand;

the reagent R2 is a mixture of a plurality of second ligands marked by detection groups;

in the reagent R2, all the second ligands are marked with the same detection group;

in both reagent R1 and reagent R2, each immune factor has a corresponding first ligand and second ligand.

Preferably, the first ligand and the second ligand are antibodies that bind different antigenic determinants of the immune factor.

Preferably, the first ligand is combined with the magnetic rare earth material coding microsphere through a modifying group;

the modifying group comprises one of carboxyl, amino, hydroxyl, avidin, epoxy, silicon base, streptomycin, aldehyde group, sulfhydryl, tosyl, chloromethyl, hydrazide, silicon hydroxyl and succinimidyl ester.

Preferably, the detection group is a luminescent substance;

the luminescent material comprises one of FITC, alexa Fluor series, cy series, rhodamine series, phycoerythrin, phycocyanin and allophycocyanin.

Preferably, the luminescent material is FITC.

Preferably, the immune factor comprises an inflammatory factor and/or a cytokine.

Preferably, the immune factor comprises C-reactive protein and serum amyloid a.

Preferably, the kit further comprises a buffer for detection and a reaction plate.

Preferably, the buffer for detection includes a sample diluent and a washing buffer.

Preferably, the reaction plate is one of a flat-bottom transparent ELISA plate, a microporous plate and a microfluidic chip.

Preferably, the reaction plate is an enzyme label plate.

The invention provides application of the kit in multiplex detection of immune factors for non-disease diagnosis purposes.

The invention provides a kit for multiple detection of immune factors, which comprises a reagent R1 and a reagent R2; the reagent R1 is a mixture of a plurality of first ligands coated magnetic rare earth material coding microspheres; in the reagent R1, magnetic rare earth material coded microspheres of one color are coated with a first ligand; the reagent R2 is a mixture of a plurality of second ligands marked by detection groups; in the reagent R2, each second ligand is labeled with one identical detection group; each immune factor has a corresponding first ligand and second ligand. The invention designs a kit for multi-index joint detection, the accuracy and the sensitivity of a detection result are relatively higher, and the use amount of a sample can be reduced when one sample is subjected to multi-index detection, and the kit has more advantages particularly like waiting for the measurement of a blood sample of an infant and a blood sample of a critically ill patient. In addition, a liquid phase reaction mode is adopted, so that the detection result is more stable and uniform.

Drawings

FIG. 1 is a standard curve for CRP in the examples;

FIG. 2 is a standard curve of SAA in the examples;

fig. 3 is the CRP clinical sample alignment data correlation in the examples;

FIG. 4 is SAA clinical sample alignment data correlation in the examples;

FIG. 5 is a photograph of two encoded microspheres taken in bright field;

FIG. 6 is a photograph of the rare earth material inside the excitation-encoded microsphere;

FIG. 7 is a color distribution plot of the microspheres of FIG. 6;

FIG. 8 is a photograph of the results of exciting FITC on the surface of yellow and blue encoded microspheres;

FIG. 9 is a plot of FITC fluorescence intensity distribution for each yellow-encoded microsphere over a field of view;

FIG. 10 is a plot of FITC fluorescence intensity distribution for each blue-encoded microsphere over the field of view.

Detailed Description

The invention provides a kit for multiple detection of immune factors, which comprises a reagent R1 and a reagent R2;

the reagent R1 is a mixture of a plurality of first ligands coated magnetic rare earth material coding microspheres;

in the reagent R1, magnetic rare earth material coded microspheres of one color are coated with a first ligand;

the reagent R2 is a mixture of a plurality of second ligands marked by detection groups;

in the reagent R2, each second ligand is labeled with one of the same detection groups;

in the reagents R1 and R2, a first ligand and a second ligand which are simultaneously combined with one immune factor are included, and different immune factors can be correspondingly combined by a plurality of first ligands and a plurality of second ligands.

In the invention, the first ligand is combined with the surface modification group of the magnetic rare earth material coding microsphere. The modifying group comprises one of carboxyl, amino, hydroxyl, avidin, epoxy, silicon base, streptomycin, aldehyde group, sulfhydryl, tosyl, chloromethyl, hydrazide, silicon hydroxyl and succinimide ester, and more preferably carboxyl. The preparation method of the magnetic rare earth material coding microsphere is preferably prepared by adding magnetic nanoparticles and rare earth element materials into a microsphere-containing system (see patent publication No. CN 102199428A). The encoded microspheres are preferably polystyrene microspheres. The average particle size of the coding microspheres is preferably 1-50 μm, and more preferably 10 μm. The excitation light wavelength of the coded microspheres is preferably 780-1100 nm, and more preferably 980nm.

In the present invention, the detecting group is preferably a luminescent substance. The luminescent material comprises one of FITC, alexa Fluor series, cy series, rhodamine series, phycoerythrin, phycocyanin and allophycocyanin. Preferably, the luminescent substance is FITC.

In the present invention, the preparation method of the first ligand-labeled magnetic rare earth material-encoded microsphere preferably comprises the following steps: cleaning the magnetic rare earth material coding microsphere, sequentially adding EDC and NHS solutions, performing oscillation activation, performing oscillation cleaning after activation, adding a first ligand for oscillation coupling, performing oscillation cleaning, oscillation sealing, cleaning and storing after coupling to obtain the magnetic rare earth material coding microsphere marked by the first ligand. The activation buffer is a solution of 20 to 100mM MES at pH5.0 to pH6.1, and more preferably a solution of 50mM MES at pH 6.1. 10-100 ten thousand magnetic rare earth material coding microspheres are added into each 200-500 mu L of the activation buffer solution. The time for the oscillation activation is preferably 15 to 60min, and more preferably 30min. The EDC or NHS solution is preferably prepared at a concentration of 1-50 mg/mL, more preferably 20mg/mL. The time for the shaking cleaning is preferably 5 to 15 seconds, and more preferably 10 seconds. Before the first ligand is added for coupling, the activation system is preferably subjected to magnetic separation and cleaning. Before the coupling, it is preferred to wash twice with coupling buffer. The coupling buffer solution is preferably one of MES buffer solution without amino group, MOPS buffer solution, phosphate buffer solution and HEPES buffer solution with pH value of 5.0-8.0 and 10-100 mM, and more preferably pH value of 6.1, 50mM MES or pH value of 7.2, 10mM PBS buffer solution. Preferably, 1-50 mu g of first ligand is added into 10-100 million magnetic rare earth material coding microspheres. The time for the oscillatory coupling is preferably 1 to 6 hours, more preferably 2 hours. The temperature of the oscillatory coupling is preferably 4 to 30 ℃ and more preferably 20 to 25 ℃. The solution for blocking is preferably a 10-50 mM PBS or Tris buffer containing 0.1-5% w/v BSA, 0-5% w/v skimmed milk powder, 0-0.05% v/v Tween 20, 0-5% w/v trehalose and 0.05-0.1% preservative of v/v Proclin 300 or Proclin 950, more preferably a 10mM PBS buffer containing 1% w/v BSA, 0.05% v/v Proclin 300 preservative. The temperature of the blocking is preferably 4 to 30 ℃, more preferably 20 to 25 ℃. The blocking time is preferably 0.5 to 6 hours, more preferably 2 hours. After the blocking, the microspheres are preferably washed once with blocking solution, and the supernatant is discarded after magnetic separation. And resuspending the magnetic rare earth material coded microspheres marked by the first ligand by using a confining liquid. In the first ligand-labeled magnetic rare earth material coding microsphere solution, the concentration of the magnetic rare earth material coding microsphere is preferably 40-400 ten thousand/mL, and the concentration of the first ligand is preferably 4-200 mug/mL.

In the present invention, the method for preparing the second ligand labeled with the detection group preferably comprises the following steps: and mixing the detection group solution with the second ligand, carrying out vibration treatment in a dark place, and purifying to obtain the detection group labeled second ligand. A second ligand: the molar ratio of the detecting groups is preferably 1. The time for the light-shielding oscillation is preferably 1 to 3 hours, and more preferably 1 hour. The temperature of the light-shielding oscillation is preferably 4 to 30 ℃, and more preferably 20 to 25 ℃. The purification method is preferably performed by removing free detection groups using a desalting column or an ultrafiltration tube or a dialysis bag. In the embodiment of the invention, NHS-FITC fluorescein is taken as an example to illustrate a method for preparing a second ligand labeled by a detection group.

In the invention, 10mg/mL NHS-FITC fluorescein mother liquor is prepared by DMF or DMSO, the mother liquor is diluted by 10 times of labeling buffer solution to obtain 1mg/mL NHS-FITC fluorescein working solution, and the fluorescein working solution and a second ligand are mixed to prepare a fluorescein-labeled second ligand. The labeling buffer is preferably 20mM boric acid buffer or 10mM phosphate buffer. The molar ratio of the second ligand to NHS-FITC is preferably 1. The temperature is preferably 25 ℃ and the time is preferably 1h by shaking in the dark.

In the present invention, the first ligand and the second ligand are preferably antibodies that bind different antigenic determinants of the immune factor. The immune factor preferably includes an inflammatory factor and/or a cytokine, and the like. In the embodiment of the invention, the composition and the detection method of the kit for detecting the multiple immune factors are explained by taking C-reactive protein and serum amyloid A as detection objects. The source of the first antibody of the C-reactive protein is preferably CRP k1016 antibody of Kyoeka Biotechnology Co. The source of the second antibody of the C-reactive protein is preferably hsCRP 12D6 antibody from Nanjing King-Story Biotechnology GmbH. The source of the primary antibody to serum amyloid a is preferably Medix SAA2203 antibody. The source of the secondary antibody for serum amyloid a is preferably Medix SAA2201 antibody.

In the present invention, the kit preferably further comprises a buffer for detection and a reaction plate. The detection buffer preferably includes a sample diluent and a washing buffer. The formulation of the sample dilutions is preferably 1% BSA and 10mM PBS (pH 7.2). The formulation of the wash buffer is preferably 0.05% Tween 20 and 10mM PBS (pH 7.2). The reaction plate is preferably one of a flat-bottom transparent ELISA plate, a microporous plate and a microfluidic chip. More preferably, the reaction plate is an ELISA plate.

The invention provides application of the kit in multiple detection of immune factors for the purpose of disease auxiliary diagnosis.

In the present invention, the detection method preferably includes a one-step method or a two-step method for detecting the sample to be detected. The one-step detection steps are as follows:

(1) Diluting a clinical sample in a sample diluent by 1-1 times to 200 times, transferring the diluted clinical sample to an unused ELISA plate, and adding a reagent R1 and a diluted reagent R2, wherein the total reaction volume is 100-300 mu L;

(2) Oscillating and incubating for 10-60 min at 18-40 ℃ and 800-1500 rpm in dark;

(3) After incubation, standing on a magnetic plate for 0.5-2 min in a magnetic separation or natural sedimentation mode, and adding 150-300 mu L of cleaning buffer solution for cleaning for 2-4 times;

(4) After 200 mu L of deionized water is used for cleaning the microspheres again and is settled at the bottom of the ELISA plate, placing the ELISA plate on an optical microscopic imaging system for photographing, wherein the coded microspheres to be measured respectively emit respective fluorescent signals under the excitation of near infrared (780 nm-1100 nm) and visible light (400 nm-780 nm) and are captured by the system, a fluorescent photo obtained by the excitation of the near infrared light is used for multi-channel qualitative detection, and a fluorescent photo obtained by the excitation of the visible light is used for multi-channel quantitative analysis; and performing statistical analysis on the near infrared excited photo and the visible light excited photo by software to complete qualitative and quantitative analysis.

In the present invention, the dilution ratio in step (1) is preferably 1. The incubation temperature in the step (2) is preferably 37 ℃, the oscillation speed is preferably 1100rpm, and the incubation time is preferably 30min; and (3) standing the magnetic plate for 1min in a sedimentation mode, and washing for 3 times under the washing condition of 200 mu L of washing buffer solution.

In the invention, the two-step detection steps are as follows:

(1) Taking a clinical sample to dilute in a sample diluent according to the proportion of 1-1;

(2) Oscillating and incubating at 18-40 ℃ and 800-1500 rpm for 10-60 min;

(3) After incubation, standing on a magnetic plate for 0.5-2 min in a magnetic or natural sedimentation separation mode, and adding 150-300 mu L of washing buffer solution for washing for 2-4 times;

(4) Adding diluted reagent R2, wherein the total reaction volume is 100-300 mu L;

(5) Oscillating and incubating for 10-60 min at 18-40 ℃ and 800-1500 rpm in dark;

(6) After incubation, standing on a magnetic plate for 0.5-2 min in a magnetic separation or natural sedimentation mode, and adding 150-300 mu L of cleaning buffer solution for cleaning for 2-4 times;

(7) Washing the microspheres with 200 mu L of deionized water again and depositing the microspheres at the bottom of the ELISA plate, placing the ELISA plate on an optical microscopic imaging system for photographing, wherein the coded microspheres to be measured respectively emit respective fluorescent signals under the excitation of near infrared (780 nm-1100 nm) and visible light (400 nm-780 nm) and are captured by the system, a fluorescent photo obtained by the excitation of the near infrared light is used for multichannel qualitative detection, and a fluorescent photo obtained by the excitation of the visible light is used for multichannel quantitative analysis; and performing statistical analysis on the near infrared excited photo and the visible light excited photo by software to complete qualitative and quantitative analysis.

In the invention, the dilution ratio in the step (1) is preferably 1; the incubation temperature of the step (2) and the step (5) is preferably 37 ℃, the oscillation speed is preferably 1100rpm, and the incubation time is preferably 30min; the sedimentation mode in the steps (3) and (6) is preferably to stand on a magnetic plate for 1min, and the washing condition is 200 mu L of washing buffer solution for washing for 3 times; the total reaction volume of the step (4) is preferably 200. Mu.L.

The kit for multiplex detection of an immune factor and the use thereof provided by the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.

The sources of the antibodies referred to in the examples of the present invention are illustrated in tables 1 and 2.

TABLE 1 CRP antibody raw Material manufacturer information

TABLE 2 SAA raw materials manufacturer information

Antibodies	Manufacturer of the product
		SAA 2203	MEDIX
SAA 2201	MEDIX
		SAA 8C7	Nanjing Kinsrui science and technology Co., ltd
SAA 6F9	Nanjing Kinsrui science and technology Co., ltd
		SAA k8d6	NANJING OUKAI BIOTECHNOLOGY Co.,Ltd.
SAA k7c5	NANJING OUKAI BIOTECHNOLOGY Co.,Ltd.
		SAA C1	CHONGQING BIOMEAN TECHNOLOGY Co.,Ltd.
SAA C2	CHONGQING BIOMEAN TECHNOLOGY Co.,Ltd.

Example 1

1. Preparation of CRP and SAA detection kit and sample detection

1. CRP and SAA capture antibody labeled magnetic rare earth material coding microsphere

(1) Cleaning microspheres: taking 2 200 mu L of 50mM MES buffer solution, putting the 2 mu L of the 50mM MES buffer solution into a 0.6mL low-adsorption centrifuge tube, respectively adding 20 ten thousand magnetic rare earth material coded microspheres 1 and coded microspheres 2 which have the diameter of 10 mu m and are modified with carboxyl groups on the surfaces, oscillating for 10s, putting the mixture on a magnetic frame for 1min, and discarding the supernatant.

Encoded microsphere 1 was an 18-yb-encoded microsphere that emitted yellow light, and encoded microsphere 2 was an 49-yb-encoded microsphere that emitted blue light.

(2) Activation of microspheres: add 200. Mu.L 50mM MES buffer and shake for 10s, add 50. Mu.L 50mg/mL ready-prepared EDC and NHS solution in turn, and shake for 30min.

(3) And (3) cleaning after activation: placing on a magnetic frame for 1min after activation, discarding the supernatant, adding 400 μ L50 mM MES buffer solution, shaking for 10s, placing on the magnetic frame for 1min, and discarding the supernatant; the washing was repeated once.

(4) Antibody coupling: mu.g of CRP capture antibody (CRP k 1016) and SAA capture antibody (SAA 2203) were added to the tubes, supplemented with 50mM MES buffer to a total volume of 200. Mu.L, and incubated for 2h with shaking.

(5) Sealing the microspheres: after the antibody couple is connected, the antibody is placed on a magnetic frame for 1min, and the supernatant is discarded. 400. Mu.L of a 10mM PBS blocking solution containing 1% BSA was added, and blocking was performed for 30min with shaking.

(6) And (3) cleaning after sealing: after blocking was completed, the mixture was placed on a magnetic rack for 1min, the supernatant was discarded, 200. Mu.L of a blocking solution containing 1% BSA (purchased from Roche) in 10mM PBS was added thereto, the mixture was shaken for 10s, the mixture was placed on a magnetic rack for 1min, and the supernatant was discarded.

(7) And (3) storage: 250. Mu.L of 1-vol% BSA-containing 10mM PBS blocking solution was added, respectively, to obtain CRP-and SAA-antibody-labeled magnetic rare earth material-encoding microspheres, the concentration of the CRP-and SAA-encoding microspheres being 80 ten thousand/mL. And combining the two tubes of coding microspheres into one tube to obtain a reagent R1 of the CRP and SAA antibody coated magnetic rare earth material coding microspheres, wherein the concentration of the CRP and SAA coding microspheres is 40 ten thousand/mL. Storing at 4 deg.C for use.

2. FITC (FITC) labeled by CRP (C-reactive protein) and SAA (serum amyloid A) detection antibodies

(1) Preparing NHS-FITC working solution: weighing 5mg of NHS-FITC fluorescein in a 2mL centrifuge tube in a dark place, adding 500 microliter of DMF organic solvent, and oscillating and dissolving to obtain 10mg/mL NHS-FITC fluorescein solution; and putting 180 mu L of 20mmol/L boric acid buffer solution into a 200 mu L centrifuge tube, adding 20 mu L of 10mg/mL NHS-FITC fluorescein solution, and uniformly mixing by shaking to obtain 1mg/mL NHS-FITC fluorescein solution.

(2) FITC labeling: 2 pieces of 200. Mu.L low adsorption centrifuge tubes were added with 100. Mu.g CRP detection antibody (CRP 12D 6) and SAA detection antibody (SAA 2201), 9.46. Mu.L of 1mg/mL NHS-FITC fluorescein solution, supplemented with 20mmol/L boric acid buffer to 50. Mu.L, protected from light and shaken at room temperature for 1h.

(3) And (3) purification: excess free NHS-FITC fluorescein was removed using a desalting column.

(4) And (3) storage: and mixing the purified FITC labeled CRP antibody and SAA antibody to obtain a mixture reagent R2 of the FITC labeled CRP detection antibody and the FITC labeled SAA antibody of 1mg/mL, and storing at 4 ℃.

3. CRP and SAA standard and clinical sample detection

Preparing: the kit is taken out in advance and is balanced at room temperature for at least 30min. 5 mu L of sample to be tested is added into 45 mu L of sample diluent for 10-fold dilution for standby. Several volumes of the fluorescent antibody reagent R2 were diluted with the sample diluent at 1.

Sample incubation: 140 mul of sample diluent and 50 mul of coding microsphere reagent R1 are added into the enzyme label plate in sequence, then 10 mul of standard substance and diluted sample are added into each hole, and the incubation is carried out for 30min at 1100rpm and 37 ℃ with oscillation.

Cleaning: after the reaction is finished, the ELISA plate is taken out, placed on a magnetic plate for magnetic adsorption for 1min, and a liquid transfer device is used for slowly sucking the supernatant from the side edge of the bottom of the reaction hole. Adding 200 μ L of washing buffer solution, placing in an oscillator, oscillating for 1min, taking out the ELISA plate, placing on a magnetic plate, magnetically adsorbing for 1min, and slowly sucking the supernatant from the side of the bottom of the reaction well by using a pipette. The washing was repeated two more times.

And (3) fluorescent antibody incubation: 200. Mu.L of diluted fluorescent-labeled antibody was added to each well, and incubated at 1100rpm and 37 ℃ for 30min with shaking in the dark.

Cleaning: after the reaction is finished, the ELISA plate is taken out, placed on a magnetic plate for magnetic adsorption for 1min, and a liquid transfer device is used for slowly sucking the supernatant from the side edge of the bottom of the reaction hole. Adding 200 μ L of washing buffer solution, placing in an oscillator, oscillating for 1min, taking out the ELISA plate, placing on a magnetic plate, performing magnetic adsorption for 1min, and slowly sucking the supernatant from the bottom side of the reaction hole by using a pipette. The washing was repeated two more times.

Add 200. Mu.L deionized water, place on magnetic plate for magnetic adsorption for 1min, use pipettor to slowly suck the supernatant from the bottom side of the reaction well.

The imaging operation is completed by the following specific steps:

and transferring the ELISA plate to an optical microscopic imaging system, and opening the optical microscopic imaging system and analysis software. And (3) turning on a white light source, searching an imaging area in a bright field, adjusting the focal length to enable the edge of the coded microsphere to be clearly visible in a visual field, clicking a software interface photographing module to finish bright field imaging and automatically storing a photo for positioning the microsphere.

And (3) turning off the white light source, turning on the 980nm laser, clicking the software interface photographing module, completing near infrared excitation imaging and automatically storing the near infrared excitation imaging for qualitatively analyzing the types and the respective quantity of the microspheres.

And closing the 980nm laser, opening the visible light 488nm laser, clicking the software interface photographing module to finish dark field imaging and automatically storing the picture for quantitative analysis of the object to be detected.

The data analysis operation is completed, and the specific steps are as follows:

and (4) loading the bright field picture by the analysis software, clicking a functional button of the software, and automatically identifying the position of the microsphere.

And (4) loading the near-infrared excited picture by the analysis software, clicking a functional button of the software, and automatically decoding the color of the microsphere where the bright field is positioned to obtain various microsphere types. Since each microsphere is coated with a specific target antibody, different colors can qualitatively analyze a plurality of analytes.

And (4) loading the dark field picture by the analysis software, clicking a functional button of the software, and automatically decoding the brightness of the microsphere where the bright field position is located to obtain different fluorescence intensities. Since the analyte contains different concentrations and the content of the fluorescent substance bound on the surface is different, the concentration of the analyte can be quantitatively analyzed according to different brightness and a standard curve.

The results are shown in tables 3 and 4. The detection result shows that the detection method provided by the invention is highly related to the clinical detection result, which shows the reliability of the detection method provided by the invention.

TABLE 3 CRP and SAA Standard Curve test results

TABLE 4 CRP and SAA clinical sample assay results

Note: the reference concentration is the clinical detection result, and the examination concentration is the result obtained by testing the same sample by the kit provided by the invention.

Example 2

Optimization experiment aiming at low CRP detection sensitivity

1. Antibody optimization protocol: the negative sample N3 and the positive sample P10 are used for respectively screening antibodies from different manufacturers to obtain a paired antibody with better signal-to-noise ratio, the detection reagent and the detection method are the same as those in example 1, and the detection signal results are shown in tables 5 and 6.

TABLE 5 negative sample N3 test results

Table 6 positive sample P10 test results:

from the above results, it can be seen that CRP k1016 was used for microsphere labeling, CRP 12D6 was used for fluorescein labeling, and the signal-to-noise ratio was superior.

2. The detection system optimization scheme comprises the following steps: the amount of the labeled protein is increased, the coupling buffer solution is replaced, the difference of the signal values of the positive and negative samples is observed, the detection method is the same as that of the example 1, and the detection signal results are shown in Table 7.

TABLE 7 results of measurements of different coupling buffers and amounts of different marker proteins

Sample(s)	CRP k1016-PBS-10μg	CRP k1016-MES-5μg	CRP k1016-PBS-5 μ g (control)
				Blank space	0.6	0.7	0.6
N5	1.7	2.2	2.0
				N6	7.1	7.1	9.3
P18	25.0	53.7	59.5
				P20	26.8	49.7	50.6
N11	5.6	4.0	4.5
				N12	3.4	5.6	6.7
P9	20.3	38.9	35.4
				P13	14.9	23.4	21.1
P16	30.7	51.7	57.0
				P28	35.6	59.6	65.5

From the above results, it was found that the labeled amount of CRP k1016 was 5. Mu.g, and the coupling buffer was MES or PBS, which was superior in signal to noise ratio.

Example 3

Optimization experiment aiming at problem of low SAA sensitivity

Antibody optimization protocol: the negative sample N2 and the positive sample P3 are used for respectively screening antibodies of different manufacturers to obtain paired antibodies with better signal-to-noise ratio, and the detection signal results are shown in tables 8 and 9.

Table 8 negative sample N2 test results:

class of antibody	SAA 8C7	SAA 6F9	SAA k7c5	SAA C1	SAA C2	SAA 2201
							SAA 8C7	2.6	9.4	0.8	6.2	13.7	6.1
SAA k8d6	12.2	0.7	5.8	12.0	2.7	3.5
							SAA C1	1.6	8.3	1.2	5.0	7.2	2.6
SAA C2	2.6	0.5	1.1	1.1	5.4	1.5
							SAA 2203	6.4	16.5	1.9	6.5	7.5	5.9

Table 9 positive sample P3 test results:

class of antibody	SAA 8C7	SAA 6F9	SAA k7c5	SAA C1	SAA C2	SAA 2201
							SAA 8C7	1.8	19.5	0.8	4.4	14.1	7.6
SAA k8d6	43.1	0.6	22.8	26.9	2.7	26.1
							SAA C1	0.9	27.9	0.7	1.1	18.7	20.3
SAA C2	11.3	0.5	3.6	6.5	0.8	5.9
							SAA 2203	1.6	40.9	1.3	24.2	13.4	43.0

From the above results, it can be seen that SAA2203 is used for microsphere labeling, SAA2201 is used for fluorescein labeling, and the signal-to-noise ratio is superior.

Example 4

Abnormal spots can be seen on the surface of the microspheres during the detection of the SAA sample, possibly due to poor microsphere sealing effect

The invention obtains better blocking effect by screening BSA from different manufacturers to block microspheres, other detection reagents and detection methods are the same as example 1, and the detection signal results are shown in Table 10.

TABLE 10 microsphere encapsulation Effect test

From the above table, it can be seen that the use of blocking agent containing Roche BSA can effectively reduce abnormal spots, and the signal value of negative samples is acceptable.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims (10)

1. The kit for multiple detection of the immune factors is characterized by comprising a reagent R1 and a reagent R2;

the reagent R1 is a mixture of a plurality of first ligand-coated magnetic rare earth material coding microspheres;

in the reagent R1, magnetic rare earth material coded microspheres of one color are coated with a first ligand;

the reagent R2 is a mixture of a plurality of second ligands marked by detection groups;

in the reagent R2, all the second ligands are marked with the same detection group;

in both reagents R1 and R2, there is a corresponding first ligand and second ligand for each immune factor.

2. The kit of claim 1, wherein the first ligand and the second ligand are antibodies that bind different epitopes of the immune factor.

3. The kit of claim 1, wherein the first ligand is bound to the magnetic rare earth material-encoded microsphere through a modifying group;

the modifying group comprises one of carboxyl, amino, hydroxyl, avidin, epoxy, silicon base, streptomycin, aldehyde group, sulfhydryl, tosyl, chloromethyl, hydrazide, silicon hydroxyl and succinimide ester.

4. The kit according to claim 1, wherein the detection group is a luminescent substance;

the luminescent material comprises one of FITC, alexaFluor series, cy series, rhodamine series, phycoerythrin, phycocyanin and allophycocyanin.

5. The kit of claim 1, wherein the immune factor comprises an inflammatory factor and/or a cytokine.

6. The kit of claim 5, wherein the immune factor comprises C-reactive protein and serum amyloid A.

7. The kit according to any one of claims 1 to 6, wherein the kit further comprises a buffer for detection and a reaction plate.

8. The kit of claim 7, wherein the assay buffer comprises a sample diluent and a wash buffer.

9. The kit of claim 7, wherein the reaction plate is one of a flat bottom, a transparent microplate, a microplate, and a microfluidic chip.

10. Use of a kit according to any one of claims 1 to 9 for multiplex detection of immune factors for non-disease diagnostic purposes.

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