CN114152743A

CN114152743A - Chemiluminescence immunoassay kit for detecting CMPF and detection method thereof

Info

Publication number: CN114152743A
Application number: CN202111461910.6A
Authority: CN
Inventors: 李祝华; 黄雪莹; 唐亚楠; 李海英; 马冰
Original assignee: Shenyang Yikangyuan Hemodialysis Technology Co ltd
Current assignee: Shenyang Yikangyuan Hemodialysis Technology Co ltd
Priority date: 2021-12-02
Filing date: 2021-12-02
Publication date: 2022-03-08

Abstract

The invention belongs to the technical field of medical analysis and detection methods, and relates to a chemiluminescence immunoassay kit for detecting CMPF and a detection method thereof. According to the invention, CMPF is respectively coupled with two different carrier proteins as an envelope (capture) antigen and an immune antigen, so that an antibody produced aiming at the immune antigen can specifically generate a specific immune reaction with a characteristic epitope on a CMPF molecular structure, and cannot generate an immune reaction with the carrier protein of the envelope (capture) antigen, thereby ensuring the accurate determination of the CMPF content. The chemiluminescence detection kit disclosed by the invention has good consistency (R) when the CMPF is detected and the liquid chromatography-mass spectrometry combination is compared²= 0.9617); the invention improves the detectionThe sensitivity and accuracy of the detection can be improved, the high investment of liquid quality equipment can be avoided, and the detection time can be greatly shortened.

Description

Chemiluminescence immunoassay kit for detecting CMPF and detection method thereof

Technical Field

The invention belongs to the technical field of medical analysis and detection methods, relates to a chemiluminescence immunoassay kit for detecting CMPF (CMPF), and a detection method thereof, and particularly relates to a chemiluminescence immunoassay kit for detecting 3-carboxyl-4-methyl-5-propyl-2-furanpropionic acid (CMPF) and a detection method thereof.

Background

Protein-bound toxoids are an important class of uremic toxins, and typically include 3-carboxy-4-methyl-5-propyl-2-furylpropionic Acid (3-carboxy-4-methyl-5-propyl-2-furylpropionic Acid, CMPF), Indoxyl Sulfate (IS), p-cresol Sulfate (PCS), Hippuric Acid (HA), and the like. The toxin substance is mainly a small molecular toxin substance (molecular weight is less than 500Da) generated by decomposing amino acid by intestinal bacteria, and the small molecular toxin substance is easy to combine with serum protein in vivo to form a medium-macromolecule substance combination, and is secreted into renal tubules by an organic anion channel of a renal proximal tubular epithelial cell basal side membrane under the normal human condition and finally discharged with urine, so that the content of the toxin substance in the serum of a normal human is very low and is about 0.5-2.0 mu g/ml, and for uremia patients, the toxin substance in the plasma can not be effectively discharged due to renal dysfunction, so that the toxin substance is retained, and the content can reach more than 5 times of that of the normal human, thereby causing damage to each organ of the body and causing a plurality of diseases.

Research shows that the detection of the level of 3-carboxyl-4-methyl-5-propyl-2 furan propionic acid (CMPF) is an important diagnostic index for chronic kidney disease and various complications thereof (particularly cardiovascular diseases). The establishment of the CMPF detection method has important clinical significance.

The CMPF detection methods reported at home and abroad at present comprise an enzyme-linked immunosorbent assay, a high performance liquid chromatography and a high performance liquid chromatography-tandem mass spectrometry. Wherein the enzyme-linked immunosorbent assay and the high performance liquid chromatography have high detection limit and low sensitivity; the influence of impurities is large, and the accuracy is not high; the liquid-mass spectrometry has accurate result, but the equipment is expensive, the detection time is long, and the liquid-mass spectrometry is not suitable for clinical popularization.

Disclosure of Invention

In order to overcome the defects of the existing detection method, the invention provides the chemiluminescence immunoassay kit for detecting the CMPF and the detection method thereof, which not only improve the sensitivity and accuracy of detection, but also avoid the investment of expensive equipment and greatly shorten the detection time.

CMPF is a small molecule substance with a molecular weight of 240Da, belongs to hapten, and only has antigenicity and no immunogenicity. To generate an immune response to an antigen, a whole antigen with immunogenicity must first be synthesized, i.e., the CMPF must first be coupled to a carrier protein.

The core of the invention is that CMPF is respectively coupled with two different carrier proteins as an envelope (capture) antigen and an immune antigen, so that an antibody produced aiming at the immune antigen can specifically generate a specific immune reaction with a characteristic epitope on the CMPF molecular structure, but can not generate an immune reaction with the carrier protein of the envelope (capture) antigen, thereby ensuring the content of the CMPF is accurately determined.

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

A chemiluminescence immunoassay kit for detecting CMPF comprises the following reagents:

m reagent, R1 reagent, R2 reagent, exciting liquid, CMPF calibrator, CMPF quality control product, CMPF binding protein dissociating agent.

The M reagent is streptavidin magnetic particles and is a streptavidin coated superparamagnetic microsphere, the magnetic microsphere is a composite microsphere formed by ferric oxide or ferroferric oxide magnetic nanoparticles and an organic polymer material, the surface of the composite microsphere is modified to have one or more active groups, and the active groups can be crosslinked with amino groups or carboxyl groups of streptavidin through amido bonds to form the streptavidin coated magnetic microsphere.

The diameter of streptavidin magnetic particles in the M reagent is 1-10um, and the concentration is 0.2-0.8 mg/ml.

The R1 reagent is CMPF-BSA conjugate (CMPF is coupled with the artificial capture antigen of bovine serum albumin) and active biotin or an active biotin derivative to form a biotin-BSA-CMPF antigen conjugate.

The concentration of CMPF-BSA conjugate in the R1 reagent was 0.1-5 ug/ml.

The R2 reagent was a solution containing an acridinium lipid-labeled anti-CMPF antibody.

The anti-CMPF antibody is monoclonal antibody or polyclonal antibody produced by immunizing animals after coupling CMPF with Keyhole Limpet Hemocyanin (KLH) or Ovalbumin (OVA).

The R2 reagent is acridinium lipid or a derivative thereof and an anti-CMPF antibody which are coupled through amido bond to form a 'acridinium lipid-anti-CMPF antibody' conjugate.

The concentration of anti-CMPF antibody in the R2 reagent is 0.1-5 ug/ml.

The buffer solution of the M reagent, the R1 reagent and the R2 reagent is one or a combination of a plurality of phosphate buffer solution, carbonate buffer solution, tris buffer solution and citrate buffer solution with the concentration of 10-100mM and the pH value of 6.0-8.0.

0.05-0.5% (v/v) of surfactant is added into the buffer solution of the M reagent, the R1 reagent and the R2 reagent.

The surfactant is Tween-20 or Triton X-100.

0.05-0.5% (w/v) bacteriostatic agent is added into the buffer solution of the M reagent, the R1 reagent and the R2 reagent.

The bacteriostatic agent is sodium azide or Proclin 300.

0.01-0.1% of bovine serum albumin is added into the buffer solution of the M reagent, the R1 reagent and the R2 reagent to be used as a protein stabilizer.

The excitation liquid is composed of an excitation liquid I and an excitation liquid II.

The exciting liquid I is formed by mixing 0.01-0.06% (w/v) of hydrogen peroxide and 0.05-1.0% (v/v) of triton X-100.

The exciting liquid II is formed by mixing 0.01-0.2M sodium hydroxide solution and 0.1-5.0% (v/v) of triton X-100.

The CMPF calibrator is prepared by adding 5% bovine serum albumin into a 3-carboxyl-4-methyl-5-propyl-2-furanpropionic acid (CMPF) standard substance, and the concentrations are respectively 0, 0.1, 0.5, 1, 2.5, 5, 7.5 and 10.0 ug/ml.

The CMPF quality control product is prepared by adding 5% bovine serum albumin into a 3-carboxyl-4-methyl-5-propyl-2 furan propionic acid (CMPF) standard product, and the concentrations are 0.5 ug/ml and 5.0ug/ml respectively.

The CMPF-binding protein dissociation agent consists of A, B solution A and 0.1% dithiothreitol (containing 0.2% acetic acid) solution; the solution B is 1M sodium hydroxide solution.

The detection method of the CMPF chemiluminescence detection kit is a competition method, and comprises the following steps:

sample preparation: fresh plasma or serum

Taking a certain amount of sample to be detected and a certain amount of CMPF binding protein dissociating agent A and B solution in the chemiluminescence detection kit, uniformly mixing, and incubating at 37 ℃ for 10-20 minutes to dissociate CMPF from binding protein in the sample and dissociate CMPF;

adding a certain amount of R2 reagent into the solution, mixing uniformly, incubating for 10-20 minutes at 37 ℃, and combining free CMPF and acridinium lipid-CMPF antibody in a sample to form an antigen-antibody complex;

adding certain amount of M reagent and R1 reagent into the reaction system, and incubating at 37 ℃ for 10-20 minutes. And (3) allowing the excessive acridinium lipid-CMPF antibody reacted in the last step in the system to form an antigen-antibody conjugate with biotin-BSA-CMPF in the R1 reagent, and forming a magnetic bead-avidin-biotin-BSA-CMPF-antibody-acridinium lipid complex with streptavidin in the M reagent.

Washing: and (3) adsorbing the magnetic particles by an external magnetic field, washing to remove other substances which are not combined with the magnetic particles in the reaction system, and keeping the magnetic bead-avidin-biotin-BSA-CMPF-antibody-acridine lipid compound.

And (3) detection: and respectively adding an excitation liquid I and an excitation liquid II into the compound, and measuring the luminous intensity of the system by using a chemiluminescence analyzer. And calculating to obtain the CMPF content in the sample.

Compared with the prior art, the invention has the beneficial effects of.

Compared with the traditional enzyme-linked immunosorbent assay, the chemiluminescence detection kit has greatly improved detection sensitivity (over 1000 times higher); compared with the liquid chromatography-mass spectrometry, the high equipment investment is greatly saved; and the detection time is greatly shortened compared with the detection time of the two.

Drawings

FIG. 15 SDS-PAGE electrophoresis of CMPF monoclonal antibody.

FIG. 25 is a linear graph of the kit for preparing the CMPF monoclonal antibody.

FIG. 3 is a comparison and comparison graph showing the CMPF content in serum detected by the chemiluminescence immunoassay kit of the present invention and the consistency of the detection result of the LC-MS method.

Detailed Description

The present invention is further described in the following examples, which are not intended to be limited to the examples but are to be accorded the widest scope consistent with the principles disclosed herein. All such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein.

Example 1.

Preparing an M reagent: taking 10ml of Streptavidin magnetic beads (Japanese JSR MS150/Streptavidin magnetic beads, particle size 1.5um, 10mg/ml), 100ml of phosphate buffer (20mM, pH7.4, containing 0.05% Tween-20, 0.05% calf serum albumin, 0.1% Proclin300), cleaning, and adsorbing the magnetic beads; repeating for 2 times, adding the buffer solution to prepare 0.5mg/ml M reagent, subpackaging, storing in 10 ml/bottle, and storing at 4 ℃.

Example 2.

Preparation of R1 reagent.

2.1 preparation of coating antigen.

The mixed anhydride method is adopted. The molar ratio of CMPF to Bovine Serum Albumin (BSA) was 100: 1. 10mg of CMPF was weighed, 200. mu.l of DMF (dimethylformamide) was added thereto and dissolved therein, and 30mg of DCC (dicyclohexylcarbodiimide) and 20mg of NHS (N-hydroxysuccinimide) were weighed and dissolved therein 200. mu.l of DMF. Dropwise adding into a conical flask under magnetic stirring, stirring at room temperature for 8h, placing in a refrigerator at 4 deg.C overnight, and centrifuging at 9000rpm/min in a high-speed centrifuge for 15min to obtain supernatant. Weighing 30mg BSA, dissolving in 100ml phosphate buffer (20mM, pH7.4), adding the above centrifugal supernatant dropwise, reacting for 4h under ice bath condition, ultrafiltering with 10KD ultrafiltration membrane, washing with the above phosphate buffer for 3 times, collecting the liquid in the ultrafiltration tube, diluting with buffer to 15ml to obtain complete antigen (2mg/ml), packaging into 200 ul/bottle, and freezing at-20 deg.C.

2.2 Biotin-coated antigen conjugation.

2.2.1 Biotin labeling solution: 1mg of the freeze-dried biotin powder was added to 300ul of anhydrous dimethyl sulfoxide and sufficiently dissolved to prepare a solution having a biotin concentration of 10 mM.

2.2.2 coating antigen solution: redissolving 1 branch of the coating antigen, washing with 200ul of a labeling buffer solution (0.1M carbonate buffer solution, pH9.0) in an ultrafiltration tube (cut-off molecular weight 10KD) for 3 times, and finally preparing a 2mg/ml coating antigen solution with the labeling buffer solution.

2.2.3 Biotin labelling: and taking 200ul of the coating antigen solution, adding 5.32ul of biotin solution, gently mixing, and placing in a 37 ℃ incubator for incubation for 30min in a dark place.

2.2.4 purification: the reaction solution was washed with a purification buffer (20mM, phosphate buffer, pH7.4) in an ultrafiltration tube (molecular weight cut-off: 10KD) and diluted with the purification buffer to a biotin-coated antigen solution stock solution having a concentration of 2mg/ml, and frozen at-20 ℃.

2.2.5 dispensing of R1 reagent: the biotin-coated antigen solution stock solution was thawed at room temperature, and diluted with a separate buffer (20mM, phosphate buffer, pH7.4, containing 1.5M sodium chloride, 0.05% Tween-20, 0.05% bovine serum albumin, 0.1% Proclin300) to a solution of 2ug/ml, dispensed into 10 ml/bottle, and stored at 4 ℃.

Example 3.

And preparing a CMPF monoclonal antibody.

3.1 preparation of Immunity antigen

The mixed anhydride method is adopted. 10mg of CMPF was weighed, 200. mu.l of DMF (dimethylformamide) was added thereto and dissolved therein, and 30mg of DCC (dicyclohexylcarbodiimide) and 20mg of NHS (N-hydroxysuccinimide) were weighed and dissolved therein 200. mu.l of DMF. Adding dropwise into an erlenmeyer flask under magnetic stirring, stirring at room temperature for reaction for 8h, placing in a refrigerator at 4 ℃ overnight, and centrifuging at 9000rpm/min for 15min to obtain a supernatant. Weighing 20mg of Keyhole Limpet Hemocyanin (KLH), dissolving in 100ml of phosphate buffer (20mM, pH7.4), dropwise adding the centrifugal supernatant, reacting in an ice bath for 4 hours, performing ultrafiltration by using a 10KD ultrafiltration membrane, cleaning by using the phosphate buffer for 3 times, collecting liquid in an ultrafiltration tube, diluting to 10ml by using buffer to obtain the whole antigen (2mg/ml), subpackaging in a centrifuge tube, and performing freezing storage at-20 ℃.

3.2 mice were immunized.

3.2.1 first needle immunization: a frozen CMPF-KLH immune antigen is taken, phosphate buffer solution (20mM, pH7.4) is added to dilute the solution into 0.25mg/ml, 2ml of the solution is taken, 4ml of complete Freund's immune adjuvant (Sigma company) with the same volume is added to emulsify the solution to make the solution milk white and not scattered in water, 5 healthy Bal b/c female mice are selected to be immunized into the abdominal cavity and the muscle, and each mouse is immunized with 0.40 ml.

3.2.2 second needle immunization: after 21 days of the first needle immunization, one frozen CMPF-KLH immune antigen was taken, phosphate buffer (20mM, pH7.4) was added to dilute the solution to 0.25mg/ml, 2ml of the above solution was taken, 4ml of an equal volume of incomplete Freund's immune adjuvant (Sigma) was added to emulsify the solution to make it milky white and not scattered by dropping in water, and then the mice immunized with the 1 st needle were immunized with a second needle, each mouse was immunized with 0.4ml of abdominal cavity and muscle.

3.2.3 third immunization: the second immunization was carried out 21 days later, and the third immunization was carried out in the same manner as in the 2 nd needle.

3.2.4 fourth needle immunization: 21 days after the third immunization, 0.4ml of CMPF-KLH immunizing antigen (0.25mg/ml) was taken for the 4 th needle intraperitoneal injection immunization.

3.3 cell fusion.

3.3.1 spleen cell suspension preparation: after 3 days of the fourth immunization, the mice were sacrificed, spleens were harvested, ground into cell suspensions on 200 mesh grids with syringe cores using an incomplete RPMI-1640 medium, centrifuged at 1000rpm for 10min, the supernatant was discarded, and the splenocytes were resuspended in 20ml of RPMI-1640 medium. (5X 10)⁷Individual cells/ml).

3.3.2 feeder cell preparation: injecting the mouse abdominal cavity with 4ml HAT culture medium containing 20% fetal calf serum RPMI-1640, repeatedly sucking for 3 times, collecting abdominal cavity liquid, and injecting the mouse abdominal cavity liquid into the mouse abdominal cavity with 20% fetal calf serum containing RPMI-1640Diluting the HAT culture medium of serum by 50ml, mixing, inoculating into 96-well microplate with each well at 100ul, 37 deg.C and 5% CO₂Culturing in an incubator for later use.

3.3.3Sp2/0 myeloma cell preparation: sp2/0 with good growth state was taken to remove the growth medium, the culture medium was blown off from the flask with RPMI-1640, the flask was placed in a sterilized 50ml centrifuge tube, centrifuged at 800rpm for 10min, and the precipitated cells were resuspended in 20ml PRMI-1640.

3.3.4 fusion of splenocytes and myeloma cells: mixing the spleen cell suspension and myeloma cell suspension, centrifuging at 800rpm for 10min to remove supernatant as much as possible, beating the bottom of the centrifuge tube with fingers to make spleen cells and myeloma cells pasty, (ratio of spleen cells to myeloma cells is 6: 1), adding 0.8ml of 50% polyethylene glycol (PEG, molecular weight 1000) in water bath at 37 deg.C within 90 sec, rotating the centrifuge tube while adding, standing for 90 sec, adding 30ml of PRMI-1640 culture medium within 150 sec, rotating the centrifuge tube while adding, centrifuging at 800rpm for 10min, discarding supernatant, adding 50ml of RPMI-1640 culture solution containing HAT of 20% calf serum into fused cells, gently suspending cells, dispersing the cells, inoculating on 96-well culture plate containing feeder cells, placing at 37 deg.C and 5% CO at 100ul per well, and removing supernatant as much as possible₂Culturing in incubator, inoculating 3 96-well culture plates to each mouse, observing and recording cell growth state, and replacing HAT culture solution containing 20% fetal calf serum once every 4-5 days.

3.3.5 hybridoma cell selection for secretion of antibodies: the hybridoma colonies were grown to a size of about 1/2-2/3 in the wells and the antibodies were measured in the culture by enzyme-linked immunosorbent assay.

3.3.6 determination of antibodies by enzyme-linked immunosorbent assay (ELISA).

(1) Coating antigen: the frozen envelope antigen obtained in example 2 was diluted to 0.05mg/ml with phosphate buffer (20mM, pH7.4) to prepare an antigen-coated solution, 100ul of the antigen-coated solution was added to a 96-well ELISA plate for coating, blocking, and blotting for future use.

(2) Sample adding: and adding 100ul of cell culture solution collected by each hole of the culture plate into each hole, enabling the serial number of the enzyme-linked plate to correspond to the serial number of the culture plate one by one, incubating, washing and patting dry.

(3) Adding an enzyme-labeled antibody: 100ul of goat anti-mouse IgG antibody (HRP-IgG) marked by horseradish peroxidase is added into each well respectively, and the mixture is incubated, washed, developed and stopped.

(4) And (3) detection: and (3) using a BIO-RAD microplate reader, determining the OD value of each well by using the dual wavelengths of 450nm and 630nm, and taking the ratio (P/N) > 2.1 of the OD value of each negative control well as the critical point for judging the positivity of the antibody.

(5) The mouse antibody titer 4# > 2# > 5#, and the 1# and 3# mice do not produce CMPF antibody by ELISA detection.

TABLE 14 determination of antibody OD values in one of the mouse hybridoma cell culture plates (4-1 plate).

Serial number	A	B	C	D	E	F	G	H
									1	0.023	0.012	0.047	0.037	0.056	0.022	0.034	0.011
2	0.069	0.897	0.066	0.091	1.032	0.546	0.443	0.189
									3	0.034	0.077	0.679	0.024	0.887	0.014	0.112	0.096
4	0.123	0.033	0.057	0.978	0.029	0.032	0.985	0.089
									5	0.357	0.086	0.889	0.031	0.017	1.363	0.067	0.045
6	0.645	0.021	0.278	0.026	1.243	0.023	0.267	0.523
									7	0.245	0.764	0.965	0.468	0.143	0.874	0.348	0.112
8	0.736	0.068	0.231	0.975	0.767	0.019	1.569	0.016
									9	0.173	1.676	0.381	0.014	0.879	0.205	1.107	0.298
10	0.056	0.275	0.675	1.109	0.077	1.007	0.256	0.017
									11	0.023	0.034	0.112	0.023	0.045	0.267	0.049	0.376
12	0.010	0.006	0.018	0.022	0.009	0.012	0.017	0.026

3.4 clonal cell selection.

3.4.1 cloning of Positive wells by enzyme-linked assay (limiting dilution method).

Taking feeder cells, diluting to 1 × 10 with HAT culture solution containing 20% calf serum⁵Feeder cell suspension of individual cells/ml was added to 96 well cell culture plates at 100ul per well.

The hybridoma cells were selected from 50 wells of 4-1, 4-2, and 4-3 plates (see Table 2) with the highest antibody titer, diluted with HAT medium containing 20% calf serum to 10 cells/ml hybridoma cell suspension, inoculated with 100ul (one cell) per well, 37 ℃, 5% CO₂Culturing in incubator for 10 days, observing the clone visible to naked eye, or observing the hybridoma cell spreading on the area of 1/3-1/2 under inverted microscope, marking the hole with only single clone growth, and taking the culture solution for antibody detection.

Table 250 wells high antibody titer hybridoma cell list.

3.4.2 second clone cell culture: the 50-well monoclonal cells with the highest potency after the first cloning culture were selected (see Table 3), diluted to 10 cells/ml hybridoma cell suspension with HAT culture medium containing 20% calf serum, and the above procedure was repeated 1 time for the second cloning culture.

Table 3 clone cell numbers of 50 wells were selected after the first clone culture.

4-1-D8-E5	4-1-F5-E6	4-1-G8-G7	4-2-E6-D5	4-3-C3-D4
					4-1-D8-F3	4-1-G4-C7	4-1-G9-B6	4-2-E6-E3	4-3-C3-D5
4-1-D8-F6	4-1-G4-D4	4-1-G9-C7	4-2-E6-E6	4-3-C3-F7
					4-1-E3-C5	4-1-G4-D8	4-1-G9-E3	4-2-F4-D7	4-3-C3-G2
4-1-E3-D6	4-1-G4-E3	4-2-C5-B3	4-2-F4-E8	4-3-C6-C7
					4-1-E6-B9	4-1-G4-E7	4-2-C5-C3	4-2-F4-F5	4-3-C6-D4
4-1-E6-D4	4-1-G4-F3	4-2-C5-D4	4-2-G6-C7	4-3-D5-E8
					4-1-E6-D7	4-1-G8-D5	4-2-C5-D7	4-2-G6-E3	4-3-D5-G3
4-1-F5-C9	4-1-G8-D6	4-2-E6-C4	4-2-G6-F8	4-3-E6-B5
					4-1-F5-D3	4-1-G8-F4	4-2-E6-C7	4-2-G6-G5	4-3-E6-D7

3.4.3 third clonal cell culture: the 50-well monoclonal cells with the highest potency after the second cloning culture (see Table 4) were selected, diluted to 10 cells/ml hybridoma cell suspension with HAT culture medium containing 20% calf serum, and the above procedure was repeated 1 time for the third cloning culture.

Table 4 clonal cell numbers of 50 wells were picked after the second clonal culture.

3.4.4 high antibody titer clonal cell screening: taking 50-hole monoclonal cell culture solution after the third-time clone culture, and diluting the culture solution into a culture solution with the ratio of 1:100 and a culture solution with the ratio of 1: 1000. 1: 10000. 100ul of each hybridoma cell with 100000 concentration is added into an enzyme-linked microplate coated with the antigen, the OD value of each well is measured by an enzyme-labeling instrument, 5 hybridoma monoclonal cells with the highest antibody titer (shown in a table 5) are selected, expanded and cultured, and the hybridoma cells are frozen.

Table 5 high titer finally selected.

Cell pores	4-1-F5-C9-D6	4-1-G8-G7-D5	4-2-C5-D7-D4	4-2-F4-F5-D6	4-3-D5-E8-D3
						Cell line	41F5C9D6	41G8G7D5	42C5D7D4	42F4F5D6	43D5E8D3

3.5 production of monoclonal antibodies.

50 healthy Bal b/c mice are selected, the mice age is 6-8 weeks, the mice are divided into 5 groups, 10 mice are selected, and the liquid paraffin is injected into the abdominal cavity of each mouse respectively by 0.5ml for 10 days for standby.

The 5 numbered hybridoma monoclonal cells in logarithmic growth phase were collected, washed twice with physiological saline at 1000rpm each, centrifuged for 10 minutes, and diluted to 2X 10 with physiological saline⁶Cell suspension of individual cells/ml, 0.5ml per mouse per group was intraperitoneally injected.

Observing the ascites of the mice every day, if the abdomen is obviously enlarged, touching with hands and the skin is stressed, killing the mice, collecting the ascites, combining the ascites of each group of mice, centrifuging at 4000rpm for 20 minutes, collecting the supernatant, respectively taking 20ml of each group, respectively purifying the antibody by a protein A chromatographic column, finally desalting by a phosphate buffer solution (20mM, pH7.4) to obtain the CMPF monoclonal antibody, and freezing and storing after protein content determination and electrophoretic purity determination (see table 6 and figure 1).

Table 65 monoclonal antibody test results.

Example 4.

Preparation of R2 reagent.

(1) Acridinium lipid labeling solution: 1mg of acridine lipid powder was dissolved in 4.0ml of anhydrous dimethyl sulfoxide to prepare a 0.25mg/ml acridine lipid labeling solution.

(2) Antibody solution: 0.2mg of each of the 5 antibodies (41F5C9D6B, 41G8G7D5B, 42C5D7D4B, 42F4F5D6B, and 43D5E8D3B) obtained in example 3 was washed 3 times with a carbonate buffer (0.1M, pH9.0) in an ultrafiltration tube (molecular weight cut-off: 10KD), and finally the carbonate buffer (0.1M, pH9.0) was prepared into an antibody solution of 2mg/ml (100 ul).

(3) Labeling acridine lipid: 100ul of each antibody solution is added with 100ul of acridine lipid labeling solution respectively, the mixture is gently mixed, and the mixture is placed into a thermostat at 37 ℃ and incubated for 30min in the dark.

(4) And (3) purification: the reaction solution was washed with a phosphate buffer (20mM, pH7.4) in an ultrafiltration tube (molecular weight cut-off: 10KD) and diluted to a stock solution of acridinium lipid-CMPF antibody solution at a concentration of 2mg/ml, and frozen at-20 ℃.

(5) And (4) subpackaging an R2 reagent: the storage solution of the acridine lipid-CMPF antibody solution is thawed at room temperature, and is released into a solution with the concentration of 2ug/ml by using a split buffer (20mM, phosphate buffer, pH7.4, containing 1.5M sodium chloride, 0.05% Tween-20, 0.05% calf serum albumin, 0.1% Proclin300), and is split packaged into 10 ml/bottle for storage at 4 ℃.

Example 5.

And (4) preparing an excitation liquid.

(1) Preparing an exciting liquid I: 0.2ml of 30% hydrogen peroxide solution and 0.5ml of triton X-100 were taken and purified water was added to 100ml to prepare a mixture containing 0.06% hydrogen peroxide and 0.5% triton X-100, which was stored at 4 ℃.

(2) Preparing an exciting liquid II: 0.4g of sodium hydroxide was weighed out and purified water was added to 100ml to obtain a 0.1M sodium hydroxide solution, which was stored at 4 ℃.

Example 6.

Preparing CMPF stock solution: a1 mg/ml stock solution of CMPF was prepared by weighing 0.0967g of 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) standard (SIGMA corporation) and adding 97ul of 5% bovine serum albumin (containing 0.1% Proclin300), and stored at 4 ℃.

Preparing a CMPF calibrator solution: the CMPF stock solution is added with 5% bovine serum albumin (containing 0.1% Proclin300) to prepare calibration solutions with the concentrations of 0, 0.1, 0.5, 1.0, 5.0, 7.5 and 10.0ug/ml CMPF respectively, and the solutions are stored at 4 ℃.

Preparing a CMPF quality control solution: CMPF stock solution is added with 5% bovine serum albumin (containing 0.1% Proclin300) to prepare CMPF quality control solution with concentration of 0.5 ug/ml and 5.0ug/ml respectively, and the CMPF quality control solution is stored at 4 ℃.

Example 7.

CMPF binding protein cleaving agent.

Solution A: 0.1g of dithiothreitol is weighed out and dissolved in 100ml of 0.2% acetic acid solution and stored at 4 ℃.

And B, liquid B: 4g of sodium hydroxide are weighed out and dissolved in 100ml of purified water and stored at 4 ℃.

Example 8.

Final screening of antibodies used in CMPF chemiluminescent immunoassay kits.

Respectively adding 50ul of each concentration calibrator solution in the example 6 into a reaction tube, respectively adding 50ul of each solution A and B of the CMPF-binding protein dissociating agent in the example 7, uniformly mixing, and incubating at 37 ℃ for 10 minutes;

respectively adding 50ul of each of the 5 CMPF antibody solutions (2ug/ml) of example 4 into the reaction tube, mixing, incubating at 37 ℃ for 10 minutes, and combining the CMPF in the solution and the acridinium lipid-CMPF antibody into an antigen-antibody complex;

50ul of each of the M reagent of example 1 and the R1 reagent of example 2 was added to the above reaction system, and the reaction was incubated at 37 ℃ for 10 minutes. So that the excessive acridinium lipid-CMPF antibody in the system forms an antigen-antibody conjugate with biotin-BSA-CMPF in the R1 reagent, and forms a "magnetic bead-avidin-biotin-BSA-CMPF-antibody-acridinium lipid" complex with streptavidin in the M reagent.

And (3) detection: 100ul of each of the excitation solution I and the excitation solution II of example 5 was added to the reaction solution, and the luminescence intensity of the solution in each reaction tube was measured by a SMART 500 full-automatic chemiluminescence analyzer, and the results are shown in Table 7 and FIG. 2). As a result, the linear curve of the 41G8G7D5B antibody is best, the lowest detection limit is lowest, the sensitivity is highest, and finally the 41G8G7D5B antibody is coupled with acridinium lipid to be used as the R2 reagent of the kit.

Table 75 conversion values of the R2 reagent prepared from the antibody strain measured on calibrators at different concentrations.

Concentration (ug/ml)	0	0.1	0.5	1	2.5	5	7.5	10
									41F5C9D6B	34698	34012	30567	26476	16754	6837	3063	1023
41G8G7D5B	107853	99747	90317	83256	60876	32708	13543	2351
									42C5D7D4B	154672	158976	157723	156388	132456	110573	92768	80643
42F4F5D6B	23899	22908	24065	23709	23687	23549	23120	22456
									43D5E8D3B	2054	2317	1897	2566	1997	1523	1764	2118

Example 9.

Determination of concentrations of R1 reagent and R2 reagent.

According to factory parameters of a SMART 500 full-automatic chemiluminescence analyzer, an optimal reaction system is 250-300ul, so that the volume adding amount of each component of the kit is determined to be 50ul, namely 50ul of each component of the M reagent, the R1 reagent, the R2 reagent, the dissociating agent A, the dissociating agent B and a sample to be detected, and the total volume is 300 ul. Referring to the procedure of example 8, R1 reagent (1.0, 1.5, 2.0ug/ml) and R2 reagent (1.0, 1.5, 2.0ug/ml) were prepared at different concentrations, and the emission values of the quality control materials were measured at 0.5 ug/ml and 5.0ug/ml, respectively, and the results are shown in Table 8. The result shows that when the concentrations of the R1 reagent and the R2 reagent are respectively 2 mug/mL, the luminous values of the high-concentration quality control product and the low-concentration quality control product are relatively proper, and when the concentration of the R1 reagent is too low, the luminous value of the low-end quality control product is relatively low; when the concentration of the R2 reagent is too low, the luminous value of the high-end quality control product is too low, and the next concentration detection cannot be carried out; therefore, the working concentration of the reagent was selected to be 2. mu.g/mL for the R1 and R2 reagents, respectively.

Table 8 effect of different concentrations of R1 and R2 reagents on luminescence values.

Example 10.

Incubation time of CMPF with antibody was screened.

Respectively taking 50ul of dissociating agent A, dissociating agent B and quality control substances, uniformly mixing, and incubating for 10 minutes at 37 ℃;

50ul of each of the R2 reagents was added thereto, mixed, and incubated at 37 ℃ for 5 minutes, 10 minutes and 15 minutes, respectively, and then the luminescence values were measured according to the procedure of example 8, and the results are shown in Table 9. As a result, the optimal reaction of CMPF with antibody was incubated for 10 minutes.

Table 9 incubation time of CMPF with antibody screen.

Incubation time (min)	Luminous value
		5	47432
10	31256
		15	32468

Example 11.

And (4) screening the optimal reaction incubation time of the R1 reagent and the R2 reagent.

50ul each of the R2 reagents was added thereto, mixed, incubated at 37 ℃ for 10 minutes, 50ul each of the M reagent and the R1 reagent was added thereto, mixed, incubated at 37 ℃ for 5, 10 and 15 minutes, respectively, and the results were measured with reference to the procedure of example 8 to determine luminescence values, and are shown in Table 10. As a result, the optimal time for incubation of the R1 reagent with the R2 reagent was 10 minutes.

Table 10 incubation times of R1 reagent and R2 reagent.

Incubation time (min)	Luminous value
		5	23678
10	33425
		15	32785

Example 12.

Optimal off-time screening for CMPF binding proteins.

Respectively adding 50ul of the serum of 5 healthy volunteers into 50ul of the dissociating agent A and 50ul of the dissociating agent B, uniformly mixing, and respectively incubating at 37 ℃ for 5 minutes, 10 minutes and 15 minutes; 50ul of each R2 reagent was added, mixed, incubated at 37 ℃ for 10 minutes, 50ul of each M reagent and R1 reagent was added, mixed, incubated at 37 ℃ for 10 minutes, the luminescence value was measured according to the procedure of example 8, and the values were substituted into the antibody curve 41G8G7D5B in Table 7 to obtain CMPF concentrations, and the results are shown in Table 11. As a result, the optimal dissociation time for the CMPF-binding protein was 10 minutes.

Table 11 CMPF binding protein optimal dissociation time screen.

Example 13.

The kit of the invention is used for performance evaluation.

13.1 Standard Curve.

With reference to the test results of example 8 and Table 7, the results of the measurement of the R2 reagent formulated with the antibody identified as 41G8G7D5B were plotted as a standard curve, R²＝0.9998。

13.2 analytical sensitivity of the kit (blank limit).

The test was repeated 20 times using 5% bovine serum as a blank sample, and the luminescence values were measured according to the procedure of example 8, and the results are shown in Table 12. The average (X) and Standard Deviation (SD) of the 20 test results were calculated, the calculated values of X + -2 SD were substituted into the standard curve to calculate the concentration values, and the obtained concentration values were used as the analytical sensitivity (blank limit) of the reagent. The assay sensitivity (blank limit) of the kit of the invention was 0.03 ug/mL.

Table 12 analysis sensitivity (blank limit).

13.3 evaluation of thermal stability.

The reagents of the kit of the present invention were placed in an incubator at 37 ℃ for 14 days, and the luminescence values of the quality control materials were measured according to the procedure of example 8, and the results are shown in Table 13. The result shows that the kit has stable measurement result at 37 ℃ in 14 days.

TABLE 1337 ℃ thermal stability results.

13.4 evaluation of the long-term stability.

Each reagent of the kit of the present invention was placed in a refrigerator at 2 to 8 ℃ and the luminescence value of the quality control product was measured according to the procedure of example 8, and the results are shown in Table 14. The result shows that the kit is placed in a refrigerator at the temperature of 2-8 ℃ for 6 months, and the determination result is stable.

TABLE 142-8 ℃ refrigerator, stability results after 6 months of storage.

13.5 interference test.

Taking specific interferent bilirubin, triglyceride and hemoglobin, preparing interferent solution with concentration of 5% bovine serum albumin to be 0.4mg/ml,12mg/ml and 4mg/ml, respectively, taking high concentration quality control solution (5ug/ml), and mixing according to the ratio of 1:1 proportion, and obtaining the interferent solution with the CMPF theoretical content of 2.5mg/ml, containing 0.2mg/ml bilirubin, 6mg/ml triglyceride and 2mg/ml hemoglobin respectively. Referring to the procedure of example 8, the luminescence value of the solution of the interfering substance was measured, and the results are shown in Table 15. The results showed that 0.2mg/ml bilirubin, 6mg/ml triglyceride, 2mg/ml hemoglobin did not interfere with the CMPF assay.

Table 15 kit interference assay results.

Example 14.

20 serum samples assayed by the CMPF kit of the invention were compared to LC/MS methods.

Clinical serum samples of 10 healthy volunteers and 10 uremia patients were collected, the CMPF content was detected by using the kit of the present invention and the LC-MS method, respectively, and the results were compared with each other in terms of consistency, as shown in Table 16 and FIG. 3. As can be seen, the CMPF in serum measured by the CMPF chemiluminescence kit of the present invention has very good consistency with the results measured by LC-MS (R)²＝0.9617)

Table 1620 example clinical serum samples the results of the two assays were compared.

Claims

1. A chemiluminescence immunoassay kit for detecting CMPF is characterized by comprising the following reagents:

m reagent, R1 reagent, R2 reagent, exciting liquid, CMPF calibrator, CMPF quality control product, CMPF binding protein dissociating agent;

the M reagent is streptavidin magnetic particles and is a streptavidin coated superparamagnetic microsphere, the magnetic microsphere is a composite microsphere formed by ferric oxide or ferroferric oxide magnetic nanoparticles and an organic polymer material, the surface of the composite microsphere is modified to have one or more active groups, and the active groups can be crosslinked with amino groups or carboxyl groups of streptavidin through amido bonds to form the streptavidin coated magnetic microsphere;

the R1 reagent is CMPF-BSA conjugate (CMPF is coupled with the artificial capture antigen of bovine serum albumin) and active biotin or an active biotin derivative to form a biotin-BSA-CMPF antigen conjugate;

the R2 reagent is a solution containing an acridinium lipid-labeled anti-CMPF antibody;

the excitation liquid consists of an excitation liquid I and an excitation liquid II;

the CMPF calibrator is prepared by adding 5% bovine serum albumin into a 3-carboxyl-4-methyl-5-propyl-2-furanpropionic acid (CMPF) standard substance, and the concentrations are respectively 0, 0.1, 0.5, 1, 2.5, 5.0, 7.5 and 10.0 ug/ml;

the CMPF quality control product is prepared by adding 5% bovine serum albumin into a 3-carboxyl-4-methyl-5-propyl-2-furanpropionic acid (CMPF) standard substance, and the concentrations are 0.5 ug/ml and 5.0ug/ml respectively;

2. The chemiluminescent immunoassay kit for detecting CMPF of claim 1, wherein the streptavidin magnetic particle in the M reagent has a particle size of 1-10um and a concentration of 0.2-0.8 mg/ml.

3. The chemiluminescent immunoassay kit of claim 1 wherein the concentration of CMPF-BSA conjugate in the R1 reagent is 0.1 ug/ml to 5 ug/ml.

4. The chemiluminescent immunoassay kit of claim 1 wherein the anti-CMPF antibody in the R2 reagent is a monoclonal or polyclonal antibody produced by immunizing an animal with CMPF conjugated to Keyhole Limpet Hemocyanin (KLH) or Ovalbumin (OVA).

5. The chemiluminescent immunoassay kit for detecting CMPF of claim 1, wherein the R2 reagent is acridinium lipid or its derivative coupled with anti-CMPF antibody via amide bond to form "acridinium lipid-anti-CMPF antibody" conjugate; the concentration of anti-CMPF antibody is 0.1-5 ug/ml.

6. The chemiluminescent immunoassay kit of claim 1 wherein the buffer solution of the M reagent, the R1 reagent and the R2 reagent is 10-100mM, pH6.0-8.0 phosphate buffer solution, carbonate buffer solution, tris buffer solution, citrate buffer solution or a combination thereof.

7. The chemiluminescent immunoassay kit for detecting CMPF of claim 1, wherein the buffer solution of the M reagent, the R1 reagent and the R2 reagent is added with 0.05% to 0.5% (v/v) of a surfactant, wherein the surfactant is tween-20 or triton X-100.

8. The chemiluminescent immunoassay kit of claim 1, wherein the buffer solution of the M reagent, the R1 reagent and the R2 reagent is added with 0.05% to 0.5% (w/v) of bacteriostatic agent, wherein the bacteriostatic agent is sodium azide or Proclin 300; 0.01-0.1% of bovine serum albumin is added into the buffer solution of the M reagent, the R1 reagent and the R2 reagent to be used as a protein stabilizer.

9. The chemiluminescent immunoassay kit of claim 1 wherein the excitation fluid I is a mixture of 0.01% to 0.06% (w/v) hydrogen peroxide and 0.05% to 1.0% (v/v) triton X-100; the exciting liquid II is formed by mixing 0.01-0.2M sodium hydroxide solution and 0.1-5.0% (v/v) of triton X-100.

10. The detection method of the chemiluminescence detection kit for detecting CMPF is characterized by comprising the following steps:

step 1, sampling: fresh plasma or serum;

adding a certain amount of M reagent and R1 reagent into the reaction system, and carrying out incubation reaction at 37 ℃ for 10-20 minutes to ensure that the excessive acridinium lipid-CMPF antibody reacted in the previous step in the system forms an antigen-antibody conjugate with biotin-BSA-CMPF in the R1 reagent and forms a 'magnetic bead-avidin-biotin-BSA-CMPF-antibody-acridinium lipid' complex with streptavidin in the M reagent;

step 2, washing: adsorbing magnetic particles by an external magnetic field, washing to remove other substances which cannot be combined with the magnetic particles in a reaction system, and reserving the magnetic bead-avidin-biotin-BSA-CMPF-antibody-acridine lipid compound;

step 3, detection: and respectively adding the excitation liquid I and the excitation liquid II into the compound, measuring the luminous intensity of the system by using a chemiluminescence analyzer, and calculating to obtain the CMPF content in the sample.