CN112326972A - Chemiluminescence quantitative detection kit for detecting complete PINP in serum - Google Patents
Chemiluminescence quantitative detection kit for detecting complete PINP in serum Download PDFInfo
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- CN112326972A CN112326972A CN202011198027.8A CN202011198027A CN112326972A CN 112326972 A CN112326972 A CN 112326972A CN 202011198027 A CN202011198027 A CN 202011198027A CN 112326972 A CN112326972 A CN 112326972A
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- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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Abstract
The invention relates to a chemiluminescence quantitative detection kit for detecting complete PINP in serum, which is based on the complete PINP in serum as a marker, and can truly and accurately reflect the synthesis of type 1 collagen and bone formation because the complete PINP metabolism in blood is not influenced by the kidney function, and a double-antibody sandwich method is adopted, an acridinium ester instant luminescence and magnetic bead separation technology is utilized, and a biotin-avidin system cascade amplification effect is utilized. The detection result has high accuracy, good linear relation, good stability, strong specificity and high sensitivity, has wider linear range (0.5-1500ng/mL) compared with the performance of Roche PINP analysis products, has wide detection range and anti-interference performance, and can more effectively meet the requirements of clinical detection reagents. The method can provide reliable clinical reference value for early diagnosis, early intervention and prognosis of osteoporosis.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a chemiluminescence quantitative detection kit for detecting complete PINP in serum.
Background
Bone is a dynamic tissue that is continuously remodeled throughout life by resorption and formation to maintain calcium homeostasis and bone mass. Osteoporosis (OP) is a systemic bone disease in which metabolic disorders of bones and minerals lead to deterioration of bone microarchitecture, resulting in decreased bone density, deterioration of bone tissue microarchitecture, increased bone fragility leading to increased bone fragility and an increased risk of fracture. According to statistics, about 2 hundred million osteoporosis patients are caused globally, and 890 fracture occurs in ten thousand times, and fracture caused by osteoporosis occurs on average every 3 seconds, and the trend is rising year by year. With the exacerbation of the aging problem in the population, the incidence of osteoporosis has stepped beyond the third most common chronic disease. Osteoporosis is becoming a serious worldwide health problem, and the incidence of osteoporosis is high, and the harm is huge. Therefore, the research and development of the early diagnosis of the osteoporosis onset have important social significance for promoting the progress of the prevention and treatment scheme of the osteoporosis onset and improving the health level of people.
At present, the detection of the dual-energy X-ray bone densitometer is a well-known method for diagnosing osteoporosis and knowing the disease progress, can also monitor the treatment effect, is the most widely used at present, is an examination means capable of truly reflecting the bone state, and is considered as the osteoporosis diagnosis gold standard. However, this method requires special equipment, is costly, and does not provide the continuous measurement required for monitoring.
Biochemical markers of bone metabolism have the potential to screen bone turnover conditions and monitor early bone response to treatment, and provide a basis for monitoring treatment of osteoporosis. Biochemical markers include markers of bone resorption and bone formation. The bone resorption marker is an osteoclastase or collagen degradation product, released into the circulation. The bone formation marker is a marker of osteoblastic enzyme, type I collagen synthesis or matrix protein product breakdown. Among them, type I collagen is synthesized by osteoblasts, and has an extended peptide at the carboxyl (C) and amino (N) termini as a precursor of type I collagen. These extensions are cleaved by proteases during the extracellular metabolism of collagen, resulting in the N-terminal (PINP) and c-terminal (PICP) propeptides of the intact type I procollagen N-terminal peptide. PINP and PICP are present in the blood as trimers, which are rapidly converted to monomers, and are markers of type I collagen total synthesis. PICP, hydrolyzed from the precursor molecule type I procollagen, can be rapidly cleared by hepatic endothelial cells via the mannose receptor regulated by growth hormone and parathyroid hormone. As a result, PICP has a serum half-life of less than 10 minutes, which limits its use as a biomarker. On the other hand, although collagen type I is rare in cartilage, dentin, skin and other tissues, PINP in the circulatory system is mainly derived from the bone formation process. Importantly, in addition to being unaffected by food intake and circadian rhythm, PINP also has longer serum stability, making it an ideal biomarker molecule for quantifying PINP to measure skeletal anabolic activity. Indeed, serum testing methods for measuring PINP concentrations have been approved for clinical studies of osteoporosis. Currently, there are only commercial diagnostic kits in china, including total PINP ECLIA (roche diagnostics) and enzyme-linked immunosorbent assays (ELISA) for total PINP measurement.
In the blood circulation system, PINP exists in various forms, such as intact trimeric PINP (intact PINP) and monomeric or dimeric degradation products. Studies have found a good correlation between integrity and total PINP measurements for healthy individuals. However, as kidney function declines, the clearance of intact and total PINP may differ, as intact PINP is cleared primarily by the liver, while monomeric forms of PINP are cleared by the kidney. In the presence of kidney disease, the total PINP value does not reliably reflect collagen type 1 synthesis and bone formation, and is therefore of limited diagnostic value. In addition, the low sensitivity of ELISA, narrow detection range and high cost of commercially available CLIA limit the clinical application of these diagnostic tests.
Based on the limitation of the means for detecting osteoporosis in the prior art, finding an accurate and effective method capable of diagnosing the occurrence of osteoporosis at an early stage is an urgent problem to be solved.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a chemiluminescence quantitative detection kit for detecting complete PINP in serum, which can realize high-flux and rapid detection of the complete PINP on a full-automatic chemiluminescence analyzer, has the advantages of simple and convenient operation, high sensitivity, strong specificity, accurate result and the like, effectively reduces the detection cost of the complete PINP, is beneficial to clinical popularization and use, and thus solves the technical problems of low sensitivity, long reaction time, high cost and the like in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme: a chemiluminescence quantitative detection kit for detecting complete PINP in serum comprises a reagent R1 containing magnetic microspheres coated by a complete PINP capture antibody, a reagent R2 containing acridinium ester labeled streptavidin and a reagent R3 containing a biotin labeled complete PINP detection antibody; the capture and detection antibodies are two different monoclonal antibodies directed against different determinants of the intact PINP antigen.
Preferably, the coating concentration of the capture antibody is 5-20 mug/mL.
Preferably, the mass ratio of the acridinium ester to the streptavidin in the acridinium ester-labeled streptavidin is 1: 25-100; the mass ratio of biotin to the detection antibody in the biotin-labeled complete PINP detection antibody is 1: 5-15.
Preferably, the device further comprises a pre-excitation solution, wherein the pre-excitation solution comprises an acidic excitation solution and an alkaline excitation solution, the acidic excitation solution is a hydrogen peroxide and nitric acid solution, and the alkaline excitation solution is a sodium hydroxide solution.
Preferably, the capture antibody coated magnetic microsphere is prepared by the following steps:
1) adding an EDC/NHS activator into the suspension magnetic microsphere liquid, rotating at 30-35 ℃ to activate carboxyl on the surface of the magnetic microsphere, adding a binding buffer solution, and cleaning the magnetic frame for multiple times to obtain an activated magnetic microsphere;
2) adding the capture antibody into the activated magnetic microspheres, adjusting the concentration of the magnetic microspheres to 15-20 mg/mL, placing the magnetic microspheres on a suspension vortex oscillator at room temperature for full oscillation, adding a sealing buffer solution for full reaction, and washing and re-suspending the magnetic microspheres coated with the capture antibody by using the sealing buffer solution after the reaction is finished to obtain the magnetic microspheres coated with the capture antibody.
Preferably, the mole ratio of EDC to NHS in the EDC/NHS activator is 5: 8.
Preferably, the blocking buffer is a Tris-base solution containing bovine serum albumin with a volume concentration of 2.5%; the binding buffer was 0.1M MES, pH 5.0.
The invention also aims to provide a use method of the detection kit, which comprises the following steps:
s1: adding 50 mu L of standard solution with concentration gradient into 60 mu L of magnetic microsphere reagent coated with complete PINP capture antibody, then adding 60 mu L of reagent containing biotin-labeled complete PINP detection antibody 7D06, lightly shaking and uniformly mixing, incubating for 15min at room temperature, then fully washing with washing buffer solution, adding Acridinium Ester (AE) -labeled Streptavidin (SA), reacting for 10min at room temperature, fully washing with washing buffer solution, finally adding pre-excitation solution, simultaneously measuring optical density value by using a luminometer, and drawing by taking logarithm of optical density value of each calibrator as ordinate and logarithm of concentration of each calibrator as abscissa to obtain a standard curve;
s2: and replacing the standard solution with the to-be-detected serum sample, and substituting the optical density value of the to-be-detected serum sample into the standard curve to obtain the complete PINP content in the to-be-detected serum in the same other steps as S1.
Further, the dilution of the biotin-labeled complete PINP detection antibody is 1: 100-500, and preferably 1: 250.
Further, the dilution of the acridinium ester-labeled streptavidin is 1: 100-1000, and preferably 1: 250.
Compared with the prior art, the invention has the following beneficial effects:
1. the kit provided by the invention is based on complete PINP in blood as a marker, and because the complete PINP metabolism in blood is not influenced by the kidney function, the synthesis and bone formation of type 1 collagen can be truly and accurately reflected, and the kit has reliable diagnostic value in osteoporosis. And the capture antibody coated magnetic microsphere, the complete PINP antigen, the biotinylation antibody and the immune complex of the Acridinium Ester (AE) marked Streptavidin (SA) form a double-sandwich structure, the acridinium ester instantaneous luminescence and magnetic bead separation technology is adopted, and the biotin-avidin system cascade amplification effect is utilized, so that the sensitivity of the detection reagent is greatly improved, the reaction time is reduced, the cost is low, the noise interference is reduced, and the like.
2. The kit disclosed by the invention takes the capture molecules and the monoclonal antibodies as detection molecules, develops a diagnostic reagent for detecting complete PINP in serum by a chemiluminescence method, evaluates the analysis performance of the diagnostic reagent in a laboratory, and shows that the detection result has high accuracy, good linear relation, good stability, strong specificity and high sensitivity, and compared with the performance of a Roche PINP analysis product, the diagnostic reagent has a wider linear range (0.5-1500ng/mL), a wide detection range and anti-interference performance, and can more effectively meet the requirements of clinical detection reagents. The method can provide reliable clinical reference value for early diagnosis, early intervention and prognosis of osteoporosis.
Drawings
Figure 1 is the formation of intact PINP and the identification of intact PINP antibodies; (A) type 1 collagen and intact PINP formation; (B) SDS-PAGE patterns of intact PINP; (C) SEM images of intact PINP molecules in osteoblasts; (D) an SDS-PAGE pattern of the monoclonal antibody 7D 13H chain; (E) SDS-PAGE of the chain of monoclonal antibody 7D 06H.
FIG. 2 is a graph based on luminescence at different PINP antibody coating concentrations.
FIG. 3 is a graph of Coefficient of Variation (CV) values for RLU at various reference standard concentrations.
FIG. 4 is the stability of the serum concentration of intact PINP in 5 healthy persons at 25 ℃ and 35 ℃; a is the measurement of intact PINP at 25 ℃; (B) assay of intact PINP at 35 ℃.
Figure 5 is a comparison between the proposed detection method in serum of patients with osteoporosis and the Roche PINP detection result (n 257).
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited thereto. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and starting materials, if not otherwise specified, are commercially available and/or may be prepared according to known methods.
Reagents referred to in the following examples: binding buffer (0.1M MES, pH 5.0); cri pyridine ester labeling buffer (0.1M phosphate buffer, pH 6.3); blocking buffer (0.1mol/L Tris-base, 2.5% bovine serum albumin); washing buffer (0.2mol/L phosphate buffered saline, 0.05% Proclin-300, 0.05% Tween-20, pH 7.2); sample buffer (0.05mol/L Tris-base, 0.85% NaCl, 0.05% Tween-20, 7.5%, bovine serum albumin, 0.05% Proclin-300, pH7.2) and TBST buffer (0.1mol/L phosphate buffer, 0.05% Proclin-300, 2.5% bovine serum albumin, cri pyridine ester, pH 7.2).
First, preparation of capture antibody 7D13 and detection antibody 7D06
The structure of a type I procollagen molecule is given in figure 1A. PINP exists in a variety of linear forms, including intact trimeric PINP (intact PINP) and monomeric or dimeric degradation products. In the extracellular space, two enzymes hydrolyze proteins and release a large globular carboxy-terminal propeptide PICP and an amino-terminal rod-like propeptide (intact PINP) domain. Like type I collagen, intact PINP consists of two pre- α 1 and one pre- α 2 chains that are not covalent to each other. The molecular weight of the entire protein was approximately 35,000kDa (FIGS. 1A and 1B). Type I collagen biosynthesis occurs mainly in osteoblasts (fig. 1C). Intact PINP is hydrolyzed from the collagen molecule primarily during the synthesis of fibrils of the collagen molecule at the collagen surface, and removal of the amino-terminal propeptide is accomplished as the thickness of the fibrils increases. Thus, intact PINP is closely associated with bone formation. In our laboratory, two paired monoclonal antibodies (7D13 and 7D06) were prepared using the intact P1NP antigen. The H chain molecular weight of the 7D13 and 7D06 monoclonal antibodies was about 50kDa (FIGS. 1D and 1E), which was confirmed by Western blotting. The two monoclonal antibodies are two different epitopes of one antibody, the former is used as a capture antibody, the latter is used as a detection antibody, and the preparation adopts a hybridoma technology.
Example 1a chemiluminescent assay kit for the detection of intact PINP in serum, comprising reagent R1 containing magnetic microspheres coated with intact PINP capture antibody 7D13, reagent R2 containing acridinium ester labeled streptavidin, and reagent R3 containing biotin labeled intact PINP detection antibody 7D 06.
1. Preparation of biotin-labeled intact PINP detection antibody 7D06
(1) Pretreatment of the PINP-Abs: because the protein has to reach a certain concentration during the labeling reaction, the protein needs to be concentrated before labeling, and an ultrafiltration centrifugation method is usually adopted at present.
(2) Ligation of biotin to PINP-Abs: covalent attachment of biotin to the PINP-Abs requires that the activity of the original antibody be maximized after attachment. To reduce the steric hindrance of Biotin binding to PINP-Abs, we selected Biotin (NHS-LC-Biotin) with an extended spacer. Firstly, balancing biotin to room temperature, adding DMSO to fully dissolve the biotin to ensure that the final concentration of the biotin is 10mg/mL, slowly adding a certain amount of biotin into the treated PINP-Abs according to the mass ratio of the biotin to the PINP-Abs, adjusting the final concentration of the antibody to be 2mg/mL by using 0.2mol/L PB, and carrying out oscillation reaction at 25 ℃ in the dark for 1 h.
(3) And (3) purification: after the crosslinking reaction, Sephadex G-50 gel chromatography is adopted for purification to remove free biotin and avoid the influence of the free biotin on the binding capacity of the biotinylated protein compound and streptavidin. The method comprises the following specific steps: 1) washing the column (1 × 25cm) with deionized water. Weighing a certain amount of chromatographic column filler Sephadex (TM) G-50, boiling with deionized water for swelling, washing with deionized water for several times, mixing uniformly, slowly injecting into a chromatographic column along the column wall, observing the filler density to be uniform by naked eyes after the filler is naturally settled, pressing the column with deionized water at the flow rate of 2mL/min after no crack or bubble, fully washing about 10 column volumes of the column bed, balancing the column bed with 0.2mol/L PB (pH7.2) buffer solution, and preparing for sample loading after the baseline of the recorder becomes stable after about 3-5 column volumes. 2) Taking down the plunger, discharging the excessive water of the gel until a layer of extremely thin liquid level is left between the gel and the gel, sucking the biotinylated antibody by a pipette gun and slowly injecting the biotinylated antibody into the column, paying attention to no need to flush the gel, installing the plunger, eluting the labeled biotinylated PINP-Abs by 0.2mol/L PB buffer solution at the flow rate of 1mL/min, starting to collect the protein when a protein peak appears on the interface of a purification system, continuously eluting by 0.2mol/L PB at the flow rate of 2mL/min until the base line is stable, eluting by 10 column volumes by deionized pure water, and finally, carefully and vertically placing the packed column in a refrigerator at the temperature of 2-8 ℃ for storage.
(4) And (3) storage: the purified biotin-labeled PINP-Abs was filtered through a 0.22 μm filter, then added with a protein stabilizer, diluted to working concentration with 2.5% TBST, dispensed into R1 reagent bottles, labeled, and stored at 4 ℃ under sealed conditions.
2. Preparation of acridinium ester-labeled streptavidin
(1) Preparation of acridinium ester and streptavidin: balancing the acridine ester to room temperature, weighing a certain amount of the acridine ester, adding DMSO to fully dissolve the acridine ester to ensure that the final concentration of the acridine ester is 10mg/mL, and storing the acridine ester in a dark place.
(2) Acridinium ester-labeled streptavidin: the acridinium ester labeled streptavidin requires that the acridinium ester can still maintain the original quantum effect after coupling, and the binding activity of the streptavidin is changed to a small extent. According to the mass ratio of the connection of the acridinium ester and the streptavidin, the acridinium ester is added into a certain amount of pretreated streptavidin, the total volume of 0.2mol/L PB (pH 6.3) is adjusted to be 500 mu L, and the vibration reaction is carried out for 12h at 25 ℃.
(3) And (3) purification: purification in the ligation step with biotin to PINP-Abs, but the purified buffer was changed to 0.2mol/L PB (pH 6.3).
(4) And (3) storage: and (4) storing the biotin and the PINP-Abs in the step of connecting, subpackaging in an R2 reagent bottle, labeling, sealing at 4 ℃ and keeping away from light.
3. Preparation of magnetic microspheres coated with complete PINP capture antibody 7D13
(1) Put 8mg of the suspended magnetic microsphere liquid into a 1.5mL EP tube, add binding buffer and wash 5 times on the magnetic frame, to activate the carboxyl groups on the surface of the magnetic microspheres, add 25. mu. LEDC (10mg/mL) freshly prepared carboxyl activating solution and 40. mu. LN-hydroxysuccinimide (NHS, 10mg/mL) and rotate at 30 ℃ for 30 minutes, add binding buffer again, and wash the magnetic frame multiple times to obtain activated magnetic microspheres.
(2) Eighty micrograms of prepared antibody (7D13) was added to the activated magnetic microspheres and the concentration of magnetic microspheres was adjusted to 20mg/mL and then placed on a suspension vortex shaker at room temperature for 12 h. The following day, blocking buffer (containing 2.5% bovine serum albumin) was added to the conjugate product and placed on a suspension vortex shaker at room temperature for 2 hours. After the reaction was completed, the reaction mixture was washed 3 times with blocking buffer, resuspended in blocking buffer, dispensed into R3 reagent bottles, labeled, and stored in sealed containers at 4 ℃.
1mg of magnetic microspheres were coupled with different concentrations (20. mu.g/mg, 10. mu.g/mg and 5. mu.g/mg) of intact PINP antibody 7D13, and the other steps were as in (1) (2), and then direct luminescence measurement was used to compare the advantages of the three envelope concentrations.
As a result, as shown in FIG. 2, the maximum luminescence value of the coating concentration of 20. mu.g/mg was close to that of the coating concentration of 10. mu.g/mg, indicating that the concentration of 10. mu.g/mg was close to saturation and that the coating of 10. mu.g/mg maintained good linearity. Therefore, we chose 10 μ g/mg as the concentration of intact PINP antibody coated by magnetic microspheres.
Example 2 method of Using a chemiluminescent assay kit for the detection of intact PINP in serum
S1: adding 50 mu L of standard solution with concentration gradient into 60 mu L of magnetic microsphere reagent coated with complete PINP capture antibody 7D13, then adding 60 mu L of reagent containing biotin-labeled complete PINP detection antibody 7D06, gently oscillating and uniformly mixing, co-incubating for 15min at room temperature, then fully washing with washing buffer solution, adding Acridinium Ester (AE) -labeled Streptavidin (SA), reacting for 10min at room temperature, fully washing with washing buffer solution, finally adding pre-excitation solution, simultaneously measuring optical density value by using a luminometer, and drawing by taking logarithm of optical density value of each calibrator as ordinate and logarithm of concentration of each calibrator as abscissa to obtain a standard curve;
s2: adding 50 mu L of a serum sample to be detected into 60 mu L of magnetic microsphere reagent coated with complete PINP capture antibody 7D13, then adding 60 mu L of reagent containing biotin-labeled complete PINP detection antibody 7D06, lightly shaking and uniformly mixing, incubating for 15min at room temperature, then fully washing with a washing buffer solution, then adding Acridinium Ester (AE) -labeled Streptavidin (SA), reacting for 10min at room temperature, then fully washing with the washing buffer solution, finally adding a pre-excitation solution, and simultaneously measuring the optical density value by using a luminometer; and substituting the optical density value of the serum sample to be detected into the standard curve to obtain the complete PINP content in the serum to be detected.
Example 3 evaluation of the Performance of the kit of the present invention
Total and intact PINPs in serum of kidney disease patients before and after hemodialysis were detected using standard methods (Roche kit) and the present invention, respectively.
The results show that: the mean concentration of total PINP (302.7 + -82.3 ng/mL) before hemodialysis was 4.63-fold greater than the mean concentration of intact PINP (65.4 + -37.5 ng/mL); the mean concentration of total PINP (125.7 + -54.6 ng/mL) after hemodialysis was 2.02-fold greater than the mean concentration of intact PINP (62.3 + -35.8 ng/mL). Overall, the mean concentration of total PINP in hemodialysis patients was significantly higher than the mean of intact PINP, and the correlation between the autoregressive and total PINP outcomes in hemodialysis patients was not linear, similar to that previously reported. This is because intact PINP is rapidly taken up by the liver, whereas the clearance of PINP monomeric forms may depend on renal function, and thus renal disease has a different clearance of the two forms of PINP. This also suggests a monomeric form of PINP in the blood of renal patients, which makes the assay value not truly reflective of the degradation status of procollagen. Thus, the intact PINP assay more closely reflects the degradation profile of procollagen in vivo than the total PINP assay.
(1) Linear analysis
The complete PINP standard was diluted with TBST buffer to give the following eight concentrations: 0.00ng/mL, 0.50ng/mL, 1.00ng/mL, 5.00ng/mL, 50.0ng/mL, 500ng/mL, 1000ng/mL and 1500 ng/mL. The assay was performed as in example 2, using the diluted concentration as the x-coordinate, and the Relative Light Units (RLUs) for each PIN as the y-coordinate, to plot a complete PINP reference standard curve.
As shown in fig. 3, the two coordinates are linear over a concentration range of 0.5-1500ng/mL, the linear equation Y is 3.94+1.04X, the linear correlation coefficient R is 0.9995, and the percentage of concentration variation coefficient of RLU per reference standard is between 3.80% and 7.10%. It can be seen that the present invention is almost linear, covering the level of medical diagnosis, in the range between very low and very high concentrations (0.5-1500 ng/mL).
(2) Specificity detection
Five antigens of bone were used using the method of example 2: alkaline phosphatase, osteocalcin, collagen type I cross-linked C-terminal peptide (β -CTX), parathyroid hormone (PTH) and 25-OH vitamin D were substituted for intact PINP and levels were determined where the level of interfering antigens was about 10-fold above the medical decision level.
TABLE 1 specificity of detection method for intact PINP
As shown in the results in Table 1, the cross-reactivity of the method of the present invention to the above five antigens was 0-0.05%, indicating that the kit did not cross-react with other bone transformation-related markers (alkaline phosphatase, osteocalcin, beta-c terminal peptide, parahormone and 25-OH vitamin D) and recognized intact PINP with high specificity.
(3) Analysis of accuracy and precision
Test samples were prepared by diluting high-value patient samples with low-value patient samples prior to assay, and after mixing equal amounts of different samples containing quantitative concentrations of intact PINP, the measured concentrations were compared to predicted concentrations, and samples of intact PINP at different concentrations were subjected to 3 sets of parallel experiments, with the results shown in table 2.
TABLE 2 test Performance of the complete PINP CLIA
② complete PINP at three quality control concentrations (1500ng/mL, 50.0ng/mL and 0.50ng/mL) was detected by the method of example 2. And the assay variation coefficient was calculated by performing 20 replicates a day and 10 replicates a day. The results are shown in Table 3.
TABLE 3 precision of the complete PINP proposed assay
③ adding intact PINP reference standards at concentrations of about 50ng/mL and 500ng/mL, respectively, to sera having intact PINP concentrations of 0.05ng/mL, 0.50ng/mL, and 5.00ng/mL, respectively. The reference standard volume added is within 10% of the sample volume and the sampling is accurate. The recovery was calculated by the formula [ measured value/expected value × 100% ]. The results are shown in Table 4.
Table 4 recovery measured by fitting a curve (n ═ 3)
a expected concentration was determined using a specific octanoic acid (BCA) kit (pierce). These values are the average of 3 independent measurements in triplicate.
b concentration measured using the CLIA kit developed in this study.
The Coefficient of Variation (CV) was determined according to the formula [ mean SD/mean X100 ], expressed as a percentage.
d percent recovery is calculated according to the formula [ measured/expected x 100 ].
As can be seen from tables 2 to 4, the within-batch coefficient of variation of 0.8 to 1.7% and the ranges of the within-batch coefficient of variation and the between-batch coefficient of variation of 2.33% to 6.61% and 1.79% to 7.56%, both less than 10%, were considered acceptable using the kit of the present invention. The recovery rate of the kit is 93.1-101.4%, and the recovery rate is linear and satisfactory.
(4) Stability test
The kit of the invention is stored for 7 days at room temperature, 25 ℃, 35 ℃ and 4 ℃ respectively, and then sample detection is carried out, and the result is shown in figure 4.
The results showed that randomly drawn 5 patient samples were stored at room temperature and 4 ℃ for 7 days with no significant change in measured concentration of intact PINP (data not shown). However, intact PINP was reduced by 6.6% in samples stored at 25 ℃, the effect on intact PINP in the samples was not significant, and the effect of intact PINP in high and low value samples was not significantly different. However, the complete P1NP drop was significant in samples stored at 35 ℃, with a 17.5% -33.5% drop over 7 days, and the effect was greater as the total P1NP content in the sample was higher. These results confirm the effectiveness of our assay in the clinical diagnosis of osteoporosis.
(5) Clinical application
To evaluate the clinical utility of this method, 257 sera (142 physical exam cases; 115 patients with osteoporosis) were tested simultaneously using the method of the invention and the Roche kit, the complete PINP chemiluminescence immunoassay developed by the invention was called CLIA, and the total PINP chemiluminescence immunoassay provided by Roche was called ECLIA. As shown in fig. 5, the correlation coefficient of the present invention is r 0.9794, the correlation equation is Y2.12 +0.97X, and p < 0.0001, consistent with the results obtained using the ECLIA method. Therefore, the method can be clinically used for measurement of serum PINP.
Further, total PINP and intact PINP in serum before and after dialysis of renal patients were measured using the method of the present invention and Roche kit simultaneously, and Spearman's rank correlation between the two methods was compared. The results are shown in Table 5.
TABLE 5 detection of total and intact PINPs in serum from Hemodialysis (HD) patients using ECLIA and CLIA methods, respectively, comparison of Spearman grade correlation between the two methods
As can be seen from table 5, the Spearman scale correlation coefficient between ECLIA and CLIA was 0.774 and 0.735 (total n 25), respectively, before and after hemodialysis in renal patients. The Spearman grade correlation between ECLIA and CLIA was less than 0.900. Therefore, the method can also be clinically used for measuring the serum PINP of patients with nephropathy.
In conclusion, the detection result of the invention has high accuracy, good linear relation, good stability, strong specificity and high sensitivity, can be clinically used for measuring the serum PINP of a nephropathy patient, and can provide reliable clinical reference value for early diagnosis, early intervention and prognosis of osteoporosis.
The above description is only exemplary of the present invention and should not be taken as limiting, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A chemiluminescence quantitative detection kit for detecting complete PINP in serum is characterized by comprising a reagent R1 containing a biotin-labeled complete PINP detection antibody, a reagent R2 containing acridinium ester-labeled streptavidin and a reagent R3 containing magnetic microspheres coated by a complete PINP capture antibody; the capture and detection antibodies are two different monoclonal antibodies directed against different determinants of the intact PINP antigen.
2. The chemiluminescence quantitative detection kit for detecting intact PINP in serum according to claim 1, wherein the coating concentration of the capture antibody is 5-20 μ g/mL.
3. The chemiluminescence quantitative detection kit for detecting complete PINP in serum according to claim 1, wherein the mass ratio of acridinium ester to streptavidin in the acridinium ester labeled streptavidin is 1: 25-100; the mass ratio of biotin to the detection antibody in the biotin-labeled complete PINP detection antibody is 1: 5-15.
4. The chemiluminescence quantitative detection kit for detecting intact PINP in serum according to claim 1, further comprising a pre-excitation solution, wherein the pre-excitation solution comprises an acidic excitation solution and an alkaline excitation solution, the acidic excitation solution is hydrogen peroxide and nitric acid solution, and the alkaline excitation solution is sodium hydroxide solution.
5. The chemiluminescent quantitative detection kit for detecting intact PINP in serum according to claim 1, wherein the magnetic microsphere coated with the capture antibody is prepared by the following steps:
1) adding an EDC/NHS activating agent into the suspension magnetic microsphere liquid, rotating at 30 ℃ to activate carboxyl on the surface of the magnetic microsphere, adding a binding buffer solution, and cleaning the magnetic frame for multiple times to obtain an activated magnetic microsphere;
2) adding the capture antibody into the activated magnetic microspheres, adjusting the concentration of the magnetic microspheres to 15-20 mg/mL, placing the magnetic microspheres on a suspension vortex oscillator at room temperature for full oscillation, adding a sealing buffer solution for full reaction, and washing and re-suspending the magnetic microspheres coated with the capture antibody by using the sealing buffer solution after the reaction is finished to obtain the magnetic microspheres coated with the capture antibody.
6. The chemiluminescent kit for detecting intact PINP in serum of claim 5, wherein the molar ratio of EDC to NHS in the EDC/NHS activator is 5: 8.
7. The chemiluminescent assay kit for the detection of intact PINP in serum of claim 5, wherein the blocking buffer is Tris-base solution containing bovine serum albumin at a volume concentration of 2.5%; the binding buffer was 0.1M MES, pH 5.0.
8. The method for using the detection kit according to any one of claims 1 to 7, comprising the steps of:
s1: adding 50 muL of standard substance solution with concentration gradient into 60 muL of magnetic microsphere reagent containing complete PINP capture antibody coating, then adding 60 muL of reagent containing biotin-labeled complete PINP detection antibody, lightly shaking and uniformly mixing, incubating for 15min under the condition of room temperature, then fully washing by using washing buffer solution, adding acridinium ester-labeled streptavidin, reacting for 10min at room temperature, fully washing by using washing buffer solution, finally adding pre-excitation solution, simultaneously using a luminometer to measure optical density value, and drawing by taking logarithm of optical density of each calibrator as ordinate and logarithm of concentration of each calibrator as abscissa to obtain a standard curve;
s2: and replacing the standard solution with the to-be-detected serum sample, and substituting the optical density value of the to-be-detected serum sample into the standard curve to obtain the complete PINP content in the to-be-detected serum in the same other steps as S1.
9. The method of using the detection kit according to claim 8, wherein the dilution of the biotin-labeled complete PINP detection antibody is 1:100 to 500, preferably 1: 250.
10. The use method of the detection kit according to claim 8, wherein the dilution of the acridinium ester-labeled streptavidin is 1: 100-1000, preferably 1: 250.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113933497A (en) * | 2021-11-03 | 2022-01-14 | 辽宁中医药大学附属医院 | I type collagen amino-terminal extension peptide magnetic particle chemiluminescence immunoassay kit and detection method thereof |
| CN115839945A (en) * | 2023-02-13 | 2023-03-24 | 上海索昕生物科技有限公司 | Photosensitive microsphere for light-activated chemiluminescence detection |
| CN116047087A (en) * | 2022-12-29 | 2023-05-02 | 宁波瑞源生物科技有限公司 | Method for judging free biotin in sample |
| CN116068206A (en) * | 2023-03-15 | 2023-05-05 | 安徽惠邦生物工程有限公司 | Myelin basic protein detection kit and detection method thereof |
| CN116203243A (en) * | 2022-09-13 | 2023-06-02 | 美康生物科技股份有限公司 | PINP monoclonal antibody, kit containing PINP monoclonal antibody and application of PINP monoclonal antibody |
| WO2023182353A1 (en) * | 2022-03-25 | 2023-09-28 | 積水メディカル株式会社 | Immunological detection method and immunological detection kit |
| WO2023182343A1 (en) * | 2022-03-25 | 2023-09-28 | 積水メディカル株式会社 | Immunological detection method and immunological detection kit |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5895746A (en) * | 1995-04-19 | 1999-04-20 | Orion-Yhtyma Oy | Antibody to aminoterminal propeptide of type 1 procollagen, and assay method using it |
| US6008325A (en) * | 1995-04-19 | 1999-12-28 | Orion Diagnostica | Antibody to aminoterminal propeptide of type 1 procollagen |
| CN105759055A (en) * | 2016-03-31 | 2016-07-13 | 广州菲康生物技术有限公司 | P1NP detection kit and preparation method thereof |
| CN108152507A (en) * | 2017-11-28 | 2018-06-12 | 泰州泽成生物技术有限公司 | A kind of I type precollagen amino terminal peptides detection kit and its detection method |
| CN108333344A (en) * | 2017-12-26 | 2018-07-27 | 南京迪格诺斯生物技术有限公司 | Highly sensitive chemical luminescence immune analysis reagent box and its preparation method and application |
| CN109061176A (en) * | 2018-06-29 | 2018-12-21 | 江南大学 | A kind of total Type I collagen aminoterminal of detection serum extends the kit and application method of peptide |
| CN111007269A (en) * | 2019-11-15 | 2020-04-14 | 迪瑞医疗科技股份有限公司 | Total I type collagen amino-terminal extension peptide chemiluminescence immunoassay kit and preparation method thereof |
-
2020
- 2020-10-30 CN CN202011198027.8A patent/CN112326972A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5895746A (en) * | 1995-04-19 | 1999-04-20 | Orion-Yhtyma Oy | Antibody to aminoterminal propeptide of type 1 procollagen, and assay method using it |
| US6008325A (en) * | 1995-04-19 | 1999-12-28 | Orion Diagnostica | Antibody to aminoterminal propeptide of type 1 procollagen |
| CN105759055A (en) * | 2016-03-31 | 2016-07-13 | 广州菲康生物技术有限公司 | P1NP detection kit and preparation method thereof |
| CN108152507A (en) * | 2017-11-28 | 2018-06-12 | 泰州泽成生物技术有限公司 | A kind of I type precollagen amino terminal peptides detection kit and its detection method |
| CN108333344A (en) * | 2017-12-26 | 2018-07-27 | 南京迪格诺斯生物技术有限公司 | Highly sensitive chemical luminescence immune analysis reagent box and its preparation method and application |
| CN109061176A (en) * | 2018-06-29 | 2018-12-21 | 江南大学 | A kind of total Type I collagen aminoterminal of detection serum extends the kit and application method of peptide |
| CN111007269A (en) * | 2019-11-15 | 2020-04-14 | 迪瑞医疗科技股份有限公司 | Total I type collagen amino-terminal extension peptide chemiluminescence immunoassay kit and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| MARJA-KAISA KOIVULA等: "Measurement of aminoterminal propeptide of type I procollagen (PINP) in serum", 《CLINICAL BIOCHEMISTRY》 * |
| MARJA-KAISA KOIVULA等: "Validation of an automated intact N-terminal propeptide of type I procollagen (PINP) assay", 《CLINICAL BIOCHEMISTRY》 * |
| PAUL D MILLER: "Chronic kidney disease and the skeleton", 《BONE RESEARCH》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113933497A (en) * | 2021-11-03 | 2022-01-14 | 辽宁中医药大学附属医院 | I type collagen amino-terminal extension peptide magnetic particle chemiluminescence immunoassay kit and detection method thereof |
| WO2023182353A1 (en) * | 2022-03-25 | 2023-09-28 | 積水メディカル株式会社 | Immunological detection method and immunological detection kit |
| WO2023182343A1 (en) * | 2022-03-25 | 2023-09-28 | 積水メディカル株式会社 | Immunological detection method and immunological detection kit |
| CN116203243A (en) * | 2022-09-13 | 2023-06-02 | 美康生物科技股份有限公司 | PINP monoclonal antibody, kit containing PINP monoclonal antibody and application of PINP monoclonal antibody |
| CN116047087A (en) * | 2022-12-29 | 2023-05-02 | 宁波瑞源生物科技有限公司 | Method for judging free biotin in sample |
| CN115839945A (en) * | 2023-02-13 | 2023-03-24 | 上海索昕生物科技有限公司 | Photosensitive microsphere for light-activated chemiluminescence detection |
| CN116380883B (en) * | 2023-02-13 | 2024-02-23 | 上海索昕生物科技有限公司 | Photosensitive microsphere for photoexcitation chemiluminescence detection |
| CN116068206A (en) * | 2023-03-15 | 2023-05-05 | 安徽惠邦生物工程有限公司 | Myelin basic protein detection kit and detection method thereof |
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