CN112269021B - IgM quality control product and preparation method thereof - Google Patents
IgM quality control product and preparation method thereof Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention provides an IgM quality control product and a preparation method thereof, wherein the IgM quality control product comprises: respectively linking a mu chain fragment and anti-2019-nCoV IgG in animal antiserum by using SMCC to obtain a compound; wherein the N-maleimide methyl-terminus of SMCC is linked to the amino-terminus of anti-2019-nCoV IgG and the succinimide-terminus of SMCC is linked to the thiol-terminus of the mu-chain fragment. The IgM quality control product has immunity and specificity, and can replace clinical anti-2019-nCoV IgM as the quality control product of an immunological diagnosis kit.
Description
Technical Field
The invention relates to the technical field of chemical detection, in particular to an IgM quality control product and a preparation method thereof.
Background
Since the outbreak of the new coronavirus epidemic situation at the end of 2019, the diagnosis method of 2019-nCoV (the new coronavirus of 2019) has great attention at home and abroad, and the method from imaging, nucleic acid detection and serum test is an effective method for diagnosing 2019-nCoV. On 4/3/2020, State health Commission released the latest version of the diagnosis and treatment of novel coronavirus pneumonia (trial seventh edition). The new diagnosis and treatment scheme adds new serum specific IgM and IgG antibodies as one of the etiological diagnosis standards. After the virus invasion, the immune response of the human body firstly reacts to IgM antibody, and generally has positive reaction 3-5 days after the virus invasion, so that the test of the specific IgM antibody of the novel coronavirus has very important significance for diagnosing the novel coronavirus.
At present, reagents capable of quickly diagnosing the specific IgM antibodies of the novel coronavirus are developed by a plurality of reagent manufacturers in China, but because the acquisition of human positive serum is very difficult, the development of clinical experiments is limited because the value of the diagnostic reagents cannot be fixed, and the marketing of products is limited or delayed, the acquisition of IgM quality control products with high yield and high safety factor has great significance.
Generally, the collection and the setting of clinical samples are usually needed to obtain human blood quality control products, and the collection and the setting are not easy to realize at present. Or the gene recombination technology can be used for directly obtaining the target protein to be used as a quality control product, but the specific IgM antibody has large molecular weight and specificity, so that the sequence cannot be accurately predicted. Therefore, there is a need to provide an artificial IgM quality control product having both immunity and specificity.
Disclosure of Invention
The invention provides an IgM quality control product and a preparation method thereof, and the IgM quality control product can replace clinical anti-2019-nCoV IgM to be used as a quality control product of an immunological diagnosis kit.
In order to achieve the purpose, the invention is realized by the following technical scheme:
in a first aspect, the present invention provides an IgM quality control comprising: respectively linking a mu chain fragment and anti-2019-nCoV IgG in animal antiserum by using SMCC to obtain a compound;
wherein the N-maleimide methyl-terminus of SMCC is linked to the amino-terminus of anti-2019-nCoV IgG and the succinimide-terminus of SMCC is linked to the thiol-terminus of the mu-chain fragment.
Optionally, the animal antisera comprises rabbit antisera or mouse antisera.
Alternatively, the μ -strand fragment is obtained by gene recombination techniques.
In a second aspect, the present invention provides a method for preparing an IgM quality control as described in any one of the first aspect above, comprising: and step one, firstly linking anti-2019-nCoV IgG in animal antiserum by using SMCC, and then linking a mu chain fragment to prepare the IgM quality control product.
Optionally, the first step includes: adding excessive SMCC into the animal antiserum according to the concentration of anti-2019-nCoV IgG in the animal antiserum, dialyzing redundant SMCC in a reaction product after reacting for a certain time at room temperature, performing the dialysis at least once, and detecting the concentration of a linking product of the SMCC and the anti-2019-nCoV IgG in the reaction product after the dialysis is finished; and adding mu chain fragments into the reaction product according to a certain proportion according to the concentration of the linking product, and reacting at room temperature for a certain time to prepare the IgM quality control product.
Optionally, before the first step, the method further comprises: a step of dialyzing the animal antiserum containing the anti-2019-nCoV IgG and the solution containing the mu chain fragment against a buffer, respectively, wherein the dialysis process is performed at least twice, and the pH of the buffer is adjusted to 7.15 in the last dialysis process.
Optionally, after the first step, the method further comprises: and (3) testing the linking efficiency of the IgM quality control product by using a colloidal gold detection card.
Optionally, before the first step, the method further comprises: culturing the mu chain fragment using a strain for amplification, extracting the mu chain fragment from the strain, and purifying the extract to obtain a purified mu chain fragment; and (3) a step of testing the activity of the purified mu chain fragment by using a colloidal gold test card.
In a third aspect, the invention provides an IgM quality control as described in any one of the first aspects above or an IgM quality control prepared by a method as described in any one of the second aspects above for use in 2019-nCoV detection.
In a fourth aspect, the invention provides a kit for detecting 2019-nCoV, comprising: an IgM quality control product according to any one of the above first aspects or an IgM quality control product produced by the method according to any one of the above second aspects.
The invention provides an IgM quality control product and a preparation method thereof, wherein the IgM quality control product comprises: respectively linking a mu chain fragment and anti-2019-nCoV IgG in animal antiserum by using SMCC to obtain a compound; wherein the N-maleimide methyl-terminus of SMCC is linked to the amino-terminus of anti-2019-nCoV IgG and the succinimide-terminus of SMCC is linked to the thiol-terminus of the mu-chain fragment. The IgM quality control product has immunity and specificity, and can replace clinical anti-2019-nCoV IgM as the quality control product of an immunological diagnosis kit.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 shows the results of the reagent diagnosis of an IgM quality control substance and a blank set according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete, the technical solutions in the embodiments of the present invention will be described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention, and based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts belong to the scope of the present invention.
Example 1
The embodiment of the invention provides an IgM quality control product, which comprises: respectively linking a mu chain fragment and anti-2019-nCoV IgG in animal antiserum by using SMCC to obtain a compound; wherein the N-maleimide methyl-terminus of SMCC is linked to the amino-terminus of anti-2019-nCoV IgG and the succinimide-terminus of SMCC is linked to the thiol-terminus of the mu-chain fragment.
In detail, the mu chain is a characteristic chain of human IgM, and the mu chain fragment in the present example is one fragment of the mu chain. The mu chain fragment can be obtained by gene recombination technology. For example, when the nucleotide sequence of the μ chain is known, the nucleotide sequence of the μ chain fragment is genetically recombined, and the artificially recombined μ chain fragment can be obtained through steps such as cell expansion culture, extraction and purification of the μ chain fragment. Because the mu chain is the characteristic chain of human IgM, when the IgM quality control product is prepared by using the mu chain fragment, the prepared IgM quality control product has immunity.
In detail, the animal antiserum in the embodiment of the present invention may be rabbit antiserum, mouse antiserum, chicken antiserum, horse antiserum, sheep antiserum, or the like. After the 2019-nCoV immunogen is used for infecting an animal, an IgG antibody resisting the 2019-nCoV, namely the anti-2019-nCoV IgG, usually exists in serum, so when the anti-2019-nCoV IgG is used for preparing an IgM quality control product, the prepared IgM quality control product has specificity.
Based on the immunity of the mu chain fragment and the specificity of anti-2019-nCoV IgG, after the two are coupled by using a cross-linking agent, the obtained coupling compound has the immunity and the specificity, and meets the quality control function of anti-2019-nCoV IgM, so that the clinical anti-2019-nCoV IgM can be replaced as the quality control material of the immune diagnostic kit.
In addition, unlike the conventional protein labeling method, which is a commonly used EDC (N-3-dimethylaminopropyl-N' -ethylcarbodiimide) labeling method, the present example uses SMCC (4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt) as a crosslinking agent to link the μ chain fragment and the anti-2019-nCoV IgG, respectively. Specifically, the N-maleimide methyl-base end of the SMCC is linked with the amino end of the anti-2019-nCoV IgG, and the succinimide end of the SMCC is linked with the sulfydryl end of the mu-chain fragment, so that the labeling can be carried out in a directional manner, and the prepared quality control product has high activity, so that the problem that the titer and the activity of the prepared quality control product are low due to self-crosslinking or polymer formation inevitably caused by the fact that one end of an EDC labeling method is linked with the amino end and the other end of the EDC labeling method is linked with the carboxyl end is avoided.
Example 2
The embodiment of the invention provides a method for preparing the IgM quality control product in embodiment 1, which comprises the following steps: and step one, firstly linking anti-2019-nCoV IgG in animal antiserum by using SMCC, and then linking a mu chain fragment to prepare the IgM quality control product.
Through multiple experimental studies, it is found that coupling fails if SMCC links the μ chain fragment first and then the IgG, which may be due to the abundant amino and thiol groups in the μ chain fragment, and if linking the μ chain fragment first leads to self-coupling, and linking the IgG first and then the μ chain fragment later leads to coupling, the N-maleimide terminal of SMCC links the amino terminal of IgG first and the succinimide terminal of SMCC links the thiol terminal of the μ chain fragment first, so that labeling can be directed, and the activity of the prepared coupling complex is high.
In one embodiment of the present invention, the first step includes: adding excessive SMCC into the animal antiserum according to the concentration of anti-2019-nCoV IgG in the animal antiserum, dialyzing redundant SMCC in a reaction product after reacting for a certain time at room temperature, performing the dialysis at least once, and detecting the concentration of a linking product of the SMCC and the anti-2019-nCoV IgG in the reaction product after the dialysis is finished; and adding mu chain fragments into the reaction product according to a certain proportion according to the concentration of the linking product, and reacting at room temperature for a certain time to prepare the IgM quality control product.
In this step, excess SMCC is removed by dialysis, thereby avoiding the reaction of excess SMCC with the mu-chain fragment to cause self-coupling, and thus avoiding the formation of the coupled complex. Thus, the amount of mu chain fragments is more accurate.
In one embodiment of the present invention, before the first step, the method further comprises: a step of dialyzing the animal antiserum containing the anti-2019-nCoV IgG and the solution containing the mu chain fragment against a buffer, respectively, wherein the dialysis process is performed at least twice, and the pH of the buffer is adjusted to 7.15 in the last dialysis process.
It is to be noted that the choice of optimal conditions for the use of SMCC as a cross-linking agent is important, and that slight deviations will result in very low reactivity and even coupling failure.
Experimental studies have shown that preferably both the animal antiserum containing anti-2019-nCoV IgG and the solution containing μ chain fragments are subjected to dialysis to change the buffer conditions (pH and ionic strength) before reaction with SMCC. The dialysis process can be performed several times and preferably, the pH of the buffer needs to be adjusted to 7.15 at the last dialysis. After the dialysis is finished, the IgM quality control product can be prepared by crosslinking with SMCC.
It was found by experimental studies that preferably the buffer used for dialysis is 100mM PB buffer and the buffer contains 50mM NaCl. Based on this, the dialysis process can be performed by first dialyzing the buffer solution with pH 7.5 at-4 ℃ for 12 hours, twice, and then dialyzing the buffer solution with pH 7.15 at-4 ℃ for 12 hours, once.
In order to optimize the pH value of the buffer solution in the last dialysis process, the pH values of the buffer solution in the last dialysis process are respectively 7.0, 7.15, 7.3 and 7.5 in the experimental research process, and IgM quality control products are respectively prepared on the basis of the pH values. After the IgM quality control was prepared, coupling efficiency of the obtained IgM quality control was checked using a finished colloidal gold test card (capture method), and the test results are shown in table 1 below. Wherein, the coupling effect is expressed by the score of the chromogenic intensity, and the value range of the score is 1 to 10. Wherein, the better the coupling effect, the clearer the color development, the higher the color development intensity and the higher the score, and if not coupled, the color development will not be carried out.
TABLE 1
From the test results shown in table 1, it was found that by adjusting the pH to 7.15 at the last dialysis, the dialyzed animal antiserum containing anti-2019-nCoV IgG and the solution containing μ chain fragments could have higher coupling efficiency when reacting with SMCC.
In addition, it should be noted that, due to the difference in osmotic pressure during dialysis, the volume of antiserum may change after dialysis with antiserum, and the concentration of anti-2019-nCoV IgG may increase or decrease. The amount of the crosslinking agent to be used is usually adjusted depending on the concentration after dialysis.
Theoretically, the molar ratio X of SMCC as a cross-linking agent to anti-2019-nCoV IgG（SMCC:Ab）The calculation can be made with reference to the following ratios:
C（Ab）＜1mg/ml，40≤X（SMCC:Ab）≤80；
1mg/ml≤C（Ab）≤4mg/ml，X（SMCC:Ab）=20；
5mg/ml≤C（Ab）≤10mg/ml，5≤X（SMCC:Ab）≤10。
it should be noted that, in the actual test process, the above theoretical ratio can be referred to and adjusted as required to achieve the desired crosslinking effect. For example, it is preferred to use the following empirical ratios as a result of experimental studies:
1) when the product is purified by a ProteinA column, C is not less than 5mg/ml（Ab）≤10mg/ml，X（SMCC:Ab）=20；
2) When purifying with an antigen affinity column, C（Ab）＜1mg/ml，XSMCC:Ab）=200。
Based on the above, the IgM quality control product prepared by the embodiment of the present invention has high activity by optimally selecting the selection of the buffer solution, the coupling order, the usage amount of the cross-linking agent, and the like.
Based on the above, in an embodiment of the present invention, after the first step, the method further includes: and (3) testing the linking efficiency of the IgM quality control product by using a colloidal gold detection card. By testing the linking efficiency of the prepared IgM quality control product, whether the preparation process of the IgM quality control product is to be optimized or not can be verified, and the preparation process can be optimized by adjusting related parameters as required so as to achieve good coupling efficiency.
In one embodiment of the present invention, before the first step, the method further comprises: culturing the mu chain fragment using a strain for amplification, extracting the mu chain fragment from the strain, and purifying the extract to obtain a purified mu chain fragment; and (3) a step of testing the activity of the purified mu chain fragment by using a colloidal gold test card.
As mentioned above, the μ -strand fragment used to link SMCCs is a fragment of the μ -strand. By verifying the activity of the mu chain fragment, it can be determined whether the selected mu chain fragment is suitable.
During the course of the experimental study, three μ -chain fragments were designed which could be used for immunodiagnosis, defined as μ 1, μ 2 and μ 3, respectively. Based on the above, after obtaining three kinds of purified μ chain fragments, the activities of the three kinds of μ chain fragments were detected using a colloidal gold assay card (NC membrane and gold particles were coated with anti- μ chain IgG, respectively). For example, a mu chain fragment can be injected into a laboratory mouse such that the mu chain fragment specifically binds to IgG in the mouse and IgG in the mouse serum is present against the mu chain fragment. The colloidal gold detection card is used for detecting the mouse serum, and the more obvious the color development is, the higher the activity of the mu chain fragment is.
The results of measurement of the activity of the μ -chain fragment are shown in Table 2 below. Wherein, the crosslinking effect is expressed by the score of the color development intensity, and the value range of the score is 1 to 10. Wherein, the better the crosslinking effect, the higher the color development intensity and the higher the score.
TABLE 2
Referring to the above scoring results, three μ chain fragments were available, indicating that μ chains can be used in immune-based diagnostic products completely in place of the IgM immunogenicity. Furthermore, the activity of the μ chain fragment corresponding to μ 2 was relatively higher compared to μ 1 and μ 3.
Based on the above, the process of preparing the IgM quality control substance will be described in detail by the following Experimental example 1.
Experimental example 1
1.1 preparation of mu chain fragments
1.1.1 design of mu chain fragments
According to the nucleotide sequence of the mu chain, the mu chain fragment which can be used for immunodiagnosis is designed, and the nucleotide chain of the mu chain fragment is obtained through a technical outsourcing service.
1.1.2 Strain expansion culture
Introducing the nucleotide chain of the mu chain fragment into the strain by gene recombination technology, and carrying out amplification culture on the strain. The strain containing the mu chain fragment is stored at-80 ℃.
1.1.3 extraction of mu chain fragments
1.1.3.1 crushing
700mL of the cells were dissolved in 50mM Tris-HCl buffer solution at pH 8.0, and the solution was sonicated in an ice bath (sonication power 400w, 6s intervals per 6s sonication, 180 sonications in total), and 7ul of 1M dtt (dithiothreitol) was added after sonication.
1.1.3.2 centrifugation
The crushed solution is separately loaded into a centrifuge tube, centrifuged for 15min at the rotating speed of 12000rmp, and the supernatant is discarded to leave a precipitate.
1.1.3.3 removal of proteins
The pellet was reconstituted with inclusion body wash, centrifuged at 12000rmp for 15min, the supernatant was discarded and the pellet was retained and the procedure repeated 2 times.
1.1.3.4 solubilization of the protein of interest (i.e., the. mu. chain fragment)
Redissolving the precipitate with 8M urea, placing in a refrigerator at 4 ℃ for 1h, centrifuging at 12000rmp for 15min, and taking the supernatant as a sample.
1.1.4 purification of mu chain fragments
1.1.4.1 connecting the nickel ion affinity chromatography column with the detector, adding 200mM imidazole to remove impure protein in the nickel ion affinity chromatography column, balancing the nickel ion affinity chromatography column with urea with pH of 8.0 and concentration of 8M, and zeroing when the detector value is stable.
1.1.4.2 collecting sample, detecting by electrophoresis, collecting flow rate of not too fast, recording value of 0.117, collecting flow-through liquid, and performing SDS-PAGE electrophoresis detection.
In this step, 0.117 is an empirical value to demonstrate that no high concentration of contaminating proteins are shed and that there is no contaminating protein at the later equilibrium.
In the step, the target protein is combined with the nickel ion affinity chromatography column, the effluent liquid should not contain the target protein, and the effluent liquid is retained for electrophoresis detection to verify whether the target protein flows out, and once the effluent liquid flows out, the nickel ion affinity chromatography column does not retain the target protein.
1.1.4.3 when the sample is about to drop below the column level, the column is rinsed to baseline with urea at pH 8.0 and 8M.
In this step, the protein of interest and possibly the remaining foreign proteins are adsorbed in the cartridge.
1.1.4.4 the column was eluted with 20mM imidazole in 8M urea buffer, peaks (0.112, 0.018) were recorded and the eluate was collected, and the column was eluted with 200mM imidazole in 8M urea buffer, peaks (0.251, 0.5, 0.16) were recorded and the eluate was collected.
In this step, the purity of the collected mu-strand fragments can be improved by eluting the column with a buffer solution of contaminating proteins that may be present. And eluting the target protein in the column by using buffer solution to complete the purification and collection of the mu chain fragment.
In this step, the eluent from the range of 0.251-0.5-0.16 is collected as the final product, and the eluent flowing out of other ranges is not used because of lower concentration.
1.1.4.5 dialysis eluent: dialyzing with buffer solution of pH 7.5 at-4 deg.C for 12 hr twice, and dialyzing with buffer solution of pH 7.15 at-4 deg.C for 12 hr once. After the completion of the three dialyzations, the concentration of the mu chain fragment in the solution containing the mu chain fragment was 0.66mg/ml as measured by spectrophotometry.
In this step, urea is removed by dialysis.
1.2 preparation of animal antiserum containing anti-2019-nCoV IgG
1.2.1 obtaining animal antiserum
After 2019-nCoV immunogen is injected into the animal, blood is taken through arterial cannula, and animal antiserum is obtained. The mouse antiserum No. 1, the rabbit antiserum No. 2 and the rabbit antiserum No. 3 are obtained in the step. Rabbit antiserum No. 2 and rabbit antiserum No. 3 were taken from different rabbit individuals, respectively.
1.2.2 purification of antisera
The Protein A affinity chromatographic column is adopted to purify a mouse antiserum No. 1, a rabbit antiserum No. 2 and a rabbit antiserum No. 3 respectively, and the concentrations of the 2019-nCoV IgG in the purified antiserum are respectively 3.95mg/ml, 6.643mg/ml and 5.76 mg/ml.
1.2.3 dialysis antisera
For the purified mouse antiserum No. 1, rabbit antiserum No. 2 and rabbit antiserum No. 3, the buffer solution with pH of 7.5 is firstly used for dialysis for 12h at-4 ℃, and the dialysis is carried out twice, and then the buffer solution with pH of 7.15 is used for dialysis for 12h at-4 ℃ and the dialysis is carried out once. After the three times of dialysis, the antiserum is detected by a spectrophotometry method, and the concentrations of the anti-2019-nCoV IgG in the mouse antiserum No. 1, the anti-rabbit antiserum No. 2 and the anti-rabbit antiserum No. 3 are respectively 5.56mg/ml, 3.5mg/ml and 3.7 mg/ml.
In this experimental example, the following empirical ratios were used for the amount of the crosslinking agent:
5mg/ml≤C（Ab）≤10mg/ml，X（SMCC:Ab）=20。
the calculation formula of the SMCC dosage is as follows:
mouse antiserum No. 1: 5.56/150 × 20 × 334.3 × 0.001=0.31 mg;
rabbit antiserum No. 2: 3.5/150 × 20 × 334.3 × 0.001=0.16 mg;
rabbit antiserum No. 3: 3.7/150 × 20 × 334.3 × 0.001=0.17 mg.
1.3 preparing IgM quality control product
1.3.1 SMCC and antisera response
Taking the SMCC and the antiserum according to the proportion, carrying out oscillation reaction at room temperature for 50min, transferring into a dialysis bag, dialyzing to remove redundant SMCC, dialyzing for 3 times, and changing the dialysate every 3 h.
After the dialysis was completed, the concentrations of SMCC and anti-2019-nCoV IgG crosslink conjugate (SMCC-Ab) were measured using an ultraviolet spectrophotometer.
1.3.2 SMCC and mu chain fragment reactions
In this step, the crosslinking conjugate (SMCC-Ab) was used in an amount of 0.25 ml. The amounts of reactants used are shown in table 3 below.
TABLE 3
Taking a molar ratio of 1:4 as an example, the calculation method of the dosage of the mu chain fragment comprises the following steps:
（m1/150）/（m2/29.8）=1/4
wherein m is1Mass of anti-2019-nCoV IgG, m2The mass of the mu chain fragment is 29.8, and the molecular weight of the mu chain fragment is shown.
Referring to the dosage in the above table 3, according to the molar ratio of the crosslinking conjugate (SMCC-Ab) to the μ chain fragment of 1:4 and 1:8, respectively, the solution containing the μ chain fragment obtained in step 1.1.4.5 (the detection concentration of the μ chain fragment is 0.66mg/ml, and is used immediately at the end of dialysis) is mixed with the crosslinking conjugate (SMCC-Ab) and reacted at room temperature for 40min with shaking, so as to obtain the analyte and test the concentration of the analyte. The substance to be detected contains a coupling complex obtained by respectively linking a mu chain fragment and anti-2019-nCoV IgG through SMCC.
1.4 verification of IgM quality control product
The 6 samples with the experiment numbers obtained in step 1.3.2 were diluted by a series of multiples of 4, 8, 16, and 50 times, respectively, and the stock solutions and the dilutions of the samples were detected by using a fluorescence immunochromatographic detection card, and the detection results are shown in table 4 below.
The results of the measurements in Table 4 are shown as T/C. In the fluorescence immunochromatographic assay, T is Test and represents a Test line, and C is control and represents a quality control line. A higher T/C value indicates a stronger positive and a higher activity.
TABLE 4
Referring to fig. 1, fig. 1 shows an example of detection. In FIG. 1, the left test card is an experimental group, an IgM quality control material prepared in this example was tested, and the right test card is a control group.
Referring to the detection result of the left detection card in fig. 1, the detection card has an upper line and a lower line, the upper line is a quality control line, the lower line is a test line, and the color development is obvious. The left detection card has high T/C value, which indicates that the detected IgM quality control substance has strong positive and high activity. Referring to the detection result of the right side detection card in fig. 1, the color development of the quality control line is obvious, and the color development of the detection line is not generated.
Referring to the detection results in table 4, it can be seen that the analytes with 6 experimental numbers obtained in step 1.3.2 can be used, i.e., can be used in immune diagnostic products instead of anti-2019-nCoV IgM. In addition, the titer of the conjugate complex was highest in the test article of experiment No. a compared with the test articles of other experiment nos.
The experimental example verifies that the fluorescent immunochromatography detection card can accurately detect the artificially synthesized IgM quality control product through a fluorescent immunochromatography technology platform, and the IgM quality control product has specificity and immunity, so the IgM quality control product can replace clinical anti-2019-nCoV IgM and serve as the quality control product of the fluorescent immunochromatography detection card.
1.5 preservation of IgM quality control product
The analyte of experiment No. A was diluted 8-fold, 16-fold and 50-fold with PBS (pH7.4, 0.1M) to serve as qualitative quality control substances at high, medium and low concentration levels, respectively.
Adding 1% goat serum, and freezing for storage.
Experimental example 2
This example was carried out to process and store the IgM quality control products prepared in example 1, and to analyze and examine their uniformity and stability.
2.1 subpackaging
Taking a proper amount of the IgM quality control product prepared in example 1, freeze-drying and subpackaging 20 bottles of 2ml each.
2.2 homogeneity test
2.2.1 test methods
And (3) numbering 10 bottles of IgM quality control products subpackaged in the step 2.1, wherein the numbers are 1-10 respectively, diluting by 8 times after redissolution, and sampling in sequence for testing. The sampling sequence is as follows: 1-3-5-7-9-2-4-6-8-10; 10-9-8-7-6-5-4-3-2-1; 2-4-6-8-10-1-3-5-7-9.
2.2.2 test results
The stability test results are shown in table 5 below. The results of the measurements in Table 5 are shown as T/C.
TABLE 5
According to the data processing principle of ISO GUIDE 35:2006, the detection results in the table 5 are subjected to data processing (calculation by using Excel2010 total number), and the processing results are as follows:
1) the intra-group coefficient of variation was 0.7%, and the inter-group coefficient of variation was not detected.
2) The results of the (regression) significance test and the (slope) significance test are: the trend was not significant (for 95% confidence level).
3) And (3) uniformity inspection: no heterogeneity was observed (for 95% confidence level).
2.3 stability test
2.3.1 test methods
And (3) storing the 3 bottles of IgM quality control products subpackaged in the step 2.1 at-18 ℃ and 4 ℃ respectively, and performing stability detection when the bottles are stored for 1 month, 2 months, 3 months and 6 months respectively.
2.3.2 test results
The results of stability testing are shown in table 6 below. The results of the measurements in Table 6 are shown as T/C.
TABLE 6
According to the data processing principle of ISO GUIDE 35:2006, the detection results in the table 6 are subjected to data processing (calculation by using Excel2010 total number), and the processing results are as follows:
both (regression) and (slope) significance test results were: no instability was observed (for 95% confidence level).
The data show that the activity of the freeze-dried IgM quality control product is not influenced by the freeze-drying process, and the uniformity and the stability of the freeze-dried IgM quality control product meet the requirements.
Example 3
The embodiment of the invention provides an IgM quality control product described in embodiment 1 or an IgM quality control product prepared by any one of the methods described in embodiment 2, and an application thereof in 2019-nCoV detection.
In detail, the IgM quality control product can be used in qualitative detection application or quantitative detection application of 2019-nCoV.
For qualitative detection, for example, the conjugate complex obtained under the condition of the number a can be diluted with PBS having a pH of 7.4 and a concentration of 0.1M, specifically 8 times, 16 times and 50 times, respectively, to obtain a high-concentration IgM quality control product, a medium-concentration IgM quality control product and a low-concentration IgM quality control product.
For quantitative determination, for example, the conjugate complex obtained under the condition of the above code A can be diluted with PBS with pH7.4 and concentration 0.1M to obtain a series of IgM quality control substances with different concentrations, and a standard curve can be prepared according to the IgM quality control substances. The standard curve can be used for quantitative detection of IgM in a serum sample by a diagnostic reagent.
Example 4
The embodiment of the invention provides a kit for detecting 2019-nCoV, which comprises: an IgM quality control product described in any one of examples 1 or an IgM quality control product produced by the method described in any one of example 2.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other similar elements in a process, method, article, or apparatus that comprises the element.
While the invention has been shown and described in detail in the drawings and in the preferred embodiments, it is not intended to limit the invention to the embodiments disclosed, and it will be apparent to those skilled in the art that various combinations of the code auditing means in the various embodiments described above may be used to obtain further embodiments of the invention, which are also within the scope of the invention.
Claims (7)
1. An IgM quality control product of the 2019-nCoV detection kit is characterized by comprising: the quality control product comprises a compound obtained by respectively linking a mu chain fragment and anti-2019-nCoVIgG in animal antiserum by using SMCC;
wherein the N-maleimide methyl-end of SMCC is linked with the amino-end of anti-2019-nCoVIgG, and the succinimide end of SMCC is linked with the sulfhydryl-end of the mu chain fragment;
the preparation method comprises the following steps: linking anti-2019-nCoV IgG in animal antiserum by using SMCC, and then linking a mu chain fragment to prepare the IgM quality control product;
before the first step, the method comprises a step of respectively dialyzing animal antiserum containing the anti-2019-nCoV IgG and a solution containing the mu chain fragments by using a buffer solution, wherein the dialysis process is performed at least twice, and the pH value of the buffer solution is adjusted to 7.15 in the last dialysis process.
2. The IgM quality control substance according to claim 1,
the animal antiserum comprises rabbit antiserum or mouse antiserum.
3. The IgM quality control substance according to claim 1,
the mu chain fragment is obtained by gene recombination technology.
4. The IgM quality control substance of claim 1, wherein said first step comprises:
adding excessive SMCC into the animal antiserum according to the concentration of the anti-2019-nCoVIgG in the animal antiserum, dialyzing redundant SMCC in a reaction product after reacting for a certain time at room temperature, performing the dialysis at least once, and detecting the concentration of a linking product of the SMCC and the anti-2019-nCoVIgG in the reaction product after the dialysis is finished;
and adding mu chain fragments into the reaction product according to a certain proportion according to the concentration of the linking product, and reacting at room temperature for a certain time to prepare the IgM quality control product.
5. The IgM quality control substance of claim 1, wherein after said first step, said method further comprises: and (3) testing the linking efficiency of the IgM quality control product by using a colloidal gold detection card.
6. The IgM quality control substance of claim 1, wherein, prior to said first step, said method further comprises:
culturing the mu chain fragment using a strain for amplification, extracting the mu chain fragment from the strain, and purifying the extract to obtain a purified mu chain fragment;
and (3) a step of testing the activity of the purified mu chain fragment by using a colloidal gold test card.
7. Use of an IgM quality control as claimed in any one of claims 1 to 6 in the manufacture of a kit for detecting 2019-nCoV.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5478753A (en) * | 1993-06-29 | 1995-12-26 | Pb Diagnostic Systems, Inc. | Positive calibrator/control composition for an IgM serology assay and an IgM serology assay |
| JP2008290964A (en) * | 2007-05-24 | 2008-12-04 | Abbott Japan Co Ltd | Antibody-antibody conjugate and application to antibody-determining diagnostic agent |
| CN104650236A (en) * | 2013-06-28 | 2015-05-27 | 英科隆生物技术（杭州）有限公司 | Heterogenous conjugate and application thereof to RV (rubella virus) assay |
| WO2016097613A1 (en) * | 2014-12-18 | 2016-06-23 | bioMérieux | Synthetic bi-epitope compound |
| CN108912223A (en) * | 2018-03-28 | 2018-11-30 | 深圳市新产业生物医学工程股份有限公司 | Insulin immunogene, preparation method, insulin antibody and kit |
| CN111273001A (en) * | 2020-02-12 | 2020-06-12 | 北京大弘生物技术有限公司 | System for rapidly detecting new coronavirus 2019-nCoV in blood sample and preparation method thereof |
| WO2020160373A1 (en) * | 2019-01-31 | 2020-08-06 | Precision Bioservices, Inc. | Methods for detection and characterization of anti-viral vector antibodies |
| CN111551743A (en) * | 2020-05-08 | 2020-08-18 | 北京贝尔生物工程股份有限公司 | Kit for rapidly and accurately detecting novel coronavirus IgM antibody and preparation method thereof |
| CN111560477A (en) * | 2020-05-25 | 2020-08-21 | 菁良基因科技(深圳)有限公司 | COVID-19 quality control product and application thereof |
| CN111690060A (en) * | 2020-07-06 | 2020-09-22 | 深圳市亚辉龙生物科技股份有限公司 | IgA antibody capable of specifically recognizing RBD protein and kit |
| CN111856004A (en) * | 2020-03-25 | 2020-10-30 | 潍坊市康华生物技术有限公司 | Reagent for detecting COVID-19 by chemiluminescence immunoassay and detection method thereof |
-
2020
- 2020-12-21 CN CN202011516149.7A patent/CN112269021B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5478753A (en) * | 1993-06-29 | 1995-12-26 | Pb Diagnostic Systems, Inc. | Positive calibrator/control composition for an IgM serology assay and an IgM serology assay |
| JP2008290964A (en) * | 2007-05-24 | 2008-12-04 | Abbott Japan Co Ltd | Antibody-antibody conjugate and application to antibody-determining diagnostic agent |
| CN104650236A (en) * | 2013-06-28 | 2015-05-27 | 英科隆生物技术（杭州）有限公司 | Heterogenous conjugate and application thereof to RV (rubella virus) assay |
| WO2016097613A1 (en) * | 2014-12-18 | 2016-06-23 | bioMérieux | Synthetic bi-epitope compound |
| CN108912223A (en) * | 2018-03-28 | 2018-11-30 | 深圳市新产业生物医学工程股份有限公司 | Insulin immunogene, preparation method, insulin antibody and kit |
| WO2020160373A1 (en) * | 2019-01-31 | 2020-08-06 | Precision Bioservices, Inc. | Methods for detection and characterization of anti-viral vector antibodies |
| CN111273001A (en) * | 2020-02-12 | 2020-06-12 | 北京大弘生物技术有限公司 | System for rapidly detecting new coronavirus 2019-nCoV in blood sample and preparation method thereof |
| CN111856004A (en) * | 2020-03-25 | 2020-10-30 | 潍坊市康华生物技术有限公司 | Reagent for detecting COVID-19 by chemiluminescence immunoassay and detection method thereof |
| CN111551743A (en) * | 2020-05-08 | 2020-08-18 | 北京贝尔生物工程股份有限公司 | Kit for rapidly and accurately detecting novel coronavirus IgM antibody and preparation method thereof |
| CN111560477A (en) * | 2020-05-25 | 2020-08-21 | 菁良基因科技(深圳)有限公司 | COVID-19 quality control product and application thereof |
| CN111690060A (en) * | 2020-07-06 | 2020-09-22 | 深圳市亚辉龙生物科技股份有限公司 | IgA antibody capable of specifically recognizing RBD protein and kit |
Non-Patent Citations (1)
| Title |
|---|
| RSeysenarcthhetic rabbit-human antibody conjugate as a control in immunoassays for immunoglobulin M specific to hepatitis E virus;Kuo Zhang 等;《Virology Journal》;20100520;第7卷;第1-5页 * |
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