CN117054657A - Combined blocking agent and application thereof in latex immunoturbidimetry detection - Google Patents
Combined blocking agent and application thereof in latex immunoturbidimetry detection Download PDFInfo
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- CN117054657A CN117054657A CN202311020635.3A CN202311020635A CN117054657A CN 117054657 A CN117054657 A CN 117054657A CN 202311020635 A CN202311020635 A CN 202311020635A CN 117054657 A CN117054657 A CN 117054657A
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of immunoassay medicine, in particular to a combined blocking agent and application thereof in latex turbidimetry detection, wherein the combined blocking agent is formed by compositing dipeptide and an animal nano antibody; the dipeptide is prepared by synthesizing two of amino acids required by a human body, and the combined blocker is added into a latex immunoturbidimetry detection kit and used for in vitro quantitative determination. The specific combined blocker prepared by combining the dipeptide with the biological nano antibody can completely and effectively shield the foreign antibody, the anti-animal antibody and the RF existing in a patient, and avoid the occurrence of false positive results.
Description
Technical Field
The invention relates to the technical field of immunoassay medicine, in particular to a combined blocker and application thereof in latex turbidimetry detection.
Background
At present, the conventional CK-MB determination mainly adopts an immunosuppression method, and the activity of the CK-MB is determined; the method is quick, concise, time-saving and high in sensitivity, but has a plurality of influencing factors and defects. The enzyme-linked immunosorbent assay (ELISA) is based on a double antibody sandwich method for detecting CK-MB existing in serum; the method has strong specificity, but needs manual operation, and has long detection time. As with conventional immunoassay techniques, latex turbidimetric detection is also susceptible to interference by various factors that may be present with the test sample. In contrast, the effects of endogenous interference, including such factors as antibodies to foreign antibodies (HA), human anti-animal antibodies (HAAA), rheumatoid Factors (RF), and autoantibodies, in immunoassays are increasingly gaining attention. Studies have demonstrated that about 3% -15% of the human population contains endogenous interfering factors. The proportion is higher in the population raised for pets and the population treated with antibodies. In summary, endogenous interference is one of the important factors that contribute to uncertainty in the results of medical immunoassays. The effective means for researching and developing and reducing and eliminating endogenous interference is an important subject for guaranteeing the reliability of medical immune test results and guaranteeing the benefits of doctors and patients.
For eliminating endogenous interference in immunodiagnosis, currently, commercial blockers currently available on the market mainly include IIR, HBR, MAB33 and the like. Reduction or elimination of interference is achieved by binding of the blocking agent to the interfering substance. However, it has been found that in some cases, the blocking effect of these blocking agents added to commercial immunoreagents is insufficient, and interference of endogenous factors on the detection results cannot be completely prevented. This is mainly because the types and concentrations of interference factors in the test sample are different, and the influence of these interference factors on different test items and different test reagents is also different.
For example, the presence of Rheumatoid Factors (RF) in patients can interfere with many immunological detection methods. IgM-type RF and IgG-type RF bind to the capture antibody in the detection reagent and the Fc of the labeled secondary antibody, resulting in detection of false positive results. The degree of RF interference to different detection methods is different and the degree of influence is not proportional to the RF concentration. Currently, most of the detection reagents used in clinic do not take good measures against RF interference (the study of interference of rheumatoid factors on immunoassays has progressed, medical review 2015 Vol21, 19:3495). Currently, strategies to prevent RF interference include: pretreating a sample to be detected by using a solid-phase adsorbent connected with heat-denatured IgG (Eurommune company), centrifuging at 4 ℃ overnight, and detecting; for antigen detection, 2-mercaptoethanol may be added to the sample dilution or sample to degrade IgM-type RF. In view of the foregoing, there remains a need in the art for a detection kit for CKMB that has high specificity, low false positives, is resistant to RF interference, and is inexpensive.
Disclosure of Invention
The invention aims to solve the technical problem of providing a combined blocking agent and application thereof in latex immunoturbidimetry detection, and the specific combined blocking agent prepared by adopting dipeptide combined biological nano-antibody can completely and effectively shield the foreign antibody, anti-animal antibody and RF existing in a patient, thereby avoiding false positive results.
In order to solve the technical problems, the invention adopts the following technical scheme:
a combined blocking agent, which is formed by compositing dipeptide and animal nano-antibody; the dipeptide is synthesized from two of the amino acids required by the human body.
Preferably, the dipeptide is one of glycine-phenylalanine, glutamine-phenylalanine, methionine-tryptophan, aspartic acid-phenylalanine.
Preferably, the animal nanobody is an alpaca anti-human IgM nanobody.
Preferably, the alpaca anti-human IgM nano-antibody is obtained by constructing an anti-IgMVHH phage display immune library after the alpaca is immunized by adopting specific human IgM, and screening to obtain the antibody with high affinity and specificity.
Use of a combination blocker according to any of the above in latex immunoturbidimetry assays, the combination blocker according to any of the above being added to a latex immunoturbidimetry assay kit and used for in vitro quantitative determination.
Preferably, the latex immunoturbidimetry detection kit comprises a first reagent and a second reagent;
wherein the first agent comprises a buffer, a joint blocker, polyethylene glycol, a surfactant, a preservative, and a protectant;
the second reagent comprises buffer solution, antibody coated particles, polystyrene latex particles, a stabilizer, a protective agent and a preservative;
the ratio of the volume of the combined blocking agent to the total volume of the first reagent is 0.01-0.03:3.
Preferably, the buffers of the first and second reagents are each independently selected from: one or more of HEPES buffer, TRIS-HCl buffer, MES buffer, TAPS buffer and glycine buffer;
the protective agents of the first and second agents are each independently selected from the group consisting of: bovine serum albumin, ovalbumin, skim milk and calf serum;
the preservatives of the first and second agents are each independently selected from the group consisting of: sodium azide, thimerosal, and ProClin300;
said polyethylene glycol of the first agent is selected from: PEG6000, PEG8000, PEG12000 and PEG20000;
the surfactant of the first agent is selected from: tween20, tween80, NP40, then;
the stabilizing agent of the second agent is selected from: sucrose, glycerol, trehalose, glucose.
The invention has the beneficial effects that:
nanobodies have the following advantages over conventional animal antibodies: (1) the nano antibody contains disulfide bonds, and can still keep stable at high temperature or under extreme pH conditions, so that the stability is strong; (2) the nano antibody has strong tissue penetrating power due to small volume, and can easily penetrate through the serum barrier; (3) the CDR3 region of about 18 amino acids can form a stable and exposed convex ring structure, can recognize some antigen hidden epitope sites and is deeply combined with the antigen hidden epitope sites, so that the affinity is high; (4) the nanometer antibody skeleton region and the human heavy chain variable domain sequence have homology of more than 80 percent and higher conformational stability, so that the nanometer antibody skeleton region and the human heavy chain variable domain sequence have weaker immunogenicity to human beings; (5) easy production and transformation.
The IgM type RF active region is in front of VL and VH, and in the narrow region between these, it is not easy to bind proteins of large molecules, but is easy to bind small peptide molecules. More than 400 dipeptide combination sequences are synthesized by arranging and combining 20 common amino acids, dipeptide sequences which are easy to combine with IgM type RF are screened out by an inhibition ELISA test, the dipeptide sequences are added to the auxiliary action of a first reagent and an animal nano antibody with high affinity, and salt ions and a surfactant with corresponding concentrations are added to weaken the nonspecific binding reaction of the RF on a detection antibody, so that the interference of the RF on a detection result is effectively eliminated.
Therefore, the specific combined blocker prepared by combining the dipeptide with the biological nano antibody can completely and effectively shield the foreign antibody, the anti-animal antibody and the RF existing in a patient, and the serum of the patient can be effectively prevented from generating false positive results by using the HRP-labeled alpaca anti-human IgM nano antibody as an enzyme-labeled secondary antibody on the basis, so that the kit is safer and more effective.
Detailed Description
The invention will be further illustrated by the following examples, which are not intended to limit the scope of the invention, in order to facilitate the understanding of those skilled in the art.
Example 1
Preparation of the combined blocker:
amino acid is selected to be dehydrated and condensed to synthesize dipeptide, and then the dipeptide is dissolved in heavy water at normal temperature and normal pressure to prepare a dipeptide supersaturated solution; if aspartic acid and phenylalanine are selected to synthesize aspartic acid-phenylalanine (Asp-Phe), asp-Phe is dissolved in heavy water to prepare an Asp-Phe supersaturated solution for later use.
Preparing alpaca anti-human IgM nanobody:
after animals are immunized by adopting specific antigens, the obtained antibody genes are displayed in a library constructed on phage, antibodies with high affinity and specificity are obtained by screening, and the antibodies are purified and prepared into a biological blocker with the concentration of 5mg/ml for standby according to a conventional method.
Selecting a healthy alpaca without an immune background, subcutaneously injecting humanized IgM4 times into the alpaca in multiple points, respectively adopting peripheral blood after each immunization to monitor immune antibody titer, and enabling the antiserum titer after 4 immunization to exceed 1.28 multiplied by 106 so as to meet the requirement of constructing an antibody library. Adopting 60mL jugular vein alpaca blood, extracting total RNA, reversely transcribing into cDNA by using a kit, adopting two-step nestedPCR to amplify VHH gene, adopting SfiI single enzyme to cut target gene VHH and carrier pComb3xss, cloning the target gene VHH and the carrier pComb3xss onto a phagemid carrier pComb3xss, electrically converting the target gene VHH and the carrier pComb3 5748 into E.coli TG I to obtain a 3.81 multiplied by 108cfu nano-antibody original library, and obtaining a 2.79 multiplied by 1013cfu/mL phage display library after M13KO7help phage rescue for subsequent specific nano-antibody screening.
Coating and fixing IgM serving as a target in an enzyme-labeled plate hole by adopting a solid-phase affinity panning technology, screening nanobodies specifically bound with IgM by reducing IgM coating concentration, phage display library input amount and antigen-antibody binding time round by round and increasing washing times round by round, identifying 133 specifically bound positive phages by using a phage-ELISA after three rounds of panning, analyzing the nanobodies with 16 unique gene sequences by using an align X comparison after sequencing, and repeatedly verifying that the nanobodies with 16 unique gene sequences are bound with IgM, and ensuring that the nanobodies are free from nonspecific binding epidemic with IgG, BSA and OVA.
The VHH genes with 16 unique gene sequences are cloned on a pET-25b vector and transformed into E.coli Rosetta competent cells, and a clone with one gene sequence M-3-70 is selected for prokaryotic intracellular expression of target proteins, and the target proteins are purified and collected by using imidazole eluents with multiple concentrations (20 mM/L, 100mM/L, 200mM/L and 300 mM/L). Prokaryotic expression of the nanobody with 16 unique gene sequences is induced for 6 hours by using LB liquid culture medium through IPTG (0.2 mM/L), and then 0.25% Triton-X-100 is added to change the permeability of cell membranes, so that the nanobody protein is secreted into the culture medium, the cells are not required to be broken by ultrasound, and the target protein with high purity can be obtained by simple purification and 300mM/L imidazole elution. By adopting indirect ELISA, the binding activity of the nano antibodies with 16 different gene sequences is compared, and the result shows that the binding activity of M-3-70VHH is best and the specificity is good for standby.
HRP-labeled M-3-70VHH nanobody by sodium periodate method:
1) 15. Mu.LHRP enzyme (19.64 mg/mL) was dissolved in 85. Mu.L of HAc-NaAc solution at pH5.6 and gently mixed;
2) 10 mu LNaIO4 solution (0.088M/L) is added, and the reaction is carried out for 20min at 4 ℃ in a dark place;
3) Adding 50 mu L0.5M/L of ethylene glycol, reacting at room temperature (20 ℃) in a dark place for 30min, boiling a disposable dialysis bag, and cleaning with ultrapure water for later use;
4) Centrifuging (2000 g,4 ℃) for 5min, collecting the supernatant, dialyzing twice against 1mM sodium acetate buffer (pH 4.4) at 4 ℃;
5) The dialyzed solution was adjusted to pH9.5 with 0.2M/L carbonate buffer;
6) 150 μg of nanobody (M-3-70) was added, and the mixture was placed in a rotary mixer and mixed at 4℃for 4 hours;
7) Adding 4 mu LCH3BNNA (5M/L) and reacting for 2 hours at room temperature;
8) Adding 15 mu L of ethanolamine (1 mM) for reaction at room temperature for 30min, blocking the binding site, and then adjusting the pH to 7.4 by using NaH2PO4 buffer;
9) Adding ammonium sulfate powder with final saturation of 45%, standing in refrigerator (4deg.C) for 2 hr, centrifuging (8000 rpm, 4deg.C) for 30min, and discarding supernatant;
10 200 mu L sodium phosphate (pH 7.2) is used for re-suspending and precipitating to obtain the HRP enzyme-coupled antibody, namely the required HRP-marked alpaca anti-human IgM nano antibody.
And mixing the Asp-Phe supersaturated solution with the alpaca anti-human IgM nano antibody solution according to the volume ratio of 5:3 to prepare the combined blocker for standby.
Example 2
Preparing a first reagent:
1. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 17g hesit, 1.0g sodium azide, 7.5g BSA and 15mL combined blocker are weighed, dissolved in 1.0L double distilled water, ph is regulated to 7.2, and the volume is fixed to 1.5L to obtain the reagent first reagent (1).
2. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 17g hesit, 1.0g sodium azide, 7.5g BSA and 10mL combined blocker are weighed, dissolved in 1.0L double distilled water, ph is regulated to 7.2, and the volume is fixed to 1.5L to prepare a first reagent (2)
3. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 17g hesit, 1.0g sodium azide, 7.5g BSA and 5mL combined blocker are weighed, dissolved in 1.0L double distilled water, ph is regulated to 7.2, and volume is fixed to 1.5L to prepare the first reagent (3).
4. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 17g sodium azide, 1.0g BSA and 7.5g BSA were weighed, dissolved in 1.0L double distilled water, ph was adjusted to 7.2, and the volume was adjusted to 1.5L to prepare a first reagent (4).
5. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 17g hesit, 1.0g sodium azide, 7.5g BSA, 15mLIIR blocker were weighed, dissolved in 1.0L double distilled water, ph adjusted to 7.2, and volume fixed to 1.5L to prepare the first reagent (5).
6. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 17thesit, 1.0g sodium azide, 7.5g BSA, 15mLHBR blocker were weighed, dissolved in 1.0L double distilled water, ph adjusted to 7.2, and volume fixed to 1.5L to prepare the first reagent (6).
7. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 17g hesit, 1.0g sodium azide, 7.5g BSA, 15mL mouse IgG blocker were weighed, dissolved in 1.0L double distilled water, ph adjusted to 7.2, and the volume was fixed to 1.5L to prepare the first reagent (7).
8. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 17g hesit, 1.0g sodium azide, 7.5g BSA, 15mLRF blocker were weighed, dissolved in 1.0L double distilled water, ph adjusted to 7.2, and volume fixed to 1.5L to prepare the first reagent (8).
Example 3
Preparation of antibody coated particles:
a. polystyrene latex particles (80 nm, carboxyl groups) were diluted to 1% (w/v) with MES buffer;
b. diluting alpaca anti-human IgM nanobody to 0.5mg/ml with MES buffer solution;
c. adding glycine buffer solution with the concentration of 0.1-1% (w/v) into the step a, and reacting for 40min at 37 ℃ in a constant temperature shaking table;
d. after the reaction is finished, adding the antibody in the step b, and reacting for 3 hours at 37 ℃ in a constant-temperature shaking table;
e. adding a sealing agent, and standing at normal temperature overnight;
f. adding cleaning solution into the particles which are sealed overnight, cleaning and centrifuging for 3 times, and preserving for later use; the order of steps a and b may be interchanged.
Preparation of the second reagent:
400mL of double distilled water is added into the prepared particles, 4.87g of TAPS, 5g of BSA, 1g of sodium azide and 50g of sucrose are added, the mixture is stirred and mixed uniformly, ph is regulated to 7.5, the double distilled water is added to 500mL, and the second reagent (1) is obtained after the main wavelength absorbance is basically unchanged after ultrasound.
Example 4
Kit assembly was performed according to the following table.
Example 5
Kit test
1. Sensitivity test 8 kits prepared in the above examples were tested using a fully automated biochemical analyzer (e.g., hitachi 7180). Measuring 660nm wavelength, sampling sample 10 μl, adding 150 μl of the first reagent, keeping the temperature at 37deg.C for 5min, adding 50 μl of the second reagent, reading absorbance A1 after 42 seconds, reading absorbance A2 after incubation for 4 min 18 seconds at 37deg.C, and reacting absorbance ΔA=A2-A1;
the performance of the 8 examples described above was verified as follows:
from the above measurement, it was found that the kits 1 to 8 each achieved good sensitivity.
Effect of blocking Agents on false Positive results
The comparative kit was prepared according to the preparation method of the kit, with the only difference that no blocking agent was included. Samples with or without the xenotropic antibody 5mg/ml, mouse IgG10mg/ml, and RF 500IU/ml were tested with each kit prepared. The results are as follows, showing that the blocking agent can significantly improve false positives of test results, avoid RF interference, and especially that the test results are optimal with the combined blocking agent of the present invention.
Kit 1 kit 2
Kit 3 kit 4
Kit 5 kit 6
Kit 7 kit 8
The results of the comparison experiments show that the combined blocking agent has good effect, can effectively control the interference of the anisotropic antibody, the anti-animal antibody and the RF in the sample, and can effectively avoid false positive results by adopting the HRP-marked alpaca anti-human IgM nano antibody as an enzyme-labeled secondary antibody to detect the serum of a patient on the basis, so that the kit is safer and more effective.
All technical features in the present embodiment can be modified in appearance according to actual needs.
The foregoing embodiments are preferred embodiments of the present invention, and in addition, the present invention may be implemented in other ways, and any obvious substitution is within the scope of the present invention without departing from the concept of the present invention.
Claims (7)
1. A combination blocker, characterized in that: the combined blocking agent is formed by compositing dipeptide and animal nano antibody; the dipeptide is synthesized from two of the amino acids required by the human body.
2. A combination blocker according to claim 1, characterized in that: the dipeptide is one of glycine-phenylalanine, glutamine-phenylalanine, methionine-tryptophan and aspartic acid-phenylalanine.
3. A combination blocker according to claim 1, characterized in that: the animal nano antibody adopts alpaca anti-human IgM nano antibody.
4. A combination blocker according to claim 3, characterized in that: after the alpaca anti-human IgM nano antibody is used for immunizing alpaca by adopting specific humanized IgM, an anti-IgM VHH phage display immune library is constructed, and the antibodies with high affinity and specificity are obtained through screening.
5. Use of a combination blocker according to any one of claims 1-4 in latex immunoturbidimetry detection, characterized in that: the combination blocker of any one of claims 1-4 added to a latex immunoturbidimetry detection kit and used for in vitro quantitative determination.
6. The use of a combination blocker according to claim 5 in latex immunoturbidimetry detection, characterized in that: the latex immunoturbidimetry detection kit comprises a first reagent and a second reagent;
wherein the first agent comprises a buffer, a joint blocker, polyethylene glycol, a surfactant, a preservative, and a protectant;
the second reagent comprises buffer solution, antibody coated particles, polystyrene latex particles, a stabilizer, a protective agent and a preservative;
the ratio of the volume of the combined blocking agent to the total volume of the first reagent is 0.01-0.03:3.
7. The use of a combination blocker according to claim 5 in latex immunoturbidimetry detection, characterized in that: the buffers of the first and second reagents are each independently selected from: one or more of HEPES buffer, TRIS-HCl buffer, MES buffer, TAPS buffer and glycine buffer;
the protective agents of the first and second agents are each independently selected from the group consisting of: bovine serum albumin, ovalbumin, skim milk and calf serum;
the preservatives of the first and second agents are each independently selected from the group consisting of: sodium azide, thimerosal, and ProClin300;
said polyethylene glycol of the first agent is selected from: PEG6000, PEG8000, PEG12000 and PEG20000;
the surfactant of the first agent is selected from: tween20, tween80, NP40, then;
the stabilizing agent of the second agent is selected from: sucrose, glycerol, trehalose, glucose.
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US20180224459A1 (en) * | 2017-02-08 | 2018-08-09 | Colorado State University Research Foundation | Nanobody conjugates and protein fusions as bioanalytical reagents |
CN111562372A (en) * | 2020-05-15 | 2020-08-21 | 深圳上泰生物工程有限公司 | Latex enhanced immunoturbidimetry kit for detecting creatine kinase isoenzyme CK-MB |
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