CN113311174A - Myoglobin antibody-enzyme marker and preparation and application thereof - Google Patents
Myoglobin antibody-enzyme marker and preparation and application thereof Download PDFInfo
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- CN113311174A CN113311174A CN202110531836.4A CN202110531836A CN113311174A CN 113311174 A CN113311174 A CN 113311174A CN 202110531836 A CN202110531836 A CN 202110531836A CN 113311174 A CN113311174 A CN 113311174A
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- 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
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
Abstract
The invention relates to the technical field of enzyme immunization, in particular to a myoglobin antibody-enzyme marker and preparation and application thereof. The myoglobin antibody-enzyme marker is obtained by cross-linking a maleimide modified myoglobin antibody MB II and a sulfhydryl modified enzyme marker; the maleimide modified myoglobin antibody MB II is prepared by taking 6- (maleimide) caproic acid succinimide ester as a cross-linking agent and cross-linking with a myoglobin antibody; the sulfhydryl modified enzyme label is prepared by using N-succinimide S-acetyl thioacetate as a cross-linking agent, cross-linking with alkaline phosphatase, and activating with hydroxylamine hydrochloride. When the myoglobin antibody-enzyme marker is diluted by a diluent and used for enzymatic chemiluminescence immunoassay, compared with a common antibody-enzyme marker, the myoglobin antibody-enzyme marker has the advantages that the background value is 3-5 times lower, and the ratio of a high-value signal to a low-value signal is more than 3 times higher.
Description
Technical Field
The invention relates to the technical field of enzyme immunity, in particular to an antibody-enzyme marker and preparation and application thereof.
Background
In the detection of enzymatic chemiluminescence immunoassay, an antigen or antibody enzyme label plays an important role in the sensitivity and specificity of immunoassay, and is a key reagent in the detection of the enzymatic chemiluminescence immunoassay. Alkaline phosphatase (ALP) is an enzyme widely applied to enzymatic chemiluminescent immunodiagnostic reagents, and is combined with an antibody by certain technical means, so that the alkaline phosphatase is an important step in the development of the enzymatic chemiluminescent immunodiagnostic reagents. Generally, the methods for cross-linking alkaline phosphatase and antibody are mainly as follows:
1. glutaraldehyde process
The method of crosslinking antibody by glutaraldehyde as a crosslinking agent is mainly divided into a one-step method and a two-step method. The one-step method can combine the antibody and the enzyme with equal probability, but has more impurities, higher background value and general crosslinking efficiency. The two-step method can control the amount of enzyme and antibody combination, but because the amount of glutaraldehyde added in the first step is difficult to control, enzyme-enzyme combination is easy to form, the cross-linking efficiency is not high, and the steps are more complicated compared with the one-step method.
2. Sodium periodate process
The method has the advantages that the sodium periodate is used for oxidizing hydroxyl of the sugar-containing part of ALP into aldehyde group, combining with the antibody to form Schiff base under certain conditions, and reducing the redundant aldehyde group by sodium borohydride.
3. Heterobifunctional methods
The reaction of succinimide and amino makes the heterobifunctional cross-linking agent respectively cross-linked to the enzyme and the antibody, and then the reaction of maleimide and sulfydryl makes the enzyme and the antibody combined.
The method has few byproducts and simple steps, can control the combination amount of the enzyme and the antibody, save raw materials, reduce cost and realize mass production, and is an ideal crosslinking method.
Disclosure of Invention
The invention aims to solve the technical problem that a novel myoglobin antibody-enzyme marker is obtained based on a heterobifunctional method, and when the myoglobin antibody-enzyme marker is used for enzymatic chemiluminescence immunoassay, compared with a common antibody-enzyme marker, the myoglobin antibody-enzyme marker has lower background value and higher sensitivity.
The invention firstly provides a myoglobin antibody-enzyme marker, which is obtained by cross-linking a maleimide modified myoglobin antibody MB II and a sulfhydryl modified enzyme marker;
the maleimide modified myoglobin antibody MB II is prepared by taking 6- (maleimide) hexanoic acid succinimide ester as a cross-linking agent and cross-linking with a myoglobin antibody;
the sulfhydryl modified enzyme label is prepared by using N-succinimide S-acetyl thioacetate as a cross-linking agent, cross-linking with alkaline phosphatase, and activating with hydroxylamine hydrochloride.
The invention secondly provides a preparation method of the myoglobin antibody-enzyme marker, which comprises the following steps:
(1) 6- (maleimide) hexanoic acid succinimide ester is taken as a cross-linking agent, and is subjected to cross-linking reaction with the myoglobin antibody MBII and then is purified to obtain a maleimide modified myoglobin antibody MB II;
(2) taking N-succinimide S-acetyl thioacetate as a cross-linking agent, carrying out cross-linking reaction with alkaline phosphatase, and activating by hydroxylamine hydrochloride to obtain a sulfydryl modified enzyme marker;
(3) carrying out sulfydryl activation on the sulfydryl modified enzyme marker to obtain an activated enzyme marker;
(4) and (4) mixing the activated enzyme marker obtained in the step (3) with the maleimide-modified myoglobin antibody MB II for reaction, and then purifying to obtain the myoglobin antibody-enzyme marker.
As one example, in step (1), the molar ratio of succinimidyl 6- (maleimido) hexanoate to myoglobin antibody was (10. + -.1) to 1.
In step (1), the crosslinking reaction is carried out at room temperature for 2. + -. 0.2 hours under protection from light.
As one example, in the step (2), the molar ratio of N-succinimide S-acetylthioacetate to alkaline phosphatase is (10. + -.1): 1.
In one example, in the step (3), the thiol group is activated by the following method: adding hydroxylamine hydrochloride buffer solution with the final concentration of 100mM into the sulfhydryl-modified enzyme label to obtain mixed solution M, adding TEA with the volume of 0.1% of the mixed solution M, and carrying out reaction at room temperature in a dark place for 1 +/-0.1 hour.
As one example, the reaction of step (4) was performed under light-shielding at room temperature for 1 ± 0.1 hour, and then the resultant was passed through a Superdex200 column, eluted using 0.1M, pH ═ 7.5 PBS buffer, and the first peak was collected to obtain the myoglobin antibody-enzyme label.
As one example, after collecting the first peak liquid, the resulting liquid was concentrated to 1-3 mL using an ultrafiltration concentrator tube.
The invention also provides a diluent matched with the myoglobin antibody-enzyme marker, wherein 1% of surfactant, 0.05% of sodium azide and 0.05% of ProClin300 are added into PBS (phosphate buffer solution) with the pH value of 7.5 and the pH value of 0.1M as a matrix according to the volume ratio; adding 10% of PEG2000, 0.5% of sodium caseinate and 3% of BSA by weight ratio, and mixing to obtain the final product.
The invention also relates to the application of the myoglobin antibody-enzyme label in preparing a myoglobin enzymatic chemiluminescence immunoassay kit.
As a case, the specific method of the application is as follows: the myoglobin antibody-enzyme marker is used as a substrate, and the myoglobin antibody-enzyme marker is diluted by the diluent according to the volume ratio of 1 to (200 +/-100) and used for enzymatic chemiluminescence detection of the myoglobin antigen.
Compared with the prior art, the technical scheme of the invention has the following advantages:
1. when the myoglobin antibody-enzyme marker is diluted by a diluent and used for enzymatic chemiluminescence immunoassay, compared with a common antibody-enzyme marker, the myoglobin antibody-enzyme marker has the advantages that the background value is 3-5 times lower, and the ratio of a high-value signal to a low-value signal is more than 3 times.
2. The myoglobin antibody-enzyme marker disclosed by the invention is diluted by a matched diluent, so that the background value is lower and the detection is more accurate.
3. Compared with the existing glutaraldehyde method and sodium periodate method, the method has the advantages of lower background value, higher sensitivity, easy operation, less by-products and obviously improved crosslinking efficiency.
4. The invention uses N-succinimidyl S-acetylthioacetate (SATA) as a cross-linking agent to be cross-linked with ALP to form an enzyme marker (ALP-SATA), uses 6- (maleimido) hexanoic acid succinimidyl ester (MHS) as a cross-linking agent to be cross-linked with a myoglobin antibody to form an antibody conjugate, and then the two are cross-linked to obtain the novel myoglobin antibody-enzyme marker prepared by a heterobifunctional method.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The following are related to abbreviations for the names explained below:
MHS: 6- (maleimido) hexanoic acid succinimidyl ester; SATA: n-succinimide S-acetylthioacetate; ALP: alkaline phosphatase; PBS: phosphate buffered saline; PEG 2000: polyethylene glycol (2000); BSA: bovine serum albumin; TEA: triethylamine; TCEP: tris (2-carboxyethyl) phosphine.
The reagents used in the following examples are not specifically described, and are all common reagents available from the market.
In the following test experiments, parameters of the same test item on the same model of instrument are the same, and specific operations are all conventional steps in the art, and are known to those skilled in the art and are not described in detail.
Example 1
The preparation method of the myoglobin antibody-enzyme marker specifically comprises the following steps:
(1) preparation and purification of maleimide-modified myoglobin antibody MB II (MB II-MHS) 500ug of myoglobin antibody MB II was weighed and dissolved in 5mL of 0.1M, pH ═ 7.5 PBS;
adding MHS with the molar ratio of the MHS to the myoglobin antibody MB II being about 10: 1 (the floating range is (10 +/-1): 1), and reacting for 2 +/-0.2 hours at room temperature in a dark place to obtain an incubation liquid;
taking out the incubation liquid, putting the incubation liquid into a dialysis bag, dialyzing the incubation liquid in 5mM PBS solution in a refrigerator at 4 ℃ for 12 +/-1.2 hours, and changing the liquid 3-4 times in the dialysis period;
collecting the dialyzed incubation liquid, and storing the incubation liquid at the temperature of 2-8 ℃ for later use;
(2) preparation and purification of sulfhydryl modified enzyme marker (ALP-SATA)
Weighing 500ug of alkaline phosphatase, dissolving in 5mL of 0.1M, pH ═ 7.5 PBS;
adding SATA with the molar ratio of alkaline phosphatase of about 10: 1 (the floating range is (10 +/-1): 1), and reacting for 2 +/-0.2 hours at room temperature in a dark place to obtain an incubation liquid;
taking out the incubation liquid, putting the incubation liquid into a dialysis bag, dialyzing the incubation liquid in 5mM PBS solution in a refrigerator at 4 ℃ for 12 +/-1.2 hours, and changing the liquid 3-4 times in the dialysis period;
collecting the dialyzed incubation liquid, and storing the incubation liquid at the temperature of 2-8 ℃ for later use;
(3) thiol activation of thiol-modified enzyme labels (ALP-SATA)
Taking the ALP-SATA obtained in the step (2), adding hydroxylamine hydrochloride buffer solution with the final concentration of 100mM (namely adding the hydroxylamine hydrochloride buffer solution until the final concentration is 100mM) to obtain mixed solution M, adding TEA with the volume of 0.1% of that of the mixed solution M, and reacting for 1 +/-0.1 hour in a dark place at room temperature to obtain an activated enzyme marker; (4) cross-linking and purifying of maleimide modified myoglobin antibody MB II (MB II-MHS) and enzyme marker (ALP-SATA)
Uniformly mixing the maleimide-modified myoglobin antibody MB II (MB II-MHS) in the step (1) and the enzyme label (ALP-SATA) activated by sulfydryl in the step (3), carrying out a dark reaction at room temperature for 1 +/-0.1 hour, then passing through a Superdex200 column, eluting by using a PBS (phosphate buffer solution) with the concentration of 0.1M, pH being 7.5, collecting the liquid of a first peak, and concentrating to 1-3 mL by using an ultrafiltration concentration tube as required to prepare the purified myoglobin antibody-enzyme label. It can be stored for more than 8 weeks at 2-8 ℃.
Example 2
The only difference from example 1 is that in step (1), the molar ratio of 6- (maleimido) hexanoic acid succinimidyl ester to myoglobin antibody was 8: 1, and the others were in agreement with example 1.
Example 3
The only difference from example 1 is that in step (1), the molar ratio of 6- (maleimido) hexanoic acid succinimidyl ester to myoglobin antibody was 13: 1, and the others were in agreement with example 1.
Example 4
The only difference from example 1 is that in step (2), the molar ratio of N-succinimidyl S-acetylthioacetate to alkaline phosphatase was 7: 1, and the rest was in accordance with example 1.
Example 5
The only difference from example 1 is that in step (2), the molar ratio of N-succinimidyl S-acetylthioacetate to alkaline phosphatase was 12: 1, and the rest was identical to example 1.
Example 6
The difference from example 1 is that the crosslinking reaction time in step (1) is 2.5h, and the rest is the same as example 1.
Example 7
The difference from example 1 is that the crosslinking reaction time in step (1) is 1.5h, and the rest is the same as example 1.
Example 8
The difference from example 1 is that in the step (3), the thiol activation method is different, and specifically the following steps are performed: to the thiol-modified enzyme label was added TCEP buffer at a final concentration of 100mM, otherwise identical to example 1.
Example 9
Provides a diluent used in combination with the myoglobin antibody-enzyme marker, and the preparation method of the diluent is as follows:
in 0.1M PBS at pH 7.5, 1% surfactant, 0.05% sodium azide, 0.05% ProClin300, 5% PEG2000, 0.5% sodium caseinate, 3% BSA were added by volume ratio. Wherein the surfactant is tween 20.
Control group: the preparation method of the common antibody-enzyme marker diluent comprises the following steps:
0.05% tween 20, 0.05% sodium azide and 2% BSA by weight were added to 0.1M PBS at pH 7.0 by volume.
Diluting the myoglobin antibody-enzyme marker by the diluent according to the volume ratio of 1: 300 respectively to be used as a marker reagent, respectively detecting background values on an enzymatic chemiluminescence immunoassay analyzer, recording the average value of RLU (relative luminescence unit), and calculating the P-average value/N-average value, wherein the P value is the background value measured by the reagent diluted by the common antibody-enzyme marker diluent; the N value is the background value measured by the reagent diluted by the antibody-enzyme marker diluent provided by the invention.
The obtained related test results are shown in a table 1, and the background value of the myoglobin antibody-enzyme marker diluent matched with the invention is compared with the background value of the common antibody-enzyme marker diluent.
TABLE 1
The test results of the above experiments show that the same myoglobin antibody-enzyme marker is diluted by different diluents, the background value mean value of the common diluent is 3.76 times of the background value mean value of the matched diluent, and the background removal effect of the matched diluent is better.
Example 10
The antibody-enzyme markers of examples 1-8 were applied to an enzymatic chemiluminescence analyzer and tested for efficacy.
The myoglobin antibody-enzyme marker is diluted by the diluent and used as a marking reagent, the myoglobin antibody-enzyme marker marked by a glutaraldehyde method and the myoglobin antibody-enzyme marker marked by a sodium periodate method are used as a control group, and the specific preparation method of each group of antibody-enzyme marker is as follows:
A1-A8: myoglobin antibody-enzyme marker prepared in examples 1 to 8 of the present invention;
b: reference patent application CN201510781611.9, wherein the glutaraldehyde method is used for cross-linking to prepare myoglobin antibody-enzyme label;
c: the sodium periodate method of the reference patent application CN201510470342.4 was cross-linked to prepare a myoglobin antibody-enzyme label.
The 3 groups are all prepared by adopting the same matched diluent according to the dilution volume ratio of 1: 300.
The background values of the A-B groups are respectively detected on an enzymatic chemiluminescence immunoassay analyzer, and the obtained RLU value results are shown in a table 2-application comparison data of the myoglobin antibody-enzyme marker obtained by the method and the antibody-enzyme marker obtained by other methods on the chemiluminescence immunoassay analyzer.
In the table, the value P is the background value obtained by the detection on the machine of each group of antibody-enzyme labeling objects, and the value N is the photometric value measured on the machine of a myoglobin pure product of 2000 ng/mL.
TABLE 2
The above experimental results show that the P mean/N mean of the group A1-A8 is more than 3 times that of the group B or the group C, namely, the application of the myoglobin antibody-enzyme marker prepared by the invention on an enzymatic chemiluminescence detection analyzer is more than 3 times higher than that of a common enzyme marker.
In addition, the preferred ratios of raw material addition in the present invention were not used in groups A2-A8, and the P-means/N-means ratio was better than that in group B or group C, but slightly worse than that in group A1.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A myoglobin antibody-enzyme marker is characterized in that the myoglobin antibody-enzyme marker is obtained by cross-linking a maleimide modified myoglobin antibody MB II and a sulfhydryl modified enzyme marker;
the maleimide modified myoglobin antibody MB II is prepared by taking 6- (maleimide) caproic acid succinimide ester as a cross-linking agent and cross-linking with a myoglobin antibody;
the sulfhydryl modified enzyme label is prepared by using N-succinimide S-acetyl thioacetate as a cross-linking agent, cross-linking with alkaline phosphatase, and activating with hydroxylamine hydrochloride.
2. A preparation method of a myoglobin antibody-enzyme marker is characterized by comprising the following steps:
(1) 6- (maleimide) caproic acid succinimide ester is taken as a cross-linking agent, and is subjected to cross-linking reaction with a myoglobin antibody MB II and then is purified to obtain a maleimide modified myoglobin antibody MB II;
(2) taking N-succinimide S-acetyl thioacetate as a cross-linking agent, carrying out cross-linking reaction with alkaline phosphatase, and activating by hydroxylamine hydrochloride to obtain a sulfydryl modified enzyme marker;
(3) carrying out sulfydryl activation on the sulfydryl modified enzyme marker to obtain an activated enzyme marker;
(4) and (4) mixing the activated enzyme marker obtained in the step (3) with the maleimide-modified myoglobin antibody MB II for reaction, and purifying to obtain the myoglobin antibody-enzyme marker.
3. The method according to claim 2, wherein in the step (1), the molar ratio of succinimidyl 6- (maleimido) hexanoate to myoglobin antibody is (10 ± 1): 1.
4. The method according to claim 2, wherein the crosslinking reaction is carried out at room temperature for 2 ± 0.2 hours in the absence of light in step (1).
5. The method according to claim 2, wherein in the step (2), the molar ratio of N-succinimide S-acetylthioacetate to alkaline phosphatase is (10. + -.1): 1.
6. The method according to claim 2, wherein in the step (3), the thiol group is activated by: adding hydroxylamine hydrochloride buffer solution with the final concentration of 100mM into the sulfhydryl-modified enzyme label to obtain mixed solution M, adding TEA with the volume of 0.1% of the mixed solution M, and carrying out reaction at room temperature in a dark place for 1 +/-0.1 hour.
7. The preparation method according to claim 2, wherein the reaction of step (4) is performed under the condition of being protected from light at room temperature for 1 ± 0.1 hour, and then is passed through a Superdex200 column, and is eluted by using PBS buffer solution with 0.1M, pH =7.5, and the liquid of the first peak is collected to obtain the myoglobin antibody-enzyme marker;
optionally, after collecting the liquid of the first peak, concentrating the obtained liquid to 1-3 mL by using an ultrafiltration concentration tube.
8. The diluted solution used in combination with the myoglobin antibody-enzyme marker of claim 1, wherein 1% of surfactant, 0.05% of sodium azide and 0.05% of ProClin300 are added by volume ratio based on PBS (pH =7.5 and 0.1M); adding 10% of PEG2000, 0.5% of sodium caseinate and 3% of BSA by weight ratio, and mixing to obtain the final product.
9. Use of the myoglobin antibody-enzyme label of claim 1 in the preparation of a myoglobin enzymatic chemiluminescent immunoassay kit.
10. The use of claim 9, wherein the myoglobin antibody-enzyme label is used for enzymatic chemiluminescence detection of myoglobin antigen after being diluted with the diluent of claim 8 according to the volume ratio of 1 (200 ± 100) as the matrix.
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| CN115993460A (en) * | 2023-03-22 | 2023-04-21 | 珠海科域生物工程股份有限公司 | Alkaline phosphatase-labeled myoglobin antibody diluent and preparation method thereof |
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| CN115993460A (en) * | 2023-03-22 | 2023-04-21 | 珠海科域生物工程股份有限公司 | Alkaline phosphatase-labeled myoglobin antibody diluent and preparation method thereof |
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