CN116008530A - Preparation method and application of HRP (HRP) conjugated antibody dilution stabilizer - Google Patents
Preparation method and application of HRP (HRP) conjugated antibody dilution stabilizer Download PDFInfo
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Abstract
The invention discloses a preparation method and application of an HRP coupled antibody dilution stabilizer, wherein the preparation method comprises the following steps: preparing PBS buffer solution, adding the protection protein, PEG, tween and preservative, mixing uniformly, filtering and sterilizing to obtain the HRP coupling antibody dilution stabilizer. The dilution stabilizer can effectively slow down the reduction of the activity of the enzyme-labeled antibody, is used for the dilution preservation of the enzyme-labeled antibody in a BSA sandwich ELISA antibody detection kit, and the activity of the diluted enzyme-labeled antibody can be basically unchanged after being preserved for at least 12 months at the temperature of 2-8 ℃. The dilution stabilizer can also be used for diluting some common enzyme-labeled antibodies, so that the biological activity of the antibodies is maintained for a long time.
Description
Technical Field
The invention belongs to the technical field of diagnostic biological products, and particularly relates to a preparation method of an HRP coupled antibody dilution stabilizer and application of the HRP coupled antibody dilution stabilizer in a BSA detection kit.
Background
The enzyme-coupled antibody, especially horseradish peroxidase (HRP) coupled antibody, can only store the stock solution at-20 ℃ or even lower temperature and can not be repeatedly frozen and thawed, and the immune activity of the enzyme-coupled antibody can not be greatly reduced within a certain time (generally one year), thereby effectively ensuring the reliability of the detection method result. In recent years, a great deal of research has shown that some protective proteins, stabilizers, etc. have a little protective effect on the activity of enzyme-coupled antibodies. However, a number of practical practices have shown that each enzyme-coupled antibody stabilizer is not broad spectrum and that the relevant formulation needs to be adjusted to verify its effect. In particular for the detection of BSA, the storage of the enzyme-conjugated antibodies does not make use of the conventional protective protein BSA.
Therefore, research and development of a dilution stabilizer capable of effectively protecting the immune activity of the BSA monoclonal antibody Mars from being greatly reduced when the Mars are stored for a long time at 2-8 ℃ and even at room temperature are explored, so that the stability of the kit is ensured, the accuracy of the result and the convenience of operation are further ensured, and the dilution stabilizer is very important for commercialized popularization of the kit.
Disclosure of Invention
Compared with the prior art, the invention provides the dilution stabilizer of the HRP conjugated antibody, which can preserve the diluted HRP conjugated antibody stock solution for at least 12 months at the temperature of 2-8 ℃. The specific implementation technology is as follows:
the preparation method of the HRP conjugated antibody dilution stabilizer comprises the following steps:
step S1: 80g NaCl, 2g KCl and Na 2 HPO 4 14.2g、KH 2 PO 4 2.7g is added into 800mL deionized water, after complete dissolution, the pH is adjusted to 7.2, and then deionized water is added to fix the volume to 1L, so as to obtain 10 XPBS solution;
step S2: taking 300mL of ultrapure water, adding 100mL of 10 XPBS solution, 1-10g of protection protein and 1-2g of high molecular polymer, fully dissolving, adding 0.5-1mL of surfactant, 500mL of antifreezing agent and 0.3-0.5mL of preservative, fully mixing, using ultrapure water to fix the volume to 1L, filtering and sterilizing by using a filter membrane with the thickness of 0.2 mu m, and obtaining the dilution stabilizer of the HRP coupling antibody.
Preferably, in step S2, the protecting protein is gelatin, the high molecular polymer is polyethylene glycol (PEG), the surfactant is tween-20, the antifreeze is glycerol, and the preservative is Proclin-300.
Preferably, the step S2 is: each 1L of HRP conjugated antibody dilution stabilizer contains 100mL of 10 XPBS solution, 2g of protection protein, 1g of high molecular polymer, 0.5mL of Tween-20, 80mL of glycerol and 0.5mL of LProclin-300.
In a preferred embodiment of the present invention, the step S2 is: 100mL of 10 XPBS solution, 2g of gelatin and 1g of high polymer are added into 300mL of ultrapure water, after the high polymer is fully dissolved, 0.5mL of Tween-20, 500mL of glycerol and 0.5mL of Proclin-300 are added, after the high polymer is fully mixed, the ultrapure water is fixed to 1L, and filtration sterilization is carried out on a filter membrane of 0.2 mu m, so as to obtain the dilution stabilizer.
The invention is based on the dilution stabilizer, and is used in a Bovine Serum Albumin (BSA) detection kit, wherein the kit comprises an anti-BSA monoclonal antibody coated micro-pore plate, an HRP labeling reagent diluent, a 15-time concentrated washing liquid, a sample diluent, a BSA standard solution, a TMB substrate solution and a stop solution. Wherein, the HRP labeling reagent is obtained by diluting HRP conjugated antibody stock solution with the HRP conjugated antibody dilution stabilizer according to 1:20.
Preferably, the specific usage method of the Bovine Serum Albumin (BSA) detection kit is as follows:
p1: the kit was removed and all reagents equilibrated at room temperature for 30min.
P2: the coated microwell plate was removed and 350 μl of wash solution was added to wash the plate per well and repeated 2 times.
P3: 0, 5, 10, 20, 30, 40, 50, 60ng/mL standard solution and sample to be tested, 50. Mu.L/well, were added well by well. .
P4: sealing the sealing plate film, and standing at 25 ℃ in a dark place for incubation for 1 hour.
P5: at the end of incubation, the wells of the microplate were discarded, and 350 μl of wash solution was added to wash the plate per well, and repeated 2 times.
P6: mu.L of HRP-labeled antibody working solution diluted with HRP-labeled reagent diluent was added to each well.
P7: sealing the sealing plate film, and standing at 25 ℃ in a dark place for incubation for 30min.
P8: at the end of incubation, the wells of the microplate were discarded, and 350 μl of wash solution was added to wash the plate per well, and repeated 4 times.
P9: 50 mu LTMB substrate solution was added to each well, and the mixture was allowed to stand at 25℃for 15 minutes in a dark place.
P10: 50. Mu.L of reaction termination solution was added to each well.
P11: absorbance was read using a microplate reader at two wavelengths (450 nm and 630 nm).
The invention solves the defects existing in the background technology, and has the following beneficial effects: the dilution stabilizer of HRP coupled antibody provided by the invention contains protective protein gelatin, high polymer PEG, surfactant Tween-20, antifreeze glycerol and preservative Proclin-300, which are mutually complemented, and the HRP marked reagent obtained by dilution of the dilution stabilizer can be stored for 12 months at 2-8 ℃. In the field of kits, the mode of combining the five reagents is not reported in the literature and patents.
Detailed Description
In order that the above-recited objects, features and advantages of the invention will be more clearly understood, a more particular description of the invention will be rendered by the following detailed description. It should be noted that, in the case of no conflict, the embodiments of the present application and the features in the embodiments may be combined with each other.
It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which are intended to be encompassed by the present invention by those of ordinary skill in the art, are intended to be within the scope of the present invention as they are not to be inventive, and numerous specific details are set forth in the following description to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those described herein, and the scope of the invention is therefore not limited to the specific embodiments disclosed below.
In the following examples and comparative examples, gelatin, PEG, proclin-300 were purchased from Sigma, and glycerol, tween-20 were purchased from the national drug group.
In the following application examples, the use method of the kit is as follows:
p1: the kit was removed and all reagents equilibrated at room temperature for 30min.
P2: the coated microwell plate was removed and 350 μl of wash solution was added to wash the plate per well and repeated 2 times.
P3: 0, 5, 10, 20, 30, 40, 50, 60ng/mL standard solution and sample to be tested, 50. Mu.L/well, were added well by well. .
P4: sealing the sealing plate film, and standing at 25 ℃ in a dark place for incubation for 1 hour.
P5: at the end of incubation, the wells of the microplate were discarded, and 350 μl of wash solution was added to wash the plate per well, and repeated 2 times.
P6: mu.L of HRP-labeled antibody working solution diluted with HRP-labeled reagent diluent was added to each well.
P7: sealing the sealing plate film, and standing at 25 ℃ in a dark place for incubation for 30min.
P8: at the end of incubation, the wells of the microplate were discarded, and 350 μl of wash solution was added to wash the plate per well, and repeated 4 times.
P9: 50 mu LTMB substrate solution was added to each well, and the mixture was allowed to stand at 25℃for 15 minutes in a dark place.
P10: 50. Mu.L of reaction termination solution was added to each well.
P11: absorbance was read using a microplate reader at two wavelengths (450 nm and 630 nm).
Example 1
The preparation method of the stabilizing diluent of the HRP conjugated antibody provided by the embodiment comprises the following steps:
step S1: 80g NaCl, 2g KCl and Na 2 HPO 4 14.2g、KH 2 PO 4 2.7g is added into 800mL deionized water, after complete dissolution, the pH is adjusted to 7.2, and then deionized water is added to fix the volume to 1L, so as to obtain 10 XPBS solution;
step S2: taking 300mL of ultrapure water, adding 100mL of 10 XPBS solution, 2g of gelatin and 1g of PEG, fully dissolving, adding 0.5mL of Tween-20, 500mL of glycerol and 0.5mL of LProclin-300, fully mixing, fixing the volume to 1L by using ultrapure water, and filtering and sterilizing by using a filter membrane with the size of 0.2 mu m to obtain the dilution stabilizer of the HRP coupling antibody.
Example 2
The preparation method of the stabilizing diluent of the HRP conjugated antibody provided by the embodiment comprises the following steps:
step S1: 80g NaCl, 2g KCl and Na 2 HPO 4 14.2g、KH 2 PO 4 2.7g is added into 800mL deionized water, after complete dissolution, the pH is adjusted to 7.2, and then deionized water is added to fix the volume to 1L, so as to obtain 10 XPBS solution;
step S2: taking 300mL of ultrapure water, adding 100mL of 10 XPBS solution, 2g of gelatin and 1g of PEG, fully dissolving, adding 0.5mL of Tween-20, 500mL of glycerol and 0.3mL of LProclin-300, fully mixing, fixing the volume to 1L by using ultrapure water, and filtering and sterilizing by using a filter membrane with the size of 0.2 mu m to obtain the dilution stabilizer of the HRP coupling antibody.
Example 3
The preparation method of the stabilizing diluent of the HRP conjugated antibody provided by the embodiment comprises the following steps:
step S1: 80g NaCl, 2g KCl and Na 2 HPO 4 14.2g、KH 2 PO 4 2.7g is added into 800mL deionized water, after complete dissolution, the pH is adjusted to 7.2, and then deionized water is added to fix the volume to 1L, so as to obtain 10 XPBS solution;
step S2: taking 300mL of ultrapure water, adding 100mL of 10 XPBS solution, 2g of gelatin and 1g of PEG, fully dissolving, adding 1mL of Tween-20, 500mL of glycerol and 0.5mL of LProclin-300, fully mixing, fixing the volume to 1L by using ultrapure water, and filtering and sterilizing by using a filter membrane with the size of 0.2 mu m to obtain the dilution stabilizer of the HRP coupled antibody.
Example 4
The preparation method of the stabilizing diluent of the HRP conjugated antibody provided by the embodiment comprises the following steps:
step S1: 80g NaCl, 2g KCl and Na 2 HPO 4 14.2g、KH 2 PO 4 2.7g is added into 800mL deionized water, after complete dissolution, the pH is adjusted to 7.2, and then deionized water is added to fix the volume to 1L, so as to obtain 10 XPBS solution;
step S2: taking 300mL of ultrapure water, adding 100mL of 10 XPBS solution, 2g of gelatin and 2g of PEG, fully dissolving, adding 0.5mL of Tween-20, 500mL of glycerol and 0.5mL of LProclin-300, fully mixing, fixing the volume to 1L by using ultrapure water, and filtering and sterilizing by using a filter membrane with the size of 0.2 mu m to obtain the dilution stabilizer of the HRP coupling antibody.
Example 5
The preparation method of the stabilizing diluent of the HRP conjugated antibody provided by the embodiment comprises the following steps:
step S1: 80g NaCl, 2g KCl and Na 2 HPO 4 14.2g、KH 2 PO 4 2.7g was added to 800mL of deionized water,after complete dissolution, adjusting the pH to 7.2, and then adding deionized water to a volume of 1L to obtain a 10 XPBS solution;
step S2: taking 300mL of ultrapure water, adding 100mL of 10 XPBS solution, 5g of gelatin and 1g of PEG, fully dissolving, adding 0.5mL of Tween-20, 500mL of glycerol and 0.5mL of LProclin-300, fully mixing, fixing the volume to 1L by using ultrapure water, and filtering and sterilizing by using a filter membrane with the size of 0.2 mu m to obtain the dilution stabilizer of the HRP coupling antibody.
Example 6
The preparation method of the stabilizing diluent of the HRP conjugated antibody provided by the embodiment comprises the following steps:
step S1: 80g NaCl, 2g KCl and Na 2 HPO 4 14.2g、KH 2 PO 4 2.7g is added into 800mL deionized water, after complete dissolution, the pH is adjusted to 7.2, and then deionized water is added to fix the volume to 1L, so as to obtain 10 XPBS solution;
step S2: taking 300mL of ultrapure water, adding 100mL of 10 XPBS solution, 10g of gelatin and 1g of PEG, fully dissolving, adding 0.5mL of Tween-20, 500mL of glycerol and 0.5mL of LProclin-300, fully mixing, fixing the volume to 1L by using ultrapure water, and filtering and sterilizing by using a filter membrane with the size of 0.2 mu m to obtain the dilution stabilizer of the HRP coupling antibody.
Comparative example 1
The preparation method of the stabilizing diluent of the HRP conjugated antibody provided by the comparative example comprises the following steps:
step S1: 80g NaCl, 2g KCl and Na 2 HPO 4 14.2g、KH 2 PO 4 2.7g is added into 800mL deionized water, after complete dissolution, the pH is adjusted to 7.2, and then deionized water is added to fix the volume to 1L, so as to obtain 10 XPBS solution;
step S2: taking 300mL of ultrapure water, adding 100mL of 10 XPBS solution, 2g of gelatin and 1g of PEG, fully dissolving, adding 0.5mL of Tween-20 and 0.5mL of LProclin-300, fully mixing, fixing the volume to 1L by using ultrapure water, and filtering and sterilizing by using a filter membrane with the size of 0.2 mu m to obtain the dilution stabilizer of the HRP coupled antibody.
Comparative example 2
The preparation method of the stabilizing diluent of the HRP conjugated antibody provided by the comparative example comprises the following steps:
step S1: naCl80g、KCl2g、Na 2 HPO 4 14.2g、KH 2 PO 4 2.7g is added into 800mL deionized water, after complete dissolution, the pH is adjusted to 7.2, and then deionized water is added to fix the volume to 1L, so as to obtain 10 XPBS solution;
step S2: taking 300mL of ultrapure water, adding 100mL of 10 XPBS solution and 1g of PEG, fully dissolving, adding 0.5mL of Tween-20, 500mL of glycerol and 0.5mL of LProclin-300, fully mixing, fixing the volume to 1L by using ultrapure water, and filtering and sterilizing by using a filter membrane with the size of 0.2 mu m to obtain the dilution stabilizer of the HRP coupling antibody.
Comparative example 3
The preparation method of the stabilizing diluent of the HRP conjugated antibody provided by the comparative example comprises the following steps:
step S1: 80g NaCl, 2g KCl and Na 2 HPO 4 14.2g、KH 2 PO 4 2.7g is added into 800mL deionized water, after complete dissolution, the pH is adjusted to 7.2, and then deionized water is added to fix the volume to 1L, so as to obtain 10 XPBS solution;
step S2: taking 300mL of ultrapure water, adding 100mL of 10 XPBS solution and 2g of gelatin, fully dissolving, adding 0.5mL of Tween-20, 500mL of glycerol and 0.5mL of LProclin-300, fully mixing, fixing the volume to 1L by using ultrapure water, and filtering and sterilizing by using a filter membrane with the size of 0.2 mu m to obtain the dilution stabilizer of the HRP coupling antibody.
Comparative example 4
The preparation method of the stabilizing diluent of the HRP conjugated antibody provided by the comparative example comprises the following steps:
step S1: 80g NaCl, 2g KCl and Na 2 HPO 4 14.2g、KH 2 PO 4 2.7g is added into 800mL deionized water, after complete dissolution, the pH is adjusted to 7.2, and then deionized water is added to fix the volume to 1L, so as to obtain 10 XPBS solution;
step S2: taking 300mL of ultrapure water, adding 100mL of 10 xPBS solution, 0.5mL of Tween-20, 500mL of glycerol and 0.5mLProclin-300, fully and uniformly mixing, fixing the volume to 1L by using the ultrapure water, and filtering and sterilizing by using a filter membrane with the thickness of 0.2 mu m to obtain the dilution stabilizer of the HRP coupling antibody.
Comparative example 5
The preparation method of the stabilizing diluent of the HRP conjugated antibody provided by the comparative example comprises the following steps:
step S1: 80g NaCl, 2g KCl and Na 2 HPO 4 14.2g、KH 2 PO 4 2.7g is added into 800mL deionized water, after complete dissolution, the pH is adjusted to 7.2, and then deionized water is added to fix the volume to 1L, so as to obtain 10 XPBS solution;
step S2: 300mL of ultrapure water is taken, 100mL of 10 XPBS solution and 0.5mL of Tween-20 are added, the mixture is fully and evenly mixed, the volume is fixed to 1L by using the ultrapure water, and then the mixture is filtered and sterilized by using a filter membrane with the thickness of 0.2 mu m, thus obtaining the dilution stabilizer of the HRP coupling antibody.
Comparative example 6
The stabilizing diluent of the HRP conjugated antibody provided in the comparative example is a commercially available stabilizing agent of the HRP conjugated antibody, and the specific formulation is not detailed.
Application example 1:
HRP conjugated antibody stock solutions prepared using the dilution stabilizers prepared in examples 1-6 and comparative examples 1-6 were prepared according to 1:20 were diluted to obtain an HRP-labeled reagent, which was subjected to an accelerated disruption assay at 37℃once every 2 days for a total of 5 assays. The dilution stabilizers prepared in examples 1 to 6 and comparative examples 1 to 6 were examined for their protection against HRP-conjugated antibodies, and the specific results are shown in Table 1.
Table 1: OD value of 60 and 0ng/mL of BSA standard solution in 37 ℃ accelerated destruction experiment
As can be seen from Table 1, the dilution stabilizer prepared in example 1 was significantly better in protection against HRP-conjugated antibodies in the accelerated disruption test at 37℃than in examples 2-6 and comparative examples 1-6. Examples 2-6, although having some protective effect, had a lower signal value at 60 ng/mL. The signal value of comparative example 1 was weak. Comparative examples 2, 4 and 5 were inferior in protection effect. Comparative examples 3 and 6 have better protection but have a higher background value at 0ng/mL, resulting in a significantly lower signal-to-noise ratio than example 1.
Application example 2: shelf life experiments at 2-8deg.C with dilution stabilizer diluted HRP-labeled reagent
Dilution stabilizers prepared in examples 1-6 and comparative examples 1-6 were selected to give HRP conjugated antibody stock solutions according to 1:20 to obtain HRP labeling reagent, and storing at 2-8deg.C for use.
Experiment 1: standard solution 60, 0ng/mL test experiment
And taking out the HRP labeling reagent every 1-2 months, and testing according to an experimental detection method for 12 months. The test results are shown in Table 2.
Table 2: results of HRP-labeled reagent test Standard solution 60, 0ng/mL stored at 4 ℃
Experiment 2: physical Property experiment
(1) Solution clarity detection: HRP-labeled reagent prepared using the dilution stabilizer prepared in example 1 was stored at 2-8 ℃. A total of 12 months were examined every 1 month to see if it was clear. The results showed clear solutions.
(2) And (3) sterile inspection: HRP-labeled reagent prepared using the dilution stabilizer prepared in example 1 was stored at 2-8 ℃. Inoculating to the plate every 1 month, and culturing at 37deg.C. The total culture was observed for 7 days, and the total examination was conducted for 12 months. The test results were all sterile.
Claims (9)
1. The preparation method of the HRP-conjugated antibody dilution stabilizer is characterized by comprising the following steps of:
step S1: 80g NaCl, 2g KCl and Na 2 HPO 4 14.2g、KH 2 PO 4 2.7g is added into 800mL deionized water, after complete dissolution, the pH is adjusted to 7.2, and then deionized water is added to fix the volume to 1L, so as to obtain 10 XPBS solution;
step S2: taking 300mL of ultrapure water, adding 100mL of 10 XPBS solution, 1-10g of protection protein and 1-2g of high molecular polymer, fully dissolving, adding 0.5-1mL of surfactant, 500mL of antifreezing agent and 0.3-0.5mL of preservative, fully mixing, using ultrapure water to fix the volume to 1L, filtering and sterilizing by using a filter membrane with the thickness of 0.2 mu m, and obtaining the dilution stabilizer of the HRP coupling antibody.
2. The method for preparing the HRP conjugated antibody dilution stabilizer according to claim 1, wherein the method comprises the following steps: the protection protein in the step S2 is gelatin.
3. The method for preparing the HRP conjugated antibody dilution stabilizer according to claim 1, wherein the method comprises the following steps: the high molecular polymer in the step S2 is polyethylene glycol (PEG).
4. The method for preparing the HRP conjugated antibody dilution stabilizer according to claim 1, wherein the method comprises the following steps: the surfactant in the step S2 is Tween-20.
5. The method for preparing the HRP conjugated antibody dilution stabilizer according to claim 1, wherein the method comprises the following steps: the antifreezing agent in the step S2 is glycerin.
6. The method for preparing the HRP conjugated antibody dilution stabilizer according to claim 1, wherein the method comprises the following steps: the preservative in the step S2 is Proclin-300.
7. The method for preparing the HRP conjugated antibody dilution stabilizer according to claim 1, wherein the step S2 is: 100mL of 10 XPBS solution, 2g of gelatin and 1g of high polymer are added into 300mL of ultrapure water, after the mixture is fully dissolved, 0.5mL of Tween-20, 500mL of glycerol and 0.5mL of LProclin-300 are added, after the mixture is fully mixed, the ultrapure water is fixed to 1L, and filtration sterilization is carried out on a filter membrane with the volume of 0.2 mu m, so as to obtain the dilution stabilizer.
8. An application of an HRP conjugated antibody dilution stabilizer, which is characterized in that: the dilution stabilizer is used in a Bovine Serum Albumin (BSA) detection kit, and the kit comprises an anti-BSA monoclonal antibody coated micro-pore plate, an HRP (high-rate fluorescent) labeled reagent, an HRP labeled reagent diluent, a 15-time concentrated washing liquid, a sample diluent, a BSA standard solution, a TMB substrate solution and a stop solution. Wherein, the HRP labeling reagent is obtained by diluting HRP conjugated antibody stock solution with the HRP conjugated antibody dilution stabilizer according to 1:20.
9. The use of an HRP conjugated antibody dilution stabilizer according to claim 8, wherein: the specific application method of the Bovine Serum Albumin (BSA) detection kit comprises the following steps:
p1: the kit was removed and all reagents equilibrated at room temperature for 30min.
P2: the coated microwell plate was removed and 350 μl of wash solution was added to wash the plate per well and repeated 2 times.
P3: 0, 5, 10, 20, 30, 40, 50, 60ng/mL standard solution and sample to be tested, 50. Mu.L/well, were added well by well. .
P4: sealing the sealing plate film, and standing at 25 ℃ in a dark place for incubation for 1 hour.
P5: at the end of incubation, the wells of the microplate were discarded, and 350 μl of wash solution was added to wash the plate per well, and repeated 2 times.
P6: mu.L of HRP-labeled antibody working solution diluted with HRP-labeled reagent diluent was added to each well.
P7: sealing the sealing plate film, and standing at 25 ℃ in a dark place for incubation for 30min.
P8: at the end of incubation, the wells of the microplate were discarded, and 350 μl of wash solution was added to wash the plate per well, and repeated 4 times.
P9: 50 mu LTMB substrate solution was added to each well, and the mixture was allowed to stand at 25℃for 15 minutes in a dark place.
P10: 50. Mu.L of reaction termination solution was added to each well.
P11: absorbance was read using a microplate reader at two wavelengths (450 nm and 630 nm).
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| CN117007791A (en) * | 2023-07-18 | 2023-11-07 | 广州市进德生物科技有限公司 | Horseradish peroxidase conjugate dilution preservation solution |
| CN117007791B (en) * | 2023-07-18 | 2024-08-09 | 广州市进德生物科技有限公司 | Horseradish peroxidase conjugate dilution preservation solution |
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