WO2023142398A1 - Coronavirus rbd protein-fluorescent microsphere complex preparation - Google Patents

Abstract

Disclosed is a coronavirus RBD protein-fluorescent microsphere complex preparation. The complex preparation comprises raw materials in the following percentages by mass: 0.5%-1% of a conjugate protein, 2.5%-5% of saccharide, 0.9%-1.3% of ions, 0.05%-0.07% of macromolecular substances, 0.05%-0.1% of a surfactant, 0.24-1.21% of a buffer and 0.02-0.05% of a preservative. The complex preparation can be used for the preservation, dilution and treatment of coronavirus RBD protein fluorescent complexes and biological samples. The biological samples comprise serum and plasma. When neutralizing antibodies in human serum, plasma and whole-blood samples are detected by means of a saturation competition method, the time-resolved fluorescent microsphere complex coupled with a coronavirus RBD protein need to be stored separately. The complex preparation can prevent the fluorescent microsphere complex from aggregation and precipitation, maintains the antigenic activity and improves the detection sensitivity.

Description

A composite preparation of novel coronavirus RBD protein fluorescent microspheres technical field

The invention belongs to the field of saturated competition, and in particular relates to a composite preparation of novel coronavirus RBD protein fluorescent microspheres.

Background technique

In the direct competition method in immunology, the labeled antigen and the antigen to be tested are both in liquid phase, and the chance of binding to the antibody is the same, and the relative binding rate of the labeled antigen is 50%. In the saturation competition method, the contact area between the solid-phase antigen and the antibody is small, and only the remaining antibody that binds to the antigen to be tested will bind to the solid-phase antigen, and the relative binding rate of the solid-phase antigen is 0%. Therefore, the slope of the inhibition curve of the indirect method will be greater than that of the direct competition method. The high sensitivity of the saturation competition method is sometimes difficult to achieve in ELISA, and the higher efficiency time-resolved fluorescence chromatography is a better choice. Using the saturation competition method to detect the new coronavirus neutralizing antibody can improve the detection sensitivity. The stability of the fluorescent microspheres plays a key role in the subsequent detection steps. If the fluorescent microspheres are not stored properly, subsequent detections will not be possible and will affect the performance of the final detection reagent. Time-resolved fluorescent microspheres labeling the new coronavirus RBD protein (hereinafter referred to as the new crown RBD protein fluorescent microsphere complex) preservation solution can maintain the stability of the complex in the liquid, so that it does not aggregate, and maintains the activity and performance of the antigen.

For the large-scale vaccination population, using simple and rapid time-resolved fluorescent microsphere immunochromatography, the level of anti-RBD antibody and neutralizing antibody in each vaccinated person can be quickly assessed. The principle of neutralizing antibody detection by the saturation competition method is to combine the sample to be tested with the new crown RBD protein fluorescent microsphere complex for 10-20 minutes, and then combine with the ACE2 protein immobilized on the nitrocellulose membrane. After reacting for 10-15 minutes, use fluorescent The analyzer reads the results, the higher the neutralizing antibody content in the sample to be tested, the weaker the fluorescent signal. The time-resolved fluorescent immunochromatography method using the saturation competition method can quickly assess the levels of anti-RBD antibodies and neutralizing antibodies in each vaccinated body, with high detection sensitivity and short detection time.

Contents of the invention

In view of this, the present invention aims to overcome the defects in the prior art, and proposes a new crown RBD protein fluorescent microsphere composite preparation.

In order to achieve the above object, technical solution of the present invention is achieved in that way:

A new crown RBD protein fluorescent microsphere composite preparation, the composite preparation is made of raw materials including the following mass percentages:

Further, the coupling protein is formed by coupling bovine serum albumin with at least one of ovalbumin (OVA), hemocyanin (KLH) or silk fibroin (SF) through EDC. The bovine serum albumin molecule contains a large number of reactive groups, such as amino groups and carboxyl groups, which can be fully dissolved in water or organic solvents. There are 17 disulfide bonds and a sulfhydryl group in the structure. The chemical reaction of the sulfhydryl group is very active. Sulfur bonds have the effect of anti-oxidation and reduction, so they can combine with various cations, anions and small molecules. Forming peptide bonds with OVA, KLH, and SF can reduce the denaturation of RBD proteins caused by adverse environmental factors, and can also prevent the adsorption of impurities by RBD proteins; it can increase the concentration of proteins in the solution, protect RBD proteins, and prevent RBD The unilateral decomposition and non-specific reaction can maintain the activity of the new crown RBD protein fluorescent microsphere complex at 2-8°C, enhance the sensitivity of the test, reduce the variability caused by changes in transportation and storage conditions, and reduce CV.

Further, the coupling protein is formed by coupling bovine serum albumin and polylysine through EDC, and the coupling sequence is polylysine-bovine serum albumin.

The coupling protein is formed by coupling bovine serum albumin and hemocyanin through EDC.

The coupling protein is formed by coupling bovine serum albumin and silk fibroin through EDC.

The coupling protein is formed by coupling bovine serum albumin and ovalbumin through EDC.

After the carboxyl group at the end of polylysine is activated by EDC, it dehydrates and condenses with the amino group of bovine serum albumin to form a peptide bond, and the only product can be obtained. Polylysine is a biomimetic functional polymer with good biocompatibility, biodegradability and rich side chain functional groups. It has outstanding advantages in protein modification. After coupling with bovine serum albumin, the protein properties can be improved. Uniformity, good repeatability, retain protein activity.

The preparation method of described coupling protein is specifically:

(1) Weigh 10mg of polylysine and add it to phosphate buffer (0.02-0.1M PBS, pH 7.2-7.4), then add 10-50mg/mL 1-ethyl-(3-dimethylaminopropyl base) carbodiimide (EDC), dark reaction 15-30min;

(2) Weighing 10 mg bovine serum albumin and adding it to the activated polylysine solution;

(3) dialyze with phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4), remove EDC;

(4) After affinity purification, the product with higher protein concentration was collected to obtain the coupled protein (ε-PL-BSA).

The preparation method of described coupling protein is specifically:

(1) Weigh 10mg ovalbumin and add it to phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4), then add 10-50mg/mL 1-ethyl-(3-dimethylaminopropyl ) carbodiimide (EDC), dark reaction 15-30min;

(2) Weigh 10 mg bovine serum albumin and 10 mg hemocyanin and add them to the activated ovalbumin solution;

(3) Dialyze with phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4) to remove EDC;

(4) After affinity purification, the product whose molecular weight was the sum of the molecular weights of the three proteins was collected to obtain the coupled protein (BSA-OVA-KLH).

The preparation method of described coupling protein is specifically:

(1) Weigh 10mg of bovine serum albumin and add it to phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4), then add 10-50mg/mL 1-ethyl-(3-dimethylaminopropyl base) carbodiimide (EDC), dark reaction 15-30min;

(2) Weigh 10 mg silk fibroin and add it to the activated bovine serum albumin solution;

(3) Dialyze with phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4) to remove EDC;

(4) After affinity purification, the product whose molecular weight is the sum of the molecular weights of the two proteins is collected to obtain the coupled protein (BSA-SF).

Further, the sugar is at least one of sucrose, trehalose or glucose. Carbohydrates are polyhydric hydrophilic compounds that can form hydrogen bonds with coupled proteins, have inherent stability and mechanical strength against temperature and humidity changes, and can form a protective film on the surface of proteins to avoid RBD proteins from being affected by temperature or enzymes. Degradation, protect RBD protein from degeneration and inactivation, and improve the stability of RBD protein.

Further, the ion is at least one of Na ⁺ , K ⁺ or Mg ²⁺ . The type of test sample includes whole blood. Adding Na ⁺ , K ⁺ or Mg ²⁺ to the preservation solution can keep the erythrocytes at an appropriate osmotic pressure, and the erythrocytes will not be broken and cause the background of the test strip to be unclear, which can make up for the blocking reagent and surfactant. Insufficient false positive elimination effect, while effectively reducing the non-specific adsorption of non-specific proteins in the sample on the coated membrane, can significantly improve the false positive problem.

The PEG4000 added in the compound preparation can effectively eliminate hyperlipidemia, reduce sample viscosity, and facilitate sample chromatography.

Further, the macromolecular substance is PEG4000. PEG4000 can effectively eliminate blood lipids, reduce sample viscosity, and facilitate sample chromatography.

Further, the buffer is Tris buffer; the concentration of the Tris buffer is 0.02-0.1M.

The isoelectric point of the RBD protein of the new coronavirus is 7.5-8.5, and the optimal buffer pH is PI+0.5, so the pH range of the buffer system is selected to be 8.0-9.0, and the Tris buffer is more suitable for this acid-base environment.

Further, the surfactant is at least one of Tween-20, Tetronin1307 or BRIJ35. Adding an appropriate amount of surfactant can change the surface charge distribution of the protein, so that the antigen and antibody have more reactive clusters exposed, and enhance the specific binding of the antigen and antibody to the labeled protein; it can block the hydrophobic groups on the surface or inside of the RBD protein to prevent RBD The interaction between proteins acts as a stabilizer; it does not destroy the structure of the protein, and can reduce the damage to the original interaction between proteins; the surfactant has a certain positive charge, which plays a role in the surface charge of the microspheres. A certain supplement makes the microspheres exist in the solution stably.

Further, the preservative is at least one of proclin 300 or KroVin300M. Preservatives can coagulate or denature the proteins of microorganisms. Preservatives can act on different parts of the KREBS cycle: pyruvate dehydrogenase, a-ketoglutarate dehydrogenase, succinate dehydrogenase and NADH dehydrogenase (NADH dehydrogenase, thereby inhibiting cell growth and metabolism, the synthesis of macromolecules, and causing a rapid decline in energy levels in cells. Due to the collapse of the energy system, cells cannot synthesize the compounds required for daily metabolism. All bacteria and fungi have at least part of the KREBS cycle, which can effectively Inhibit the growth and proliferation of microorganisms. It is easy to be biodegraded into non-toxic and non-polluting substances at the recommended concentration. It has good compatibility with various emulsifiers, surfactants and protein components, and has good compatibility. Influences the binding of coupled fluorescent microsphere complexes and coupled proteins.

Compared with the prior art, the present invention has the following advantages:

The new crown RBD protein fluorescent microsphere composite preparation described in the present invention can be used to detect neutralizing antibodies in human serum, plasma and whole blood samples in the saturation competition method, and improve detection sensitivity; the saturation competition method needs to be coupled with the new crown RBD protein The time-resolved fluorescent microsphere complex is stored separately, and the complex preparation can prevent the fluorescent microsphere complex from aggregation and precipitation, maintain antigen activity, and improve detection sensitivity.

The new crown RBD protein fluorescent microsphere composite preparation described in the present invention can be directly used to process samples, adding ions to the preservation solution can effectively improve the ionic environment of the sample, keep the red blood cells with a suitable osmotic pressure, and the red blood cells will not be broken and affect the background of the test strip. Effectively reduce the adsorption of non-specific proteins, effectively eliminate false negative results or invalid results.

The novel coronavirus RBD protein fluorescent microsphere composite preparation described in the present invention can be used for sample dilution. Add the required samples into the compound preparation, mix and incubate for 15 minutes, and then add the sample directly, reducing the use of operating steps and consumables, saving detection time and steps, and facilitating operation.

The new crown RBD protein fluorescent microsphere compound preparation of the present invention can be used as a microsphere complex solution in the preparation of other fluorescent microsphere immunochromatography test strips, and the prepared fluorescent pad has good stability and high sensitivity; it can be used for hyperlipidemia The treatment of the sample reduces the viscosity of the sample; it can be used for sample dilution; it can be used for the preservation solution of the calibrator/quality control product. The new crown RBD protein fluorescent microsphere composite preparation can maintain the activity of fluorescent microspheres and antigen/antibody, and improve detection sensitivity.

Description of drawings

Fig. 1 is a histogram of fluorescence values of the compound preparations described in Examples 1-3 and Comparative Examples 1-7 of the present invention.

Detailed ways

Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods, unless otherwise specified, are conventional methods.

The present invention will be described in detail below in conjunction with examples.

Example 1

A new crown RBD protein fluorescent microsphere composite preparation, the preparation method is as follows:

(1) Weigh 10mg ovalbumin and add it to phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4), then add 10-50mg/mL 1-ethyl-(3-dimethylaminopropyl ) carbodiimide (EDC), dark reaction 15-30min;

(2) Weigh 10 mg bovine serum albumin and 10 mg hemocyanin and add them to the activated ovalbumin solution;

(3) Dialyze with phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4) to remove EDC;

(4) After affinity purification, the product whose molecular weight is the sum of the molecular weights of the three proteins is collected to obtain coupling protein 1 (BSA-OVA-KLH);

(5) Weigh 2.5g trehalose, 0.9g NaCl, 0.05g PEG4000, 0.05g Tween20, 0.05g proclin 300, 0.24g Tris, 100g process water, add coupling protein 1 to make the final concentration 1%, and adjust the pH to 8.5, to obtain the compound preparation.

Example 2

A new crown RBD protein fluorescent microsphere composite preparation, the preparation method is as follows:

(1) Weigh 10mg of bovine serum albumin and add it to phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4), then add 10-50mg/mL 1-ethyl-(3-dimethylaminopropyl base) carbodiimide (EDC), dark reaction 15-30min;

(2) Weigh 10 mg silk fibroin and add it to the activated bovine serum albumin solution;

(3) Dialyze with phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4) to remove EDC;

(4) After affinity purification, the product whose molecular weight was the sum of the molecular weights of the two proteins was collected to obtain coupling protein 2 (BSA-SF).

(5) Weigh 2.5g trehalose, 0.9g NaCl, 0.05g PEG4000, 0.05g Tween 20, 0.05g proclin 300, 0.24g Tris, 100g process water, add coupling protein 2 to make the final concentration 1%, adjust the pH It is 8.5, obtains compound preparation.

Example 3

A new crown RBD protein fluorescent microsphere composite preparation, the preparation method is as follows:

(1) Weigh 10mg of polylysine and add it to phosphate buffer (0.02-0.1M PBS, pH 7.2-7.4), then add 10-50mg/mL 1-ethyl-(3-dimethylaminopropyl base) carbodiimide (EDC), dark reaction 15-30min;

(2) Weighing 10 mg bovine serum albumin and adding it to the activated polylysine solution;

(3) Dialysis with phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4) to remove EDC;

(4) After affinity purification, the product with higher protein concentration was collected to obtain coupled protein 3 (ε-PL-BSA);

(5) Weigh 5g sucrose, 1.3g MgCl ₂ , 0.05g PEG4000, 0.02g Tetronin1307, 0.02g KroVin300M, 0.24g Tris, 100g process water, add coupling protein 3 to make the final concentration 1%, adjust the pH to 8.5, Obtain compound preparation.

Comparative example 1

Weigh 2.5g trehalose, 0.9g NaCl, 0.05g PEG4000, 0.05g Tween 20, 0.05g proclin 300, 0.24g Tris, 0.33g bovine serum albumin, 0.33g ovalbumin, 0.33g hemocyanin, 100g process water , adjust the pH to 8.5 to obtain the compound preparation.

Comparative example 2

Weigh 2.5g trehalose, 0.9g NaCl, 0.05g PEG4000, 0.05g Tween 20, 0.05g proclin 300, 0.24g Tris, 0.5g bovine serum albumin, 0.5g silk fibroin, 100g process water, adjust the pH to 8.5, Obtain compound preparation.

Comparative example 3

Weigh 0.9g NaCl, 0.05g PEG4000, 0.05g Tween 20, 0.05g proclin 300, 0.24g Tris, 100g process water, add coupling protein 1 to make the final concentration 1%, and adjust the pH to 8.5 to obtain a compound preparation.

Comparative example 4

Weigh 2.5g trehalose, 0.05g PEG4000, 0.05g Tween 20, 0.05g proclin 300, 0.24g Tris, 100g process water, add coupling protein 1 to make the final concentration 1%, and adjust the pH to 8.5 to obtain a compound preparation.

Comparative example 5

(1) Weigh 10mg of bovine serum albumin and add it to phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4), then add 10-50mg/mL 1-ethyl-(3-dimethylaminopropyl base) carbodiimide (EDC), dark reaction 15-30min;

(2) Weighing 10 mg of polylysine and adding it to the activated bovine serum albumin solution;

(3) Dialysis with phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4) to remove EDC;

(4) After affinity purification, the product with higher protein concentration was collected to obtain coupling protein 4 (BSA-ε-PL);

(5) Weigh 5g sucrose, 1.3g MgCl ₂ , 0.05g PEG4000, 0.02g Tetronin1307, 0.02g KroVin300M, 0.24g Tris, 100g process water, add coupling protein 4 to make the final concentration 1%, adjust the pH to 8.5, Obtain compound preparation.

Comparative example 6

Weigh 2.5g trehalose, 0.9g NaCl, 0.05g PEG4000, 0.05g proclin 300, 0.24g Tris, 100g process water, add coupling protein 1 to make the final concentration 1%, and adjust the pH to 8.5 to obtain a compound preparation.

Comparative example 7

A compound preparation, the preparation method is as follows: Weigh 0.15% glucose, 0.6% HEPES, 1% NaCl, 0.5% KCl, 0.09% EDTA-2Na, 0.03% SDS, 1% BSA, 0.05% Triton 100, 0.02% Tween-20, 0.05% Proclin, adjust the solution to pH8.5 after mixing.

The composite preparations prepared in Examples 1-3 and Comparative Examples 1-7 are detected, and the specific methods are as follows:

1. The operation process of labeling RBD protein with time-resolved fluorescent microspheres: after activation of fluorescent microspheres, resuspend in HEPES buffer, add RBD protein, label overnight, add blocking solution for blocking for 2 hours, centrifuge and use examples 1-3 and paired samples respectively after centrifugation. The compound preparation prepared in proportion 1-7 is stored, and the obtained new crown RBD protein fluorescent microsphere complex is divided into 50 μL/tube, and compared with the results after 0 days and 2-8°C for 30 days, if the CV of the fluorescence value≤15 %, absolute value of relative deviation (absolute value of relative deviation=|average value of fluorescence value on day 0-average value of fluorescence value on day 30|/average value of fluorescence value on day 0)≤10%, negative and positive coincidence rate≥95%, Then the composite preparation meets the requirements, and the repeatability, stability and detection sensitivity are good.

2. Chicken IgY antibody labeling and conjugate release pad preparation process: After activation of fluorescent microspheres, resuspend in HEPES buffer, add chicken IgY antibody, label overnight, add blocking solution for blocking for 2 hours, centrifuge and resuspend in microsphere preservation solution 4μL/cm was sprayed on the conjugate release pad (0.7*30cm), and dried at 37°C for 4h.

3. Sample pad treatment process: sample pad treatment solution: 0.02M Tris buffer containing 1% sucrose, 1% BSA, 0.2% Tween-20, 0.4mg/mL RBC antibody, adjust pH to 8.0-8.5, 6cm/mL Cloth sample pad (2.5*30cm), dry at 37°C for 4h.

4. T-line fixation: use 0.01MPB buffer containing 0.5-1% BSA to dilute the ACE2 protein of the new coronavirus to 0.1-0.5mg/mL, 1μL/cm, 50mm/s and fix it on the nitrocellulose membrane (2*30cm ) at 37°C for 2 hours.

5. C-line fixation: Dilute the anti-chicken IgY antibody to 0.1-0.5 mg/mL with 0.01 MPB buffer containing 0.5-1% BSA, fix on nitrocellulose membrane at 1 μL/cm, 50 mm/s, and bake at 37 °C Dry for 2 hours.

6. Large plate assembly and test strip packing: according to the water absorption pad (3*30cm)-nitrocellulose membrane-chicken IgY conjugate release pad-sample pad, they are overlapped and pasted on the 8*30cm PVC backboard in sequence , cut into 4mm ± 0.4mm test strips, and put them into the card case.

7. Sample preparation: Add 50 μL of sample to the aliquoted new crown RBD protein fluorescent microsphere compound preparation, shake and mix; whole blood sample is mixed upside down and gently, and incubated at room temperature for 10-15 minutes.

8. Sample detection: add 85-95μL/well, incubate at room temperature for 10-15min.

9. Result processing: Use a dry-type fluorescence analyzer to read the value to obtain the fluorescence value or inhibition rate. The specific results are shown in Table 1.

Table 1 Fluorescence value detection results

Remarks: Absolute value of relative deviation (absolute value of relative deviation=|average value of fluorescence value on day 0-average value of fluorescence value on day 30|/average value of fluorescence value on day 0)

Table 2 Sample negative and positive coincidence rate detection results

The CV of Example 1, Example 2 and Example 3≤15%, the absolute value of relative deviation≤10%, the negative-positive coincidence rate≥95%, and the repeatability, stability, and sample coincidence rate are good. The difference between the fluorescence values of Example 1 and Example 3 within a small and controllable range does not mean that there is an upward trend, but is just a normal fluctuation of the methodology.

In Comparative Example 1 and Comparative Example 2, proteins were directly added without coupling, and no peptide bonds were formed between proteins. After 30 days of storage, the complete structure may not be maintained, and the protective effect on the RBD protein fluorescent microsphere complex was reduced, resulting in RBD protein The lower the fluorescence value of the saturation competition method, the stronger the positive, so the positive coincidence rate increases and the negative coincidence rate decreases.

In Comparative Example 3, no sugar was added, and the absence of the protective film on the surface of the RBD protein led to the denaturation of the RBD protein, and the repeatability, stability, and coincidence rate of the test results deteriorated.

In Comparative Example 4, no ions were added, and the blood cells could not remain intact due to the osmotic pressure of the buffer solution when detecting the whole blood sample, the NC membrane background was not clear due to the broken blood cells, and the fluorescence analyzer could not obtain the highest peak fluorescence value of the sample, resulting in false negative results; The absence of ions will also cause non-specific adsorption of RBD protein on the coated membrane, which will deteriorate the repeatability, stability and sample coincidence rate of the test results.

In Comparative Example 5, the coupling sequence between BSA and polylysine is different. After EDC activates the carboxyl group of BSA, it can combine with one or more amino groups of BSA to obtain the product BSA-n*BSA (n=N+); it can also be combined with One or more amino groups of polylysine are combined to obtain the product BSA-n*polylysine (n=N+), and the difference between batches cannot be controlled. Only the coupling combination of BSA-polylysine can make the RBD protein have uniform properties and good activity. The results of Comparative Example 5 are by chance.

In Comparative Example 6, no surfactant was added, and the RBD proteins may bind to each other, resulting in the aggregation of fluorescent complexes; the specific binding between the labeled antigen and the coated antibody is reduced, resulting in poor repeatability, stability, and sample coincidence rate.

In Comparative Example 7, the existing compound preparation was used, the protein concentration was low, and the ionic surfactant SDS would destroy the protein structure. After 30 days of storage, the signal value of the sample was the same as the background value, and the repeatability, stability and coincidence rate of the sample changed. Difference.

The signal value of the compound preparation prepared in comparative examples 1-7 decreased by 20-90% after being stored for 30 days, and at least one of the relative deviation absolute value, CV and sample compliance rate did not meet the requirements (required CV≤15%, relative deviation absolute value ≤10%, negative and positive coincidence rate ≥95%), poor stability and repeatability. The signal of the composite preparation prepared in Examples 1-3 did not decrease substantially after being stored for 30 days, and the repeatability was good.

It can be seen from the results that there is no significant difference between Example 1, Example 2 and Example 3, so Example 1 is selected for the precision evaluation experiment.

Experimental protocol

Take 4 serum samples (1 negative sample, 1 borderline positive sample, 1 moderately positive sample, 1 strong positive sample) and test them separately with 3 models, 2 operators for each model, a total of 6 operations Personnel, use 3 batches of kits for each model, test for 5 days, and do 5 repetitions for each sample every day (3 models × 3 kit batches × 5 days × 5 repetitions/day = 225 results/sample).

data analysis method

(1) (moderate or strong) positive sample data analysis

The positive detection rate is required to be 100%, the repeatability ≤ 15%, the indoor precision ≤ 15%, and the inter-batch precision ≤ 20%.

(2) Data analysis of critical positive samples

Calculate the positive detection rate. The positive detection rate is required to be ≥95%.

(3) Negative sample data analysis

Calculate the negative detection rate. It is required that the negative detection rate should be 100%.

test results

Table 3 (moderate or strong) positive sample data analysis results

Table 4 Data Analysis Results of Critical Positive Samples

instrument	Inhibition rate	Number of results with ≥20% inhibition	Number of results with inhibition rate < 20%	positive coincidence rate
Model 1	23.92%	73	2	97.26%
Model 2	21.78%	74	1	97.33%
Model 3	22.32%	74	1	97.33%

Table 5 negative sample data analysis results

instrument	Inhibition rate	Number of results with ≥20% inhibition	Number of results with inhibition rate < 20%	Negative coincidence rate
Model 1	1.24%	0	75	100.00%
Model 2	1.04%	0	75	100.00%
Model 3	1.27%	0	75	100.00%

From the data analysis results in the above table, it can be seen that the repeatability, indoor precision and inter-assay precision CV of the positive samples and strong positive samples in the detection of the three models are all less than 15%, which meets the requirements (repeatability CV≤15%, inter-assay CV≤20%); the positive compliance rate of borderline positive samples is ≥95%, which meets the requirements; the negative compliance rate of negative samples is 100%, which meets the requirements.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (9)

1. A new crown RBD protein fluorescent microsphere composite preparation, characterized in that: the composite preparation is made of raw materials including the following mass percentages:

   
2. The new crown RBD protein fluorescent microsphere composite preparation according to claim 1, characterized in that: the coupling protein is at least the combination of bovine serum albumin and polylysine, hemocyanin, silk fibroin or ovalbumin. One is formed by EDC coupling.
3. The new crown RBD protein fluorescent microsphere composite preparation according to claim 2, characterized in that: the coupling protein is formed by coupling bovine serum albumin and polylysine through EDC, and the coupling sequence is polylysine Acid - Bovine Serum Albumin.
4. The new crown RBD protein fluorescent microsphere composite preparation according to claim 1, wherein the sugar is at least one of sucrose, trehalose or glucose.
5. The new crown RBD protein fluorescent microsphere composite preparation according to claim 1, wherein the ion is at least one of Na ⁺ , K ⁺ or Mg ²⁺ .
6. The new crown RBD protein fluorescent microsphere composite preparation according to claim 1, characterized in that: the macromolecular substance is PEG4000.
7. The new crown RBD protein fluorescent microsphere composite preparation according to claim 1, characterized in that: the buffer is Tris buffer; the concentration of the Tris buffer is 0.02-0.1M.
8. The new crown RBD protein fluorescent microsphere composite preparation according to claim 1, wherein the surfactant is at least one of Tween-20, Tetronin1307 or BRIJ35.
9. The new crown RBD protein fluorescent microsphere composite preparation according to claim 1, characterized in that: the preservative is at least one of proclin 300 or KroVin300M.

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