CN111721935A

CN111721935A - Sugar chain antigen CA153 detection kit

Info

Publication number: CN111721935A
Application number: CN202010236601.8A
Authority: CN
Inventors: 熊争平; 果玮; 刘瑶; 刘希
Original assignee: Beijing Strong Biotechnologies Inc
Current assignee: Beijing Strong Biotechnologies Inc
Priority date: 2019-03-29
Filing date: 2020-03-30
Publication date: 2020-09-29

Abstract

The present application relates to a sugar chain antigen CA153 detection kit. The kit comprises a first reagent and a second reagent. The first reagent comprises buffer solution, electrolyte, preservative, surfactant and blocking agent; the second reagent comprises a buffer solution, a stabilizing agent, a preservative, a protective agent, polystyrene latex particles and a sugar chain antigen CA153 antibody. The kit has the advantages of good stability, strong specificity and the like, is suitable for various large types of biochemical instruments, and can detect the carbohydrate chain antigen CA153 at a higher speed.

Description

Sugar chain antigen CA153 detection kit

This application claims priority to chinese patent application 201910246917.2 filed on 29/3/2019.

Technical Field

The present application relates to the field of clinical testing. In particular to a quantitative detection reagent of CA 153.

Background

The short name of CA, Carcinoma Antigen, namely cancer Antigen 153, is a marker of various tumors, belongs to a broad-spectrum marker, and is remarkably shown in breast cancer patients.

CA153 is a transmembrane protein, expressed as the apical membrane of highly differentiated epithelial cells. In epithelial tumors that are overexpressed in the case of breast or ovarian cancer, CA153 contains two subunits, an alpha subunit and a beta subunit.

The breast cancer is often accompanied by CA153, but the sensitivity is lower in the early stage of the breast cancer, about 60 percent, and the positive rate of metastatic breast cancer can reach 80 percent. In Europe, CA153 is generally used as an auxiliary diagnosis index of breast cancer, and is also used as an index for monitoring tumor recurrence and metastasis after postoperative follow-up. The literature indicates that when the CA153 level exceeds 30, 40 and 50U/ml, the breast cancer patients are judged to have postoperative local region recurrence or distant metastasis, the sensitivity of the breast cancer patients exceeds 90 percent, the specificity of the breast cancer patients is respectively 95 percent, 99 percent and 100 percent, and the correct judgment rate of the breast cancer patients respectively reaches 56 percent, 83 percent and 100 percent.

In addition, breast cancer patients with elevated CA153 levels develop metastases much earlier than those with normal CA153 levels. According to research analysis, the consistency of the change of the serum CA153 level of a breast cancer patient with the local lymph node and distant metastasis conditions is changed, particularly in the case of distant metastasis, the CA153 expression level and the positive rate are obviously increased, so the CA153 has the effect of monitoring the breast cancer metastasis, and if the serum level is continuously increased, chemotherapy, radiotherapy or endocrine treatment is started or enhanced.

At present, most of the chemiluminescence detection adopted by hospitals is basically monopolized by foreign IVD industries, such as Roche, Siemens, Yapeh and the like, and has no autonomy in price, so that the medical cost is easily increased, and the detection burden of patients is increased.

Therefore, it is necessary to develop a kit for detecting CA153, which is effective and widely applicable to various hospitals.

Disclosure of Invention

According to some embodiments of the present application, there is provided a CA153 detection kit comprising a first reagent and a second reagent,

wherein the first reagent comprises:

the second reagent comprises:

in some embodiments, the buffer is selected from: glycine buffer, Tris-HCl buffer, HEPES buffer.

In some embodiments, the buffers in the first and second reagents may be the same or different.

In some embodiments, the preservative is selected from: sodium azide, thimerosal, ProClin 300.

In some embodiments, the polyethylene glycol is selected from: PEG6000, PEG8000, PEG12000, PEG20000, preferably PEG 20000.

In some embodiments, the surfactant is selected from: tween 20, Tween 80, NP40, thesit, preferably Tween 20.

In some embodiments, the protective agent is selected from: bovine serum albumin, ovalbumin, skim milk, calf serum.

In some embodiments, the blocking agent is goat anti-mouse IgG.

In some embodiments, the stabilizing agent is selected from: sucrose, glycerol, trehalose, glucose, preferably trehalose.

In some embodiments, the surface functional groups of the latex particles are selected from: amino, carboxyl, chloromethyl, epoxy, preferably carboxyl.

In some embodiments, the latex particles have an average particle size of 200nm to 500nm, preferably 400 nm.

In some embodiments, the CA153 antibody is a monoclonal antibody.

In some embodiments, the kits of the present application comprise a first reagent and a second reagent,

wherein the first reagent comprises:

the second reagent comprises:

the CA153 antibody is a murine monoclonal antibody; and the polystyrene latex particles have an average particle size of 400 nm; the polystyrene latex particles are carboxyl modified.

Drawings

FIG. 1 is a calibration curve of the kit of the present application.

Detailed Description

EXAMPLE 1 preparation of the first reagent

1. Weighing 17.87g of HEPES, 13.15g of NaCl, 3g of PEG20000, 7.5g of Tween 20, 1.5g of sodium azide, 7.5g of BSA and 15mL of blocking agent (5mg/mL), dissolving in 1.0L of double distilled water, adjusting the ph to 7.0, and carrying out constant volume to 1.5L to obtain a first reagent (1):

2. alternatively, 5.63g glycine, 13.15g NaCl, 3g PEG20000, 7.5g Tween 80, 1.5g sodium azide, 7.5g BSA, 10mL blocking agent (same as above) were weighed, dissolved in 1.0L double distilled water, adjusted to ph 7.0, and made up to 1.5L to prepare the first reagent (2):

3. alternatively, 9.09g of Tris, 13.15g of NaCl, 3g of PEG20000, 7.5g of Tween 20, 1.5g of sodium azide, 7.5g of BSA, and 10mL of a blocking agent (the same as above) were weighed, dissolved in 1.0L of double distilled water, adjusted to ph 7.0, and made up to 1.5L to prepare the first reagent (3):

example 2 preparation of antibody-coated particles

1. Particles (400nm, carboxyl) were diluted to 1% (w/v) with HEPES buffer;

2. diluting the antibody (murine monoclonal antibody) to 0.5mg/ml with HEPES buffer;

3. adding EDAC aqueous solution with concentration of 0.1% to 1% (w/v) into the granules in the step 1, and reacting for 30min at 37 ℃ in a constant temperature shaking table;

4. after the reaction is finished, adding the antibody in the step 2, and reacting for 3 hours in a constant temperature shaking table at 37 ℃;

5. adding a sealing agent, and standing overnight at normal temperature;

6. and adding a cleaning solution into the particles sealed overnight, cleaning and centrifuging for 3 times, and storing for later use.

Step 1 and step 2 sequences may be interchanged or in parallel.

EXAMPLE 3 preparation of the second reagent

Adding 400mL of double distilled water into the granules prepared in the embodiment 2, adding 18.76g of glycine, 2.5g of BSA, 0.5g of sodium azide and 50g of trehalose, stirring and uniformly mixing, adjusting the ph to 7.2, adding the double distilled water to 500mL, and carrying out ultrasonic treatment until the absorbance of the main wavelength is basically unchanged to obtain a second reagent:

example 4 preparation of the kit

The foregoing reagents were assembled into kits according to the following table.

TABLE 1 kit Assembly

	A first reagent	Second reagent
			Kit 1	First reagent (1)	Second reagent
Kit 2	First reagent (2)	Second reagent
			Kit 3	First reagent (3)	Second reagent

Test example

Test example 1 detection Limit test

The 3 kits prepared in the above examples were tested using a fully automatic biochemical analyzer (e.g., Hitachi 7180).

The measurement wavelength is 660nm, the sample sampling amount is 5 mu L, 150 mu L of first reagent is added, the temperature is kept constant for 5min at 37 ℃, 50 mu L of second reagent is then added, the absorbance A1 is read after 42 seconds, the absorbance A2 is read after 4 minutes and 18 seconds of incubation at 37 ℃, and the reaction absorbance delta A is A2-A1; the performance of the 3 kits was verified, and the results were as follows:

TABLE 2 detection limit results

According to the determination, the kits 1 to 3 are only distinguished by different buffers, however, only the kit 1 presents an unexpected detection limit, which can reach 2.0U/ml.

Test example 2 screening of surfactants

According to the preparation method of the kit 1, the following kits were prepared, except that the surfactant Tween 20 was replaced with Tween 80, NP40, and thesit, respectively, and the anti-interference effect on triglyceride interferents was tested.

TABLE 3 surfactant screening

It is known to those skilled in the art that in the field of formulation, surfactants function to promote uniform dispersion of components in a reagent system or substances in a test sample. Thus, surfactants having solubilizing properties in theory can be used, e.g., Tween 20, Tween 40, Triton X-100, ethylphenylpolyethylene glycol, etc. (see Experimental materials and methods 2013, 3: 163, page 3). The inventors have unexpectedly noted that while Tween 20, Tween 80, NP40 and Thesit are all assigned to non-ionic surfactants, only Tween 20 achieved a significant advantage in anti-triglyceride interference, forming a statistically significant difference with the other three surfactants.

Test example 3 Effect of blockers on false Positive results

The control kit was prepared according to the preparation method of kit 1, except that no blocking agent was included. Samples with or without 500IU/ml RF were tested with each kit prepared. The results are as follows, showing that blockers can significantly improve the false positives of the test results, avoiding RF interference.

TABLE 4 influence of blockers on false positive results

Claims

1. A CA153 detection kit comprising a first reagent and a second reagent, wherein: the first reagent comprises:

the second reagent comprises:

the buffer is selected from one or a combination of the following: glycine buffer solution, Tris-HCl buffer solution and HEPES buffer solution;

the preservative is selected from: sodium azide, thimerosal, ProClin 300;

the polyethylene glycol is selected from: PEG6000, PEG8000, PEG12000, PEG20000, preferably PEG 20000;

the surfactant is selected from: tween 20, Tween 80, NP40, thesit, preferably Tween 20;

the protective agent is selected from: bovine serum albumin, ovalbumin, skim milk, calf serum;

the blocking agent is goat anti-mouse IgG;

the stabilizer is selected from: sucrose, glycerol, trehalose, glucose, preferably trehalose;

the surface functional groups of the latex particles are selected from: amino, carboxyl, chloromethyl, epoxy, preferably carboxyl;

the latex particles have an average particle diameter of 200nm to 500nm, preferably 400 nm;

preferably, the CA153 antibody is a monoclonal antibody.

2. The CA153 assay kit of claim 1, wherein the buffer of the first reagent is not a glycine buffer or a Tris buffer.

3. The CA153 detection kit of claim 1, comprising a first reagent and a second reagent,

wherein the first reagent comprises:

the second reagent comprises:

the CA153 antibody is a murine monoclonal antibody;

the polystyrene latex particles have an average particle size of 400 nm;

the polystyrene latex particles are carboxyl-modified.

4. The CA153 detection kit of claim 1, comprising a first reagent and a second reagent,

wherein the first reagent comprises:

the second reagent comprises:

the CA153 antibody is a murine monoclonal antibody;

the polystyrene latex particles have an average particle size of 400 nm;

the polystyrene latex particles are carboxyl-modified.