阅读说明：本技术 一种去除血清中抗原的方法 (Method for removing antigen in serum ) 是由 李娟� 张雪连 于 2021-07-20 设计创作，主要内容包括：本发明涉及生物技术领域,特别涉及一种去除血清中抗原的方法。本发明所述的去除血清中抗原的方法包括如下步骤：(1)向待处理的含有抗原的血清样品中加入乙醇,混合均匀；(2)2℃～8℃冷藏6h～12h；(3)离心,吸取上清液；(4)将上清液在50℃～60℃条件下加热20min～40min；(5)冷却至室温得到处理后的血清样品。本发明所提供方法可以在保证血清质量的同时快速简便的去除血清中的多种抗原。(The invention relates to the technical field of biology, in particular to a method for removing an antigen in serum. The method for removing the antigen in the serum comprises the following steps: (1) adding ethanol into the serum sample containing the antigen to be treated, and uniformly mixing; (2) refrigerating for 6-12 h at 2-8 ℃; (3) centrifuging and sucking supernatant; (4) heating the supernatant at 50-60 deg.c for 20-40 min; (5) and cooling to room temperature to obtain a treated serum sample. The method provided by the invention can rapidly, simply and conveniently remove a plurality of antigens in the serum while ensuring the quality of the serum.)

1. A method for removing an antigen from serum, comprising the steps of:

(1) adding ethanol into a serum sample to be treated and containing the antigen, uniformly mixing, and refrigerating for 6-12 h at the temperature of 2-8 ℃;

(2) centrifuging and sucking supernatant;

(3) heating the supernatant at 50-60 ℃ for 20-40 min, and cooling to 20-30 ℃ to obtain a treated serum sample.

2. The method of claim 1, wherein the antigen in the antigen-containing serum sample comprises one or more of triiodothyronine, free triiodothyronine, thyroxine, free thyroxine, thyroid stimulating hormone, luteinizing hormone, human chorionic gonadotropin, follitrogenic hormone, prolactin, progesterone, testosterone, and estradiol.

3. The method according to claim 1, wherein the serum sample in step (1) is human serum, bovine serum, horse serum or sheep serum.

4. The method according to claim 1, wherein the ethanol in step (1) has a purity of 95% or more, and the volume of the ethanol is 15-50% of the volume of the serum sample containing the antigen.

5. The method according to claim 1, wherein the centrifugation in step (2) is performed at a rotation speed of 1000r/min to 10000r/min for 5min to 60 min.

6. The method according to claim 1, wherein the heating temperature of the step (3) is 56 ℃ and the heating time is 30 min.

7. The method according to claim 1, wherein the heating manner of the step (3) is water bath heating.

8. The method according to any one of claims 1 to 7, characterized in that a protective agent is added to the obtained treated serum sample, wherein the protective agent comprises 0-5% of protein and 0-10% of carbohydrate, and the percentages refer to the mass volume percentage of the treated serum sample; the protein is selected from bovine serum albumin or human serum albumin; the saccharide is selected from one or more of sucrose, trehalose, glucose and mannitol.

Technical Field

The invention relates to the technical field of biology, in particular to a method for removing an antigen in serum.

Background

When the serum is used as part of projects of in vitro diagnostic reagents, the antigen content in most of the serum directly obtained is higher and is generally about the median value of a linear range, but some products of manufacturers, such as serum in a working calibrator, need a low value, and the serum directly used for physical examination cannot meet the requirement of the linear range; and the low value part of the partial items is also meaningful for clinical diagnosis, but the low value serum of the part is difficult to collect.

The method for removing the antigen in the prior art generally adds a certain proportion of active carbon or passes through a column, and the method of adding the active carbon needs filtration, but the subsequent complete removal of the active carbon is difficult, which can cause the serum to be slightly black after the treatment; the column passing mode is complex, and the acquisition of a large amount of serum is difficult and cannot be universally adopted.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for removing the antigen in the serum, thereby solving the problems of the prior art that the quality of the serum is reduced or the operation is complicated.

In order to achieve the purpose, the invention specifically adopts the following technical scheme:

a method for removing an antigen from serum comprising the steps of:

(1) adding ethanol into a serum sample to be treated and containing the antigen, uniformly mixing, and refrigerating for 6-12 h at the temperature of 2-8 ℃;

(2) centrifuging and sucking supernatant;

(3) heating the supernatant at 50-60 ℃ for 20-40 min, and cooling to 20-30 ℃ to obtain a treated serum sample.

Preferably, the antigen in the antigen-containing serum sample comprises one or more of triiodothyronine (TT3), free triiodothyronine (FT3), thyroxine (TT4), free thyroxine (FT4), Thyroid Stimulating Hormone (TSH), Luteinizing Hormone (LH), Human Chorionic Gonadotropin (HCG), Follicle Stimulating Hormone (FSH), Prolactin (PRL), progesterone (P), testosterone (T), estradiol (E2).

Preferably, the serum sample in step (1) is human serum, bovine serum, horse serum or sheep serum.

Preferably, the ethanol purity in the step (1) is more than or equal to 95 percent, and the volume of the ethanol is 15 to 50 percent of the volume of the serum sample containing the antigen.

Preferably, the rotation speed of the centrifugal operation in the step (2) is 1000 r/min-10000 r/min, and the centrifugal time is 5 min-60 min.

Preferably, the heating temperature of the step (3) is 56 ℃, and the heating time is 30 min.

Preferably, the heating mode of the step (3) is water bath heating.

Preferably, a protective agent is added into the obtained treated serum sample, wherein the protective agent comprises 0-5% of protein and 0-10% of carbohydrate, and the percentages refer to the mass volume percentage of the treated serum sample, namely the ratio of g/ml; the protein is selected from bovine serum albumin or human serum albumin; the saccharide is selected from one or more of sucrose, trehalose, glucose and mannitol. For example, the treated serum sample is 500ml, the content of the added protein is required to be 0 g-25 g; the amount of the saccharides to be added is 0 to 50 g.

Advantageous effects

The method provided by the invention can remove various antigens in the serum rapidly, simply and simultaneously, and ensures the quality of the serum.

The method skillfully adopting the water bath heating not only removes the residual ethanol, but also has the other two functions: 1. inactivating serum to remove heat-sensitive substances such as complement in serum; 2. besides complement inactivation, the method can also remove mycoplasma pollution in serum, thereby ensuring the use safety.

Drawings

FIG. 1 is a diagram of a sample of the product obtained in example 1 of the present invention after lyophilization.

FIG. 2 is a diagram of a sample of the product obtained in example 4 of the present invention after lyophilization.

Detailed Description

The following will clearly and completely describe the technical solutions in the specific embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. 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.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The present invention will be described in detail with reference to examples below to facilitate understanding of the present invention by those skilled in the art.

Human serum used in the following examples of the invention was collected from xu state general mine;

bovine serum was purchased from Hangzhou Biotechnology GmbH, Zhejiang;

horse serum and sheep serum were purchased from Beijing Borxi technology, Inc.;

nanodrop (ultra-micro uv-vis spectrophotometer) used for protein concentration detection of samples was purchased from Thermo Fisher Scientific;

the detection method of the antigen concentration comprises the following steps: chemiluminescence immunoassay (using a basic egg biological full-automatic chemiluminescence determinator and a matched reagent);

the ethanol used in the examples of the present invention had a purity of 99.7% and was purchased from Nanjing chemical reagents, Inc.

Example 1

Taking a serum sample of a normal human body examination, wherein the protein concentration of the test sample is 286mg/ml, and the concentrations of 12 antigen items in the sample are respectively as follows:

item	Measured value (concentration) before treatment
		TT3(nmol/L)	1.73
FT3(pmol/L)	4.51
		TT4(nmol/L)	118.06
FT4(pmol/L)	19.15
		TSH(μIU/ml)	1.58
LH(mIU/ml)	3.74
		HCG(mIU/ml)	18.97
FSH(mIU/ml)	4.36
		PRL(ng/ml)	3.41
P(ng/ml)	1.25
		T(ng/ml)	0.64
E2(pg/ml)	29.73

Step (1): taking 50ml of the human serum sample, adding 15ml of ethanol into the human serum sample, and putting the mixed solution obtained by uniformly mixing the human serum sample and the ethanol in a refrigerator at the temperature of 2-8 ℃ for 8 hours;

step (2): centrifuging the placed mixed solution at the speed of 5000r/min for 30min, sucking supernatant for later use after centrifugation is finished, and treating residual residues in a local biological garbage mode; the residual residue is that part of redundant fibrin is separated out after the ethanol is added, so that the subsequent use is not influenced;

and (3): putting the supernatant obtained in the step (2) into a water bath kettle, wherein the water bath temperature is 56 ℃, the water bath time is 30min, taking out the supernatant after the water bath is finished, cooling the supernatant to room temperature, adding 3% of human serum albumin and 5% of mannitol in mass volume ratio, and storing the mixture in a refrigerator for later use after the human serum albumin and the mannitol are dissolved and mixed uniformly;

and (4): the color of the sample obtained in the step (3) is visually detected to be no different from the color before the treatment, but the sample looks clearer, the protein concentration of the sample after the treatment by using the nanodrop test is 138mg/ml, and meanwhile, the antigen concentration of each item of the product obtained in the step (3) is detected, and the detection results are shown in the following table.

The formula for calculating the relative deviation in the above table is: (measured value before treatment-measured value after treatment)/measured value before treatment.

As can be seen from the above table, the method of the present invention can remove 12 antigens simultaneously, simply and rapidly, and the removal rate of most antigens reaches more than 90%.

The treated product of this example was lyophilized to obtain a lyophilized product, which was shown in fig. 1, and the product obtained in this example was lyophilized three times, and it can be seen that the product obtained by three-time lyophilization in this example all maintained the volume before lyophilization, had a shape that was also substantially unchanged from that before lyophilization, had a porous (micro-pore) structure, and the lyophilized powder did not collapse, expand or leave liquid.

Example 2

The amount of ethanol added in step (1) of example 1 was changed to 5ml, and the other operations were exactly the same as in example 1, to finally obtain a treated serum sample. The products obtained after the treatment were tested, and the test results are shown in the following table.

As can be seen from the above table, when the amount of ethanol is too small, the removal effect of the antigen in the serum is not good, and the removal rate of all the antigens is less than 50%.

Example 3

The amount of ethanol added in step (1) of example 1 was changed to 30ml, and the other operations were exactly the same as in example 1, to finally obtain a treated serum sample. The products obtained after the treatment were tested, and the test results are shown in the following table.

As can be seen from the above table, the removal rate of all antigens is excellent, which results in the measured value after treatment being too low, and the color of the sample obtained after treatment is light, the protein concentration of the sample after treatment by nanodrop test is 24mg/ml, and the protein concentration is too low compared with the sample before treatment, which is reduced by more than 11 times, and the serum quality after default treatment is greatly different from that before treatment.

Example 4

The same procedures as in example 1 were carried out except that human serum albumin and mannitol were not added in step (3) of example 1, to thereby obtain a treated serum sample. Then, the serum samples were extracted into three portions with the same volume as that of example 1 and freeze-dried for three times, and the effect after freeze-drying is shown in fig. 2, which shows that the freeze-drying effect is very poor without adding human serum albumin and mannitol, the shape of the product of the third freeze-drying of this example which is visible to the naked eye is not full, the shape and volume of the freeze-dried powder are greatly different from those before freeze-drying, and part of the product has liquid residue.

Example 5

Taking a bovine serum sample, wherein the protein concentration in the test sample is 289mg/ml, and the antigen concentrations of 12 items in the sample are respectively as follows:

item	Measured value (concentration) before treatment
		TT3(nmol/L)	1.75
FT3(pmol/L)	4.55
		TT4(nmol/L)	120.51
FT4(pmol/L)	19.56
		TSH(μIU/ml)	1.55
LH(mIU/ml)	3.78
		HCG(mIU/ml)	19.10
FSH(mIU/ml)	4.16
		PRL(ng/ml)	3.32
P(ng/ml)	1.31
		T(ng/ml)	0.66
E2(pg/ml)	28.45

Step (1): taking 50ml of the bovine serum sample, adding 7.5ml of ethanol into the bovine serum sample, and putting the mixed solution after the two are uniformly mixed in a refrigerator at the temperature of 2-8 ℃ for 12 hours;

step (2): centrifuging the placed mixed solution at the speed of 1000r/min for 60min, sucking supernatant for later use after centrifugation is finished, and treating residual residues in a local biological garbage mode;

and (3): and (3) putting the supernatant obtained in the step (2) into a water bath kettle, wherein the water bath temperature is 56 ℃, the water bath time is 30min, taking out the supernatant after the water bath is finished, cooling the supernatant to room temperature, adding 0% of protein and 0% of saccharide, and storing the supernatant in a refrigerator for later use after the supernatant is completely dissolved and uniformly mixed.

And (4): the color of the obtained sample was not different from that before the treatment, but appeared clearer, and the protein concentration of the sample after the treatment by the nanodrop test was 182mg/ml, and the antigen concentration of each item in the product obtained in step (3) was measured, and the results of the measurements are shown in the following table.

Example 6

Taking a horse serum sample, wherein the concentration of the protein in the test sample is 274mg/ml, and the antigen concentration of 12 items in the sample is respectively as follows:

item	Measured value (concentration) before treatment
		TT3(nmol/L)	1.76
FT3(pmol/L)	4.54
		TT4(nmol/L)	118.14
FT4(pmol/L)	19.58
		TSH(μIU/ml)	1.51
LH(mIU/ml)	3.60
		HCG(mIU/ml)	19.05
FSH(mIU/ml)	4.48
		PRL(ng/ml)	3.57
P(ng/ml)	1.24
		T(ng/ml)	0.63
E2(pg/ml)	30.36

Taking 50ml of the horse serum sample, adding 25ml of ethanol into the horse serum sample, and uniformly mixing the horse serum sample and the ethanol for 6 hours in a refrigerator at the temperature of 2-8 ℃;

centrifuging the placed mixed solution at the centrifugation speed of 10000r/min for 5min, sucking supernatant for later use after centrifugation is finished, and treating residual residues in a local biological garbage mode;

and (3) putting the supernatant obtained in the step (2) into a water bath kettle, taking out and cooling to room temperature after the water bath is finished at the water bath temperature of 56 ℃ for 30min, adding 5% of protein and 10% of sugar, and storing in a refrigerator for later use after the protein and the sugar are dissolved and mixed completely and uniformly.

The color of the sample obtained in the step (4) is not different from the color before treatment, but looks clearer, the protein concentration of the sample after treatment by the nanodrop test is 118mg/ml, and meanwhile, the antigen concentration of each item in the product obtained in the step (3) is detected, and the detection results are shown in the following table.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.