Preparation method and detection kit for hormone-free serum

Abstract

This invention discloses a method for preparing hormone-free serum and a detection kit. The method for preparing hormone-free serum includes the following steps: centrifuging human or animal serum and collecting the supernatant; adding a protein dissociation agent to the supernatant to react and release small molecule hormones bound to proteins, obtaining an intermediate sample; and dialysis the intermediate sample to remove the small molecule hormones, obtaining the hormone-free serum. This invention not only improves the removal rate of small molecule hormones but also avoids the influence of activated carbon on detection.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and more specifically, to a method for preparing hormone-free serum and a detection kit. Background Technology

[0002] Small molecule hormones, as an important component of human hormones, play a role in regulating bodily functions. For example, they can regulate metabolism and maintain homeostasis; promote cell proliferation and differentiation, control growth, development, and reproductive functions, and influence the aging process; and work closely with the nervous system to enhance the body's adaptation to the environment, etc. Common small molecule hormones include estradiol (E2), testosterone (T), progesterone (P), thyroid hormone (T4), and triiodothyronine (T3). Quantitative detection of small molecule hormone concentrations in samples helps clinicians assess patients' conditions and guide subsequent treatment and follow-up.

[0003] Currently, in the development of small molecule hormone assay kits, the preparation of calibrators and quality control samples often faces the challenge of matrix selection. Conventional buffer matrices often exhibit significant matrix differences from the samples, making them unsuitable as substrates for testing. Furthermore, ordinary human serum and animal serum matrices often contain a certain concentration of small molecule hormones, which can affect the test results of the kit. Therefore, the development of small molecule hormone assay kits often selects hormone-free serum as the matrix for calibrators and quality control samples during sample preparation.

[0004] Currently, the preparation process of hormone-free serum is quite complex, often involving the use of activated carbon for adsorption. However, activated carbon tends to stain the serum black during preparation, and this black residue is difficult to remove. Furthermore, activated carbon has a relatively large pore size, and since small molecule hormones typically have a molecular weight less than 1000D, they are difficult for activated carbon to adsorb completely. Using such serum as a matrix for calibrators and quality control samples in the development of small molecule hormone detection kits will adversely affect the performance of the kits. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects in the prior art and provide a new method for preparing hormone-free serum and a detection kit, which not only improves the removal rate of small molecule hormones, but also avoids the influence of activated carbon on detection.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A method for preparing hormone-free serum includes the following steps:

[0008] Human or animal serum is centrifuged and the supernatant is collected.

[0009] A protein dissociation agent is added to the supernatant to react and remove the small molecule hormones bound to the protein, thus obtaining an intermediate sample.

[0010] The intermediate sample was subjected to dialysis to remove small molecule hormones, resulting in hormone-free serum.

[0011] The present invention also provides a test kit comprising hormone-free serum prepared by the above-described preparation method.

[0012] Implementing the embodiments of the present invention will have the following beneficial effects:

[0013] This invention first uses a protein dissociation agent to detach small molecule hormones bound to proteins, converting them into a free state. Then, dialysis is used to remove the free small molecule hormones, thus effectively removing both free and bound small molecule hormones from the serum. Compared to existing technologies, this invention not only significantly improves the removal rate of small molecule hormones but also avoids the influence of activated carbon on detection. Detailed Implementation

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] This invention discloses a method for preparing hormone-free serum, comprising the following steps:

[0016] 1) Centrifuge human or animal serum to remove insoluble macromolecules and collect the supernatant.

[0017] In this invention, human or animal serum refers to the pale yellow, transparent liquid separated from blood plasma after fibrinogen is removed after blood coagulation. Serum is mainly composed of water and various chemical components, including nutrients required by the body and metabolic products produced by the body, such as albumin, globulin, plasma protein, polypeptide, triglycerides, cholesterol, transaminase, hormones, growth factors, carbohydrates, inorganic substances, etc.

[0018] 2) Add a protein dissociation agent to the supernatant to react and remove the small molecule hormones bound to the protein to obtain an intermediate sample.

[0019] Serum contains both free and bound small molecule hormones. This step uses a protein dissociation agent to dissociate the bound small molecule hormones from their respective transport proteins, making them free.

[0020] In one specific embodiment, the protein dissociation agent comprises ammonium 8-phenylamino-1-naphthalenesulfonate and / or sodium salicylate. This protein dissociation agent can dissociate bound small molecule hormones from their respective transport proteins without causing denaturation, decomposition, or recombination of other components in the serum. Furthermore, the protein dissociation agent is also a small molecule, and excess protein dissociation agent can be removed in subsequent dialysis steps.

[0021] Preferably, the mass of the protein dissociation agent is 0.01% to 1% of the mass of serum.

[0022] 3) The intermediate sample was dialyzed to remove small molecule hormones, resulting in hormone-free serum.

[0023] Dialysis separation utilizes osmotic pressure for separation. The difficulty of this invention lies in preparing a dialysate with essentially the same composition as serum. This dialysate not only provides osmotic pressure but also needs to maintain the original various nutrients in the serum as much as possible, as well as the original water, electrolyte, and acid-base balance in the serum.

[0024] In one specific embodiment, dialysis separation uses dialysis bags of 1000D to 5000D, which can remove small molecule hormones while retaining other chemical components in the serum.

[0025] Furthermore, the dialysate used for dialysis separation includes a pH of 6.8–7.5 and Na+. + Sodium carbonate-sodium bicarbonate buffer solution with a concentration of 10 mmol / L to 100 mmol / L not only provides osmotic pressure and removes small molecule hormones, but also maintains the original water and acid-base balance in the serum as much as possible.

[0026] Furthermore, the dialysate also includes 0.1 mmol / L to 4 mmol / L potassium chloride, 0.5 mmol / L to 4 mmol / L calcium chloride, 0.5 mmol / L to 2 mmol / L magnesium chloride, and 50 mmol / L to 150 mmol / L sodium chloride, which are basically the same as the concentrations of inorganic salt ions such as potassium, calcium, chloride, magnesium, and sodium in serum, in order to maintain the balance of inorganic salt ions in serum as much as possible.

[0027] In one specific embodiment, dialysis separation includes the following process: the intermediate sample is placed into a dialysis bag of 1000D to 5000D, the dialysis bag is placed in the above-mentioned dialysis solution for dialysis for 12h to 24h, the dialysis solution is replaced, and the dialysis is repeated 2 to 10 times.

[0028] 4) The hormone-free serum is treated with preservatives and sterilized for easy preservation.

[0029] Specifically, in one embodiment, the anti-corrosion treatment includes the following processes:

[0030] Add 0.05% to 0.2% of a preservative, including sodium azide and / or ProClin 300, to the hormone-free serum.

[0031] In one specific embodiment, the sterilization process includes the following steps:

[0032] The hormone-free serum is filtered sequentially through a series of filter membranes with decreasing pore size gradients to remove impurities and bacteria.

[0033] In one specific embodiment, the hormone-free serum is filtered sequentially through filter membranes with pore sizes of 0.8 μm, 0.45 μm, and 0.22 μm.

[0034] In the above embodiments, the small molecule hormones mainly include estradiol, testosterone, progesterone, thyroid hormones, triiodothyronine, free thyroxine, and free triiodothyronine.

[0035] The present invention also provides a test kit comprising hormone-free serum prepared by the above-described method.

[0036] Furthermore, the present invention also provides a small molecule hormone detection kit, comprising hormone-free serum prepared by the above method. The hormone-free serum is used as a matrix for calibrators and / or quality control products, and is substantially the same as the matrix of the blood sample to be tested, thereby improving the detection precision and accuracy.

[0037] The following are specific examples.

[0038] Example 1

[0039] A method for preparing hormone-free serum includes the following steps:

[0040] 1. Take 15000g of fetal bovine serum, centrifuge for 30 minutes, and collect the supernatant.

[0041] 2. Add 0.1% sodium salicylate protein dissociation agent (based on the mass of fetal bovine serum) and stir for 40 minutes.

[0042] 3. Add serum to a pre-treated cellulose dialysis bag (3000D). Seal both ends of the dialysis bag and place it into the dialysis solution (pH 7.40, Na+, etc.) for dialysis. + The dialysis was performed using a sodium carbonate-bicarbonate buffer solution with a concentration of 20 mmol / L, potassium chloride 4 mmol / L, calcium chloride 2 mmol / L, magnesium chloride 0.75 mmol / L, and sodium chloride 120 mmol / L for 24 hours. The dialysis was repeated twice with the dialysis solution changed to obtain hormone-free serum.

[0043] 4. Add sodium azide to the hormone-free serum at a mass of 0.05% of the hormone-free serum mass.

[0044] 5. The serum obtained in step 4 is filtered sequentially through 0.8μm, 0.45μm and 0.22μm filter membranes to obtain the final hormone-free serum.

[0045] Example 2

[0046] A method for preparing hormone-free serum includes the following steps:

[0047] 1. Take 12000g of horse serum, centrifuge for 60min, and collect the supernatant.

[0048] 2. Add 0.1% of 8-phenylamino-1-naphthalenesulfonate ammonium salt protein dissociation agent (by weight of horse serum) and stir for 30 min.

[0049] 3. Add serum to a pre-treated cellulose dialysis bag (1000D). Seal both ends of the dialysis bag and place it into the dialysis solution (pH 7.40, Na+, etc.) for dialysis. + The dialysis solution consisted of 30 mmol / L sodium carbonate-sodium bicarbonate buffer, 2 mmol / L potassium chloride, 2 mmol / L calcium chloride, 0.75 mmol / L magnesium chloride, and 130 mmol / L sodium chloride. The dialysis time was 12 h, and the dialysis was repeated 4 times with different dialysis solutions to obtain hormone-free serum.

[0050] 4. Add 0.1% of ProClin 300 (by weight of the hormone-free serum) to the hormone-free serum.

[0051] 5. The serum obtained in step 4 is filtered sequentially through 0.8μm, 0.45μm and 0.22μm filter membranes to obtain the final hormone-free serum.

[0052] Example 3

[0053] A method for preparing hormone-free serum includes the following steps:

[0054] 1. Take 15000g of fetal bovine serum, centrifuge for 30 minutes, and collect the supernatant.

[0055] 2. Add 0.1% sodium salicylate protein dissociation agent (based on the mass of fetal bovine serum) and stir for 40 minutes.

[0056] 3. Add serum to a pre-treated cellulose dialysis bag (3000D). Seal both ends of the dialysis bag and place it into the dialysis solution (pH 7.40, Na+, etc.) for dialysis. +The dialysis solution consisted of 10 mmol / L sodium carbonate-sodium bicarbonate buffer, 4 mmol / L potassium chloride, 2 mmol / L calcium chloride, 0.75 mmol / L magnesium chloride, and 120 mmol / L sodium chloride. The dialysis time was 24 h, and the dialysate was changed and the dialysis was repeated twice to obtain hormone-free serum.

[0057] 4. Add sodium azide to the hormone-free serum at a mass of 0.05% of the hormone-free serum mass.

[0058] 5. The serum obtained in step 4 is filtered sequentially through 0.8μm, 0.45μm and 0.22μm filter membranes to obtain the final hormone-free serum.

[0059] Example 4

[0060] A method for preparing hormone-free serum includes the following steps:

[0061] 1. Take 15000g of fetal bovine serum, centrifuge for 30 minutes, and collect the supernatant.

[0062] 2. Add 0.1% sodium salicylate protein dissociation agent (based on the mass of fetal bovine serum) and stir for 40 minutes.

[0063] 3. Add serum to a pre-treated cellulose dialysis bag (5000D). Seal both ends of the dialysis bag and place it into the dialysis solution (pH 7.40, Na+, etc.) for dialysis. + The dialysis solution consisted of 100 mmol / L sodium carbonate-sodium bicarbonate buffer, 4 mmol / L potassium chloride, 2 mmol / L calcium chloride, 0.75 mmol / L magnesium chloride, and 120 mmol / L sodium chloride. The dialysis time was 24 h, and the dialysate was changed and the dialysis was repeated twice to obtain hormone-free serum.

[0064] 4. Add sodium azide to the hormone-free serum at a mass of 0.05% of the hormone-free serum mass.

[0065] 5. The serum obtained in step 4 is filtered sequentially through 0.8μm, 0.45μm and 0.22μm filter membranes to obtain the final hormone-free serum.

[0066] Example 5

[0067] A method for preparing hormone-free serum includes the following steps:

[0068] 1. Take 15000g of fetal bovine serum, centrifuge for 30 minutes, and collect the supernatant.

[0069] 2. Add 0.1% of 8-phenylamino-1-naphthalenesulfonate ammonium salt protein dissociation agent (based on the mass of fetal bovine serum) and stir for 40 min.

[0070] 3. Add serum to a pre-treated cellulose dialysis bag (3000D). Seal both ends of the dialysis bag and place it into the dialysis solution (pH 7.40, Na+, etc.) for dialysis. + The dialysis was performed using a sodium carbonate-bicarbonate buffer solution with a concentration of 20 mmol / L, potassium chloride 4 mmol / L, calcium chloride 2 mmol / L, magnesium chloride 0.75 mmol / L, and sodium chloride 120 mmol / L for 24 hours. The dialysis was repeated twice with the dialysis solution changed to obtain hormone-free serum.

[0071] 4. Add sodium azide to the hormone-free serum at a mass of 0.05% of the hormone-free serum mass.

[0072] 5. The serum obtained in step 4 is filtered sequentially through 0.8μm, 0.45μm and 0.22μm filter membranes to obtain the final hormone-free serum.

[0073] Example 6

[0074] A method for preparing hormone-free serum includes the following steps:

[0075] 1. Take 15000g of fetal bovine serum, centrifuge for 30 minutes, and collect the supernatant.

[0076] 2. Add 0.1% sodium salicylate protein dissociation agent (based on the mass of fetal bovine serum) and stir for 40 minutes.

[0077] 3. Add serum to a pre-treated cellulose dialysis bag (1000D). Seal both ends of the dialysis bag and place it into the dialysis solution (pH 7.40, Na+, etc.) for dialysis. + The dialysis was performed using a sodium carbonate-bicarbonate buffer solution with a concentration of 20 mmol / L, potassium chloride 4 mmol / L, calcium chloride 2 mmol / L, magnesium chloride 0.75 mmol / L, and sodium chloride 120 mmol / L for 24 hours. The dialysis was repeated twice with the dialysis solution changed to obtain hormone-free serum.

[0078] 4. Add sodium azide to the hormone-free serum at a mass of 0.05% of the hormone-free serum mass.

[0079] 5. The serum obtained in step 4 is filtered sequentially through 0.8μm, 0.45μm and 0.22μm filter membranes to obtain the final hormone-free serum.

[0080] Test Example 1

[0081] Hormone concentrations in the dehorned serum prepared in Examples 1–6 were determined using the following kits: Roche's estradiol assay kit (electrochemiluminescence method) Elecsys Estradiol III, testosterone assay kit (electrochemiluminescence method) Elecsys Testosterone II, progesterone assay kit (electrochemiluminescence method) Elecsys Progesterone III, free thyroxine assay kit (electrochemiluminescence method) Elecsys FT4 III, thyroxine assay kit (electrochemiluminescence method) Elecsys T4, free triiodothyronine assay kit (electrochemiluminescence method) Elecsys FT3 III, and triiodothyronine assay kit (electrochemiluminescence method) Elecsys T3. The results are shown in Table 1.

[0082] Using commercially available serum that removes small molecule hormones using existing activated carbon adsorption technology as a comparative example, the hormone-removing serum was purchased from scantibodies, product code 3SH009.

[0083] Table 1: Small molecule hormone residues in the hormone-free serum obtained in Examples 1-6

[0084]

[0085] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing hormone-free serum, characterized in that, The process includes the following: Human or animal serum is centrifuged and the supernatant is collected. A protein dissociation agent is added to the supernatant to react and dissociate the small molecule hormone bound to the protein, thereby obtaining an intermediate sample; the protein dissociation agent includes ammonium 8-phenylamino-1-naphthalenesulfonate and / or sodium salicylate, and the mass of the protein dissociation agent is 0.01% to 1% of the mass of the serum; The intermediate sample was subjected to dialysis to remove small molecule hormones, resulting in hormone-free serum. The dialysis separation uses dialysis bags with a density of 1000D to 5000D. The dialysis solution used for the dialysis separation includes a pH of 6.8-7.5 and Na+. + Sodium carbonate-sodium bicarbonate buffer solutions with concentrations of 10 mmol / L to 100 mmol / L, and potassium chloride (0.1 mmol / L to 4 mmol / L), calcium chloride (0.5 mmol / L to 4 mmol / L), magnesium chloride (0.5 mmol / L to 2 mmol / L), and sodium chloride (50 mmol / L to 150 mmol / L).

2. The method for preparing hormone-free serum according to claim 1, characterized in that, The dialysis separation includes the following process: the intermediate sample is placed into the dialysis bag, the dialysis bag is placed in the dialysis solution for dialysis for 12h to 24h, the dialysis solution is replaced, and the dialysis is repeated 2 to 10 times.

3. The method for preparing hormone-free serum according to claim 1, characterized in that, Also includes: The hormone-free serum was subjected to preservation and sterilization treatment.

4. The method for preparing hormone-free serum according to claim 3, characterized in that, The corrosion protection treatment includes the following processes: Add 0.05% to 0.2% of a preservative, comprising sodium azide and / or ProClin 300, to the hormone-free serum.

5. The method for preparing hormone-free serum according to claim 3, characterized in that, The sterilization process includes the following steps: The hormone-free serum was filtered sequentially through a series of filter membranes with decreasing pore size gradients.

6. A test kit, characterized in that, This includes hormone-free serum prepared by the preparation method described in any one of claims 1 to 5.

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