A non-specific agglutination inhibitor removing reagent and a method for preparing the same

Abstract

The application discloses a non-specific agglutination inhibitor removing reagent and a preparation method thereof. The method comprises the following steps: (1) adding kaolin and montmorillonite powder into acid liquid respectively, uniformly mixing, standing, and removing supernatant; (2) adding the same amount of acid liquid as that in step (1) into the product obtained in step (1), uniformly mixing, standing, and removing supernatant; (3) adding buffer liquid into the product obtained in step (2), and then high-pressure sterilizing; (4) after the sterilized product is cooled to 2-8 DEG C, adding trypsin solution, and the non-specific agglutination inhibitor removing reagent is prepared by compounding kaolin, montmorillonite powder and porcine trypsin, and the non-specific agglutination inhibitor removing reagent can effectively remove non-specific agglutination inhibitors in serum. After the serum is treated by the non-specific agglutination inhibitor removing reagent, the hemagglutination inhibition antibody titer of the treated serum is equivalent to that of the serum treated by receptor blocking enzyme.

Description

Technical Field

[0001] This invention belongs to the technical field of diagnostic toxicology products, specifically relating to a non-specific agglutination inhibitor removal reagent and its preparation method. Background Technology

[0002] The hemagglutination inhibition (HI) test is the most commonly used serological method for assessing infection status and detecting antibody levels for various viruses (such as swine influenza virus, Newcastle disease virus, and swine parvovirus). Non-specific agglutination factors and non-specific agglutination inhibitors exist in the sera of many animals (pigs, guinea pigs, chickens, and rabbits). If these factors are not removed, they can cause deviations in HI results, leading to false negatives (low antibody titers) or false positives (high antibody titers). Therefore, certain animal sera require pretreatment before the HI test.

[0003] The commonly used reagent for removing nonspecific agglutination inhibitors is 25% kaolin. However, kaolin used according to conventional methods cannot effectively remove nonspecific agglutination inhibitors in the hemagglutination inhibition test or can cause instability due to the removal of nonspecific agglutination inhibitors, resulting in high HI antibody titers or unstable HI titers.

[0004] As our understanding of nonspecific agglutination inhibitors deepens, we have discovered that receptor-blocking enzymes can effectively remove these inhibitors from serum. However, the high cost of receptor-blocking enzymes necessitates the development of a cheaper reagent for removing nonspecific agglutination inhibitors. Summary of the Invention

[0005] To address the aforementioned shortcomings in the existing technology, this invention provides a non-specific agglutination inhibitor removal reagent and its preparation method, which can ensure the stability of the results while maintaining the HI antibody titer.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0007] A method for preparing a nonspecific agglutination inhibitor removal reagent includes the following steps:

[0008] (1) Add kaolin and montmorillonite powder to the acid solution respectively, mix evenly, let stand, and remove the supernatant; the final concentration of kaolin in the acid solution is 0.01-0.1 g / mL, and the final concentration of montmorillonite powder in the acid solution is 0.01-0.1 g / mL.

[0009] (2) Add the same amount of acid solution as used in step (1) to the product obtained in step (1), mix well and let stand, and remove the supernatant; repeat the above process 2 to 3 times.

[0010] (3) Add buffer solution to the product obtained in step (2) and adjust its pH value to 7-9, and then autoclave at 100-130℃ for 30-50 min;

[0011] (4) After the sterilized product has cooled to 2-8°C, add trypsin solution and stir at 2-8°C for 30-60 minutes.

[0012] The technical principle of this invention is as follows:

[0013] Montmorillonite powder has strong ion exchange capacity and adsorption properties; after absorbing water, it expands in volume and becomes colloidal. Kaolin has weak ion exchange capacity, good adsorption properties, and exhibits suspension and dispersibility in water. Furthermore, a trypsin-serine proteolytic enzyme was extracted from pig pancreas. This enzyme can cleave the carboxyl groups of lysine and arginine residues in the receptor polypeptide chain, thereby blocking the receptor's role in hemagglutination inhibition.

[0014] This invention utilizes the complementary ion exchange capacity and dual adsorption properties of montmorillonite powder and kaolin, the gelling properties of montmorillonite powder and the suspension properties of kaolin, and the property of trypsin to cleave the carboxyl groups of lysine and arginine in the receptor, to formulate a non-specific aggregation inhibitor removal reagent.

[0015] Furthermore, the acid solution is dilute hydrochloric acid with a concentration of 1–3 mol / L.

[0016] Furthermore, the final concentration of kaolin in the acid solution was 0.03 g / mL, and the final concentration of montmorillonite powder was 0.02 g / mL.

[0017] Furthermore, the buffer is PBS buffer, which comprises the following components at final concentrations:

[0018] 0.001~0.01g / mL NaCl, 0.001~0.005g / mL Na2HPO4, 0.0001~0.001g / mL KCl and 0.001~0.005g / mL KH2PO4.

[0019] Furthermore, the pH value was adjusted to 7.2 using a sodium hydroxide solution with a concentration of 2–5 mol / L.

[0020] Furthermore, the sterilization temperature is 121℃.

[0021] Furthermore, the volume concentration of the trypsin solution is 1-5%, and the amount added is 0.5-1% of the volume of the sterilized product.

[0022] Furthermore, the volume concentration of the trypsin solution is 1%.

[0023] Furthermore, the trypsin solution used is porcine trypsin.

[0024] Furthermore, trypsin is a serine proteolytic enzyme.

[0025] A non-specific agglutination inhibitor removal reagent is prepared using the method described above.

[0026] The beneficial effects of this invention are:

[0027] This invention utilizes a composite adsorbent of kaolin, montmorillonite powder, and porcine trypsin to prepare a nonspecific agglutination factor removal reagent, which can effectively remove nonspecific agglutination inhibitors from serum. After treatment with this reagent, the hemagglutination inhibition antibody titer of the treated serum is comparable to that of serum treated with receptor-blocking enzymes. Detailed Implementation

[0028] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0029] Example 1

[0030] A non-specific agglutination inhibitor removal reagent, the preparation method of which is as follows:

[0031] (1) Dissolve 8g NaCl, 1.44g Na2HPO4, 0.2g KCl and 0.2g KH2PO4 in 800mL of deionized water. After dissolving completely, add deionized water to make up to 1000mL to prepare PBS buffer.

[0032] (2) Take 43 mL of 36% concentrated hydrochloric acid and add it to 457 mL of double-distilled water to prepare a 1 mol / L dilute hydrochloric acid solution;

[0033] (3) Weigh 40g of NaOH and add it to 200mL of double-distilled water to prepare a 5mol / L sodium hydroxide solution;

[0034] (4) Weigh 1g of porcine trypsin and add it to 100mL of 0.01mol / L PBS.

[0035] (5) Take one 500 mL of 1 mol / L dilute hydrochloric acid solution, add 15 g of kaolin and 10 g of montmorillonite powder to each part of the dilute hydrochloric acid solution, mix well and let stand. After sedimentation, carefully remove the supernatant. Add another 500 mL of 1 mol / L dilute hydrochloric acid solution, mix well and let stand. After sedimentation, carefully remove the supernatant. Repeat twice.

[0036] (6) Add 500 mL of PBS buffer to the precipitate, mix well and let stand. After sedimentation, remove the supernatant and repeat 3 times.

[0037] (7) After the third removal of PBS buffer, the pH was adjusted to 7.2 with 5 mol / L sodium hydroxide solution.

[0038] (8) Add an appropriate amount of PBS buffer to bring the volume up to 100 mL, then autoclave at 121 °C for 30 min and cool to 5 °C.

[0039] (9) Add 1 mL of 1% trypsin and stir with a magnetic stirrer at 4°C for 30 min to obtain the non-specific agglutination inhibitor removal reagent for the hemagglutination inhibition test.

[0040] Example 2

[0041] A non-specific agglutination inhibitor removal reagent, the preparation method of which is as follows:

[0042] (1) Dissolve 8g NaCl, 1.44g Na2HPO4, 0.2g KCl and 0.2g KH2PO4 in 800mL of deionized water. After dissolving completely, add deionized water to make up to 1000mL to prepare PBS buffer.

[0043] (2) Take 43 mL of 36% concentrated hydrochloric acid and add it to 457 mL of double-distilled water to prepare a 1 mol / L dilute hydrochloric acid solution;

[0044] (3) Weigh 40g of NaOH and add it to 200mL of double-distilled water to prepare a 5mol / L sodium hydroxide solution;

[0045] (4) Weigh 1g of porcine trypsin and add it to 100mL of 0.01mol / L PBS.

[0046] (5) Take one 500 mL of 1 mol / L dilute hydrochloric acid solution, add 20 g of kaolin and 15 g of montmorillonite powder to each part of the dilute hydrochloric acid solution, mix well and let stand. After sedimentation, carefully remove the supernatant. Add another 500 mL of 1 mol / L dilute hydrochloric acid solution, mix well and let stand. After sedimentation, carefully remove the supernatant. Repeat twice.

[0047] (6) Add 500 mL of PBS buffer to the precipitate, mix well and let stand. After sedimentation, remove the supernatant and repeat 3 times.

[0048] (7) After the third removal of PBS buffer, the pH was adjusted to 7.2 with 5 mol / L sodium hydroxide solution.

[0049] (8) Add an appropriate amount of PBS buffer, bring the volume to 100 mL, then autoclave at 121 °C for 30 min and cool to 4 °C.

[0050] (9) Add 2 mL of 1% trypsin and stir at 4°C with a magnetic stirrer for 30 min to obtain the non-specific agglutination inhibitor removal reagent for the hemagglutination inhibition test.

[0051] Example 3

[0052] A non-specific agglutination inhibitor removal reagent, the preparation method of which is as follows:

[0053] (1) Dissolve 8g NaCl, 1.44g Na2HPO4, 0.2g KCl and 0.2g KH2PO4 in 800mL of deionized water. After dissolving completely, add deionized water to make up to 1000mL to prepare PBS buffer.

[0054] (2) Take 43 mL of 36% concentrated hydrochloric acid and add it to 457 mL of double-distilled water to prepare a 1 mol / L dilute hydrochloric acid solution;

[0055] (3) Weigh 40g of NaOH and add it to 200mL of double-distilled water to prepare a 5mol / L sodium hydroxide solution;

[0056] (4) Weigh 1g of porcine trypsin and add it to 100mL of 0.01mol / L PBS.

[0057] (5) Take one 500 mL of 1 mol / L dilute hydrochloric acid solution, add 10 g of kaolin and 10 g of montmorillonite powder to each part of the dilute hydrochloric acid solution, mix well and let stand. After sedimentation, carefully remove the supernatant. Add another 500 mL of 1 mol / L dilute hydrochloric acid solution, mix well and let stand. After sedimentation, carefully remove the supernatant. Repeat twice.

[0058] (6) Add 500 mL of PBS buffer to the precipitate, mix well and let stand. After sedimentation, remove the supernatant and repeat 3 times.

[0059] (7) After the third removal of PBS buffer, the pH was adjusted to 7.2 with 5 mol / L sodium hydroxide solution.

[0060] (8) Add an appropriate amount of PBS buffer, bring the volume to 100 mL, then autoclave at 121 °C for 30 min and cool to 5 °C.

[0061] (9) Add 3 mL of 1% trypsin and stir with a magnetic stirrer at 5°C for 40 min to obtain the non-specific agglutination inhibitor removal reagent for the hemagglutination inhibition test.

[0062] Experimental Example

[0063] The properties and efficacy of the nonspecific agglutination inhibitor removal reagent prepared in Example 1 were evaluated as follows:

[0064] 1. In terms of properties, the non-specific agglutination inhibitor removal reagent prepared with 25% kaolin and 25% montmorillonite powder was compared to determine whether it could form a colloid and its stability as a colloid, as well as the precipitation time after being added to PBS and shaken. Serum from guinea pigs untreated with porcine parvovirus inactivated vaccine and guinea pigs vaccinated with porcine parvovirus inactivated vaccine were treated with the non-specific agglutination inhibitor removal reagent and compared with serum not treated with the non-specific agglutination inhibitor removal reagent. The results of the property evaluation are shown in Table 1.

[0065] Table 1. Trait Assessment

[0066]

[0067]

[0068] As shown in Table 1, the suspension properties of kaolin combined with the gelling properties of montmorillonite powder and the ability of porcine trypsin to cleave the carboxyl side of lysine and arginine residues in the receptor polypeptide chain make the prepared non-specific aggregation inhibitor removal reagent more stable in colloids. It settles more slowly when added to PBS, which is more conducive to fully binding with some non-specific factors in the solution and degrading the non-specific factors.

[0069] 2. Detection of ability to remove non-specific agglutination inhibitors

[0070] (1) The serum was placed in a 56°C constant temperature water bath for 30 minutes.

[0071] (2) Add 0.1 mL serum + 0.3 mL PBS, add 0.4 mL non-specific agglutination inhibitor removal reagent or 25% kaolin, incubate at room temperature for 20-30 minutes, then centrifuge at 10000 r / min for 10 minutes, take 350 μL of supernatant, then add 50 μL of 1% guinea pig red blood cells and shake well, incubate at room temperature for about 30 minutes, then centrifuge at 10000 r / min for 10 minutes, and take the supernatant as the serum diluted 1:8.

[0072] (3) Determination of porcine parvovirus hemagglutination titer (HA)

[0073] The hemagglutination titer of the virus to be tested is the highest viral dilution that causes 100% agglutination of red blood cells.

[0074] (4) Dilute the virus solution used to determine the HA titer to 4HA / 50μL.

[0075] (5) HI test

[0076] Add 50 μL of PBS to wells 2-11 of a 96-well V-type microplate, and 100 μL of PBS to well 12. Add 50 μL of serum to well 1 (if well 1 is positive, the HI titer of the serum is 1:8), and another 50 μL of serum to well 2. Mix thoroughly in well 2, then add 50 μL to well 3. Serially dilute up to well 10, discarding 50 μL from well 10. Add 50 μL of viral antigen solution containing 4 HA units to wells 1-11 and incubate at 20-25°C for 60 minutes. Add 50 μL of guinea pig red blood cell suspension to each well 1-12, mix gently, and incubate at 20-25°C for approximately 60-120 minutes. Control red blood cells will settle to the bottom of the wells in a button-like pattern. The highest dilution of serum that completely inhibits the four hemagglutination unit antigens was used as the hemagglutination inhibition (HI) titer of the tested serum. The test results are shown in Tables 2-4.

[0077] Table 2 Serum HI antibody titers after treatment with reagents for removing nonspecific agglutination inhibitors.

[0078]

[0079]

[0080] Table 3 Serum HI antibody titers after treatment with reagents for removing nonspecific agglutination inhibitors.

[0081]

[0082]

[0083] Table 4. HI antibody titers in serum samples treated with nonspecific agglutination inhibitor removal reagent at different time points.

[0084]

[0085] As shown in Tables 2-4, treating guinea pig serum with a reagent for removing nonspecific agglutination inhibitors can effectively remove nonspecific agglutination inhibitors.

Claims

1. A method for preparing a non-specific agglutination inhibitor removing reagent, characterized by, Includes the following steps: (1) Add kaolin and montmorillonite powder to the acid solution respectively, mix evenly, let stand, and remove the supernatant; the final concentration of kaolin in the acid solution is 0.01~0.1g / mL, and the final concentration of montmorillonite powder in the acid solution is 0.01~0.1g / mL. (2) Add the same amount of acid solution as in step (1) to the product obtained in step (1), mix well and let stand, and remove the supernatant; repeat the above process 2 to 3 times. (3) Add buffer solution to the product obtained in step (2) and adjust its pH value to 7~9, and then sterilize at 100~130℃ for 30~50min; (4) After the sterilized product has cooled to 2~8℃, add trypsin solution and stir at 2~8℃ for 30~60min; the volume concentration of the trypsin solution is 1~5%, and the amount added is 0.5~1% of the volume of the sterilized product.

2. The preparation method according to claim 1, characterized in that, The acid solution is dilute hydrochloric acid with a concentration of 1~3 mol / L.

3. The preparation method according to claim 1, characterized in that, The final concentration of kaolin in the acid solution is 0.03 g / mL, and the final concentration of montmorillonite powder is 0.02 g / mL.

4. The preparation method according to claim 1, characterized in that, The buffer solution is PBS buffer, which comprises the following components at final concentrations: 0.001~0.01 g / mL NaCl, 0.001~0.005 g / mL Na2HPO4, 0.0001~0.001 g / mL KCl, and 0.001~0.005 g / mL KH2PO4.

5. The preparation method according to claim 1, characterized in that, The pH value was adjusted to 7.2 using a sodium hydroxide solution with a concentration of 2-5 mol / L.

6. The preparation method according to claim 1, characterized in that, The sterilization temperature is 121°C.

7. The preparation method according to claim 1, characterized in that, The volume concentration of the trypsin solution is 1%.

8. The preparation method according to claim 1, characterized in that, The trypsin solution used is porcine trypsin.

9. A reagent for removing non-specific agglutination inhibitors, characterized in that, It is prepared by the method described in any one of claims 1 to 8.

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