Method for purifying rabbit serum against pneumococcal polysaccharide based on polysaccharide conjugate and tt protein purified liquid

Abstract

The present application relates to the biomedical technology field, provide a kind of based on polysaccharide and carrier protein purification liquid's anti-pneumococcal polysaccharide rabbit serum purification method, including the best dilution multiple of the to-be-purified anti-pneumococcal rabbit serum is determined by rate turbidimetry, carries out pre-absorption specificity detection, and the serum after dilution is reacted with blank / positive / negative control respectively to judge whether cross exists, if exists, determine cross type, lock cross type by monotype detection and add T test, and join the same type polysaccharide conjugate and TT protein purification liquid.In the present application, the antiserum uses polysaccharide conjugate as immunogen, and the carrier protein combined with polysaccharide has certain immunogenicity, can induce the body to produce certain antibody, therefore, the present application uses polysaccharide conjugate and TT protein purification liquid to carry out absorption purification simultaneously, can effectively remove cross type antibody and carrier antibody, reach the purpose of high-efficiency purification.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for purifying rabbit serum containing anti-pneumococcal capsular polysaccharide based on polysaccharide conjugates and TT protein purification solution. Background Technology

[0002] The current Chinese Pharmacopoeia requires the use of rabbit serum to detect the polysaccharide content of 23-valent pneumococcal vaccines, but imported serum is expensive and time-consuming. Traditional bacterial suspension adsorption methods introduce impurities and pose high biosafety risks, while gel filtration / affinity chromatography is complex and costly. The shortcomings of existing technologies include:

[0003] 1. Introducing impurities (such as bacterial lipopolysaccharides) through bacterial suspension adsorption.

[0004] 2. Chromatography takes >48 hours, and antibody recovery rate <70%;

[0005] 3. Carrier proteins (such as TT)-induced heterologous antibodies cannot be effectively removed.

[0006] Therefore, based on the above problems, a method for purifying rabbit serum containing anti-pneumococcal capsular polysaccharide based on polysaccharide conjugates and TT protein purification solution is proposed. Summary of the Invention

[0007] The present invention aims to develop a rapid, efficient, stable and simple method for purifying rabbit serum containing anti-pneumococcal capsular polysaccharides, which can both rapidly purify serum and save costs.

[0008] This invention is achieved through the following technical solution: a method for purifying rabbit serum containing anti-pneumococcal capsular polysaccharide based on polysaccharide conjugates and TT protein purification solution, comprising the following specific operational steps:

[0009] S1. Determination of serum dilution factor: A standard curve was plotted using the rate turbidimetric method to determine the optimal serum dilution factor;

[0010] S2. Pre-absorption specificity test: The diluted serum was tested with blank control, positive control and negative control to determine whether there was cross-reaction;

[0011] The blank control was a diluent, the positive control was a mixture of pneumococcal capsular polysaccharides, and the negative control was a mixture of polysaccharides other than the target type.

[0012] S3. Cross-type determination: For serum with cross-reactivity, it is tested with polysaccharide solutions of other types besides this type and TT protein purification solution. The cross-type and vector are determined by T test.

[0013] S4. Absorption treatment: Add pneumococcal polysaccharide stock solution of the same cross-type and an appropriate amount of TT protein purification solution to the serum for absorption treatment;

[0014] S5. Post-absorption specificity test: Repeat S2 on the post-absorption serum to verify whether the cross-reactivity has been eliminated;

[0015] S6. Ultrafiltration Concentration: Concentrate qualified serum using an ultrafiltration tube;

[0016] S7. Quality Assay: The antibody level, specificity, and linearity of concentrated serum were determined using a rate turbidimetric method.

[0017] S8. Aliquoting and Storage: Aliquot the qualified serum and store it at low temperature.

[0018] Preferably, the detections described in S2, S3, and S7 are performed on a specific protein analyzer.

[0019] Preferably, in step S2, the presence of serum crossover is determined by calculating the serum crossover rate and the P-value, using the following specific formula:

[0020] Serum crossover rate = (Mean negative control value - Blank control value) / (Mean positive control value - Blank control value) * 100%

[0021] Judgment criteria: Cross-reactivity exists: serum cross-reactivity rate > 5%, P-value (T-test) < 0.05;

[0022] No cross-reactivity: serum cross-reactivity rate < 5%, P value (T test) > 0.05.

[0023] Preferably, the amount of polysaccharide conjugate stock solution added in S4 is 0.1-0.5 mg / mL serum, and the amount of TT protein purification solution added is 1 mg / mL serum.

[0024] Preferably, the absorption process described in S4 includes:

[0025] S41. Mix the serum and polysaccharide conjugate stock solution thoroughly;

[0026] S42. Incubate the mixture in a constant temperature water bath;

[0027] S43. Centrifuge the mixture, discard the precipitate, and collect the supernatant;

[0028] S44. Filter the supernatant using a filter membrane.

[0029] Preferably, the constant temperature water bath temperature in S42 is 37°C and the incubation time is 30 min; the centrifugation conditions in S43 are 10000 r / min and centrifugation for 15 min.

[0030] The S44 uses a 0.22μm filter for filtration.

[0031] Preferably, the pneumococcal polysaccharide conjugate in S4 is a covalently bound capsule polysaccharide and a carrier protein. Tetanus toxoid (TT) is preferred as the carrier protein. Other optional carrier proteins include diphtheria toxoid (DT), CRM197, etc., but TT is the best choice because it has stable immunogenicity and good synergy with TT protein purification solution.

[0032] This invention provides a method for purifying rabbit serum containing anti-pneumococcal capsular polysaccharides based on polysaccharide conjugates and TT protein purification solution. It has the following beneficial effects:

[0033] 1. This antiserum uses a polysaccharide conjugate as an immunogen. The carrier protein bound to the polysaccharide has a certain immunogenicity and can induce the body to produce certain antibodies. Therefore, this invention uses the polysaccharide conjugate and TT protein purification solution for simultaneous absorption and purification, which can effectively remove cross-type antibodies and carrier antibodies, achieving the purpose of high-efficiency purification.

[0034] 2. This invention uses rate turbidimetry to determine serum cross-linking, which provides more objective and accurate results compared to traditional slide agglutination.

[0035] 3. This invention is quick and easy to operate, saves costs, and is conducive to large-scale production. Attached Figure Description

[0036] Figure 1 This is an overall flowchart of the present invention;

[0037] Figure 2 This is a flowchart of the targeted absorption and purification process in this invention. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0039] The rabbit serum used in this invention is derived from New Zealand white rabbits. Pneumococcal capsular polysaccharide conjugate is used as an immunogen. After immunization according to the conventional immunization program (French adjuvant emulsification, booster immunization every 2 weeks, for a total of 3-4 times), blood is collected from the heart to obtain the serum.

[0040] The TT protein purification solution used in this invention is prepared according to the above method, or it is prepared by using commercially available tetanus toxoid protein (such as Sigma-Aldrich) in 0.01M PBS (pH 7.2) to a stock solution concentration of 10 mg / mL.

[0041] The specific protein analyzer used in this invention is the Beckman Coulter IMMAGE 800 or an equivalent device.

[0042] The method for determining the optimal dilution factor of serum described in this invention is as follows: after serially diluting the serum, react it with the corresponding type polysaccharide antigen on a specific protein analyzer, and select the dilution factor with the reaction value at the midpoint of the linear range (300-500 mg / dL) as the optimal dilution factor.

[0043] The positive control described in this invention is a mixture of polysaccharides from the capsular region of 23-valent pneumococci (final concentration of 1 μg / mL for each type), and the negative control is a mixture of polysaccharides from the remaining 22 types excluding the target type (final concentration of 1 μg / mL for each type).

[0044] The ultrafiltration tube described in this invention has a molecular weight cutoff of 100 kDa.

[0045] Example 1

[0046] Serum cross-type determination

[0047] (1) Mixed cross-detection: Pneumococcal serum was diluted to the optimal dilution factor and then tested separately with a blank control (diluent), a positive control, and a negative control (a mixture of polysaccharides from 23 serotypes other than this serotype) on a specific protein analyzer to determine whether cross-contamination occurred. The cross-contamination rate and P-value (T-test) were calculated to determine whether cross-contamination occurred. The specific details are as follows:

[0048] Serum crossover rate = (Mean negative control value - Blank control value) / (Mean positive control value - Blank control value) × 100%

[0049] P-value: T-test, P < 0.05, the difference is statistically significant, indicating cross-reactivity.

[0050] Judgment criteria: Cross-reactivity present: serum cross-reactivity rate > 5%, P value (T test) < 0.05; No cross-reactivity: serum cross-reactivity rate < 5%, P value (T test) > 0.05.

[0051] (2) Single-type cross-reaction detection: For serums that are found to have cross-reaction, the rate values ​​of polysaccharide solutions of 23 other types (excluding the original type) and TT protein purification solution are measured on a specific protein analyzer. The cross-type is determined by calculating the P value.

[0052] Example 2

[0053] Absorption test

[0054] Based on the results of the cross-reaction, take 1 mL of the target serum and add 0.1-0.5 mg of monopolysaccharide conjugate stock solution of the same type as the cross-reaction and 1 mg of TT protein purification solution (i.e., add 0.1 mL of 10 mg / mL stock solution).

[0055] After mixing, place the mixture in a constant temperature water bath and incubate. Centrifuge at 10,000 r / min for 15 min, discard the precipitate, and collect the supernatant.

[0056] Add the filtrate into a 100kD ultrafiltration tube in multiple portions and centrifuge at 10000 r / min for 15 min.

[0057] Discard the filtrate, gently blow and mix the liquid in the filter tube, then recover it, filter it using a 0.22μm filter, and transfer it to a new centrifuge tube. Store at -80℃ and temporarily store at 4℃.

[0058] The polysaccharide conjugate stock solution was added at a concentration of 0.1-0.5 mg / mL serum, and the TT protein purification solution was added at a concentration of 1 mg / mL serum. These concentrations were determined based on the following preliminary experimental data:

[0059] Experimental Design: Cross-reactive rabbit serum (targeting pneumococcal serotype 4, showing cross-reactivity with serotype 9V) was collected and divided into 6 groups. Group 1 was the control group without absorbent; Groups 2, 3, and 4 were treated with 0.1, 0.3, and 0.5 mg / mL of serotype 9V polysaccharide-TT conjugate, respectively; Group 5 was treated with 1 mg / mL TT protein purification solution; and Group 6 was treated with a combination of 0.3 mg / mL polysaccharide conjugate and 1 mg / mL TT protein purification solution. After absorption treatment, the cross-reactivity rate of serum with serotype 9V was measured.

[0060] The results showed that when the amount of polysaccharide conjugate was 0.3 mg / mL, the cross-reactivity rate decreased to 8.7%, and further decreased to 4.1% (below the critical value of 5%) after combining with TT protein, indicating that TT protein can effectively remove anti-carrier protein antibodies and synergistically enhance the absorption effect.

[0061] Therefore, the preferred dosage is 0.3 mg / mL of polysaccharide conjugate and 1 mg / mL of TT protein in serum. This ratio is based on the balance of antigen-antibody reactions, ensuring an excess of antigen to bind all impurities.

[0062] Example 3

[0063] To verify the synergistic effect of the TT protein purification solution, the following comparative examples were designed:

[0064] Polysaccharide absorption alone vs. polysaccharide + TT in combination

[0065] Serum sample: Rabbit anti-pneumococcal serotype 19F serum, which showed cross-reactivity with serotype 19A (cross-reactivity rate 18.5%).

[0066] Group treatment: Group A was given 0.3 mg / mL of type 19A polysaccharide-TT conjugate alone; Group B was given 1 mg / mL of TT protein purification solution alone; Group C was given both 0.3 mg / mL of type 19A polysaccharide-TT conjugate and 1 mg / mL of TT protein purification solution; Group D was the control group without absorption treatment.

[0067] Absorption method: Same as step S4 of the present invention, incubate at 37°C for 30 minutes, then centrifuge and filter.

[0068] Detection indicators: cross-reactivity rate and type 19F antibody recovery rate.

[0069] The results showed that: Group A (polysaccharide alone) had a cross-reactivity rate of 9.2% and an antibody recovery rate of 82.3%; Group B (TT protein alone) had a cross-reactivity rate of 15.6% and an antibody recovery rate of 94.1%; Group C (combined use) had the lowest cross-reactivity rate of 3.8% and an antibody recovery rate of 91.5%; and Group D (control) had a cross-reactivity rate of 18.5%.

[0070] Conclusion: Both polysaccharide absorption and TT protein alone can partially reduce cross-reactivity, but the combined use (group C) resulted in the lowest cross-reactivity rate, higher antibody recovery rate, and lowest total protein residue, demonstrating a significant synergistic effect.

[0071] Using TT protein alone primarily removes anti-carrier antibodies, but it is insufficient for removing cross-linked polysaccharide antibodies; polysaccharide uptake alone may be inefficient due to interference from carrier proteins. The combined approach can simultaneously target both polysaccharides and carrier contaminants, achieving highly efficient purification.

[0072] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for purifying rabbit serum containing anti-pneumococcal capsular polysaccharide based on polysaccharide conjugates and TT protein purification solution, characterized in that: The specific operating steps are as follows: S1. Determination of serum dilution factor: A standard curve was plotted using the rate turbidimetric method to determine the optimal serum dilution factor; S2. Pre-absorption specificity test: The diluted serum was tested with blank control, positive control and negative control to determine whether there was cross-reaction; The blank control was a diluent, the positive control was a mixture of pneumococcal capsular polysaccharides, and the negative control was a mixture of polysaccharides other than the target type. S3. Cross-type determination: For serum with cross-reactivity, it is tested with polysaccharide solutions of other types besides this type and TT protein purification solution. The cross-type and vector are determined by T test. S4. Absorption treatment: Add the same pneumococcal polysaccharide conjugate stock solution and an appropriate amount of TT protein purification solution to the serum for absorption treatment; S5. Post-absorption specificity test: Repeat S2 on the post-absorption serum to verify whether the cross-reactivity has been eliminated; S6. Ultrafiltration Concentration: Concentrate non-crosslinked serum using an ultrafiltration tube; S7. Quality testing: The antibody level, specificity, and linearity of concentrated serum were detected using rate turbidimetry. S8. Aliquoting and Storage: Aliquot the qualified serum and store it at low temperature.

2. The method for purifying rabbit serum containing anti-pneumococcal capsular polysaccharide based on polysaccharide conjugate and TT protein purification solution according to claim 1, characterized in that: The detections described in S2, S3, and S7 are performed on a specific protein analyzer.

3. The method for purifying rabbit serum containing anti-pneumococcal capsular polysaccharide based on polysaccharide conjugates and TT protein purification solution according to claim 1, characterized in that: In step S2, the presence of serum crossover is determined by calculating the serum crossover rate and the P-value. The specific formula is as follows: Serum crossover rate = (Mean negative control value - Blank control value) / (Mean positive control value - Blank control value) * 100% Judgment criteria: Cross-reactivity exists: serum cross-reactivity rate > 5%, P-value (T-test) < 0.05; No cross-reactivity: serum cross-reactivity < 5%, P value (T test) > 0.

05.

4. The method for purifying rabbit serum containing anti-pneumococcal capsular polysaccharide based on polysaccharide conjugate and TT protein purification solution according to claim 1, characterized in that: The amount of polysaccharide conjugate stock solution added in S4 is 0.1-0.5 mg / mL serum, and the amount of TT protein purification solution added is 1 mg / mL serum.

5. The method for purifying rabbit serum containing anti-pneumococcal capsular polysaccharide based on polysaccharide conjugate and TT protein purification solution according to claim 1, characterized in that: The absorption process described in S4 includes: S41. Mix the serum and polysaccharide conjugate stock solution thoroughly; S42. Incubate the mixture in a constant temperature water bath; S43. Centrifuge the mixture, discard the precipitate, and collect the supernatant; S44. Filter the supernatant using a filter membrane.

6. The method for purifying rabbit serum containing anti-pneumococcal capsular polysaccharide based on polysaccharide conjugate and TT protein purification solution according to claim 5, characterized in that: In S42, the constant temperature water bath temperature is 37℃ and the incubation time is 30 min; in S43, the centrifugation conditions are 10000 r / min and centrifugation time is 15 min. The S44 uses a 0.22μm filter for filtration.

7. The method for purifying rabbit serum containing anti-pneumococcal capsular polysaccharide based on polysaccharide conjugate and TT protein purification solution according to claim 1, characterized in that: The pneumococcal polysaccharide conjugate described in S4 is a covalently bound compound of capsular polysaccharide and carrier protein, wherein the carrier protein includes TT protein.

8. The method for purifying rabbit serum containing anti-pneumococcal capsular polysaccharide based on polysaccharide conjugates and TT protein purification solution according to claim 1, characterized in that... The TT protein purification solution is tetanus toxoid protein produced by Clostridium tetani, which is purified and prepared in 0.01M PBS (pH 7.2) with a stock solution protein concentration of 10 mg / mL.

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