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CN-121994978-A - Antibody titer detection method

CN121994978ACN 121994978 ACN121994978 ACN 121994978ACN-121994978-A

Abstract

The invention provides a method for detecting antibody titer. The detection method comprises the steps of loading a standard substance protein on an affinity chromatography column, eluting while carrying out ultraviolet detection on an elution product of the standard substance protein to obtain a standard substance protein elution curve, establishing a standard curve of the elution curve peak area and the titer of the standard substance protein, loading a sample to be detected on the affinity chromatography column, eluting while carrying out ultraviolet detection on the elution product of the sample to be detected, and obtaining the antibody titer of the sample to be detected by utilizing the elution curve peak area of the standard curve and the elution curve peak area of the sample to be detected, wherein the filler of the affinity chromatography column is protein A, eluting by utilizing a mobile phase A and a mobile phase B, and arginine is contained in the mobile phase A and the mobile phase B. By using a mobile phase containing arginine for elution of protein a affinity chromatography, arginine can affect protein surface tension or amino acid solubility, and by affecting the hydrophobicity of the sample, the sensitivity and accuracy of antibody titer detection are improved.

Inventors

  • ZHAO PUYA
  • WEI LAN
  • GAO KAI

Assignees

  • 上海凯莱英生物技术发展有限公司
  • 上海凯莱英生物技术有限公司
  • 上海凯莱英生物制药有限公司

Dates

Publication Date
20260508
Application Date
20260130
Priority Date
20250415

Claims (10)

  1. 1. A method for detecting antibody titer, the method comprising: Loading the standard protein on an affinity chromatographic column, eluting while carrying out ultraviolet detection on an eluted product of the standard protein to obtain a standard protein elution curve, and establishing a standard curve of the peak area and the titer of the elution curve of the standard protein; loading a sample to be detected on the affinity chromatographic column, eluting while carrying out ultraviolet detection on an elution product of the sample to be detected, and obtaining the antibody titer of the sample to be detected by utilizing the peak areas of the standard curve and the elution curve of the sample to be detected; the packing of the affinity chromatographic column is protein A, and the elution is carried out by using a mobile phase A and a mobile phase B, wherein the mobile phase A and the mobile phase B both contain arginine.
  2. 2. The method according to claim 1, wherein the mobile phase A comprises 45-55mM Tris-HAc, 140-160 mM NaCl and 0.45-0.55M arginine; Preferably, the mobile phase B comprises 110-130 mM HAc and 0.45-0.55M arginine.
  3. 3. The method according to claim 2, wherein the mobile phase A comprises 50mM Tris-HAc, 150mM NaCl and 0.5M arginine.
  4. 4. The method according to claim 2, wherein the mobile phase B comprises 120 mM HAc and 0.5M arginine.
  5. 5. The method according to claim 2, wherein the mobile phase a has a pH of 6.8-7.2.
  6. 6. The method according to claim 2, wherein the mobile phase B has a pH of 2.9-3.3.
  7. 7. The method according to claim 1, wherein the eluting comprises equilibration with mobile phase A at 0-1min, protein elution with mobile phase B at 1-3min, and rinsing with mobile phase A at 3-6 min.
  8. 8. The method of claim 1, wherein loading the standard onto an affinity chromatography column comprises: And carrying out gradient concentration dilution on the standard protein by using a buffer solution to obtain 7-8 standard proteins with different concentrations, respectively loading the standard proteins on the affinity chromatographic column, and respectively carrying out elution and ultraviolet detection.
  9. 9. The method according to claim 8, wherein the concentration of the standard protein obtained by dilution is in the range of 0.1 to 10 g/L.
  10. 10. The method according to claim 1, wherein the sample protein, the sample to be tested, the mobile phase a and the mobile phase B are subjected to filtration treatment using a 0.22 μm filter, respectively, before the loading or the elution is performed.

Description

Antibody titer detection method The present application is based on and claims priority from chinese application CN number 2025104718090, application day 2025, month 04, 15, the disclosure of which is incorporated herein by reference in its entirety. Technical Field The invention relates to the technical field of protein purification, in particular to a detection method of antibody titer. Background In recent years, the worldwide biomedical industry has rapidly developed, and the market scale of the antibody industry has grown. Protein A is a Protein on the wall of Staphylococcus aureus and is capable of specifically binding to immunoglobulins of humans and various mammals, in particular to the Fc portion of IgG. Affinity chromatography of Protein A is a main means for detecting antibody titer with Fc fragment in cell culture process, however, the components in cell culture supernatant are complex, wherein the components comprise the feed, cell fragments, host cell proteins and other impurities added in the culture process, and part of antibodies can generate double peak or split peak phenomenon in the Protein A affinity chromatography analysis process due to the existence of a large amount of aggregates and fragments, so that the detection result of antibody titer is affected. In order to solve the problem of double peak or split peak phenomenon in the process of detecting antibody titer, the existing method comprises adding hydrophobic electrolyte (such as tetramine chloride and tetraethylamine chloride) into a mobile phase, adding 0.5% -1% Tween, or increasing the concentration of buffer solution to 1.0-2.0M, and also reducing the eluting pH to ease the detection abnormality. However, the above-mentioned methods have drawbacks of toxicity, flammability (addition of hydrophobic electrolyte or addition of tween), and instability (increase of buffer concentration and decrease of elution pH), and cannot be stably used for a long period of time. Therefore, how to provide a detection method for the antibody titer, which is simple to operate and has good effect and can avoid the phenomenon of double peaks or split peaks in the process of affinity chromatography analysis, has great significance for the accuracy of antibody titer detection. Disclosure of Invention The invention mainly aims to provide a method for detecting antibody titer, which solves the problem that in the prior art, the phenomenon of double peaks or split peaks exists in the process of antibody titer. In order to achieve the above object, according to a first aspect of the present invention, there is provided a method for detecting antibody titer, comprising loading a standard protein onto an affinity chromatography column, eluting while ultraviolet detecting an eluted product of the standard protein to obtain a standard protein elution curve, establishing a standard curve of an elution curve peak area and titer of the standard protein, loading a sample to be detected onto the affinity chromatography column, eluting while ultraviolet detecting an eluted product of the sample to be detected, and obtaining antibody titer of the sample to be detected by using the peak areas of the elution curve of the standard curve and the elution curve of the sample to be detected, wherein the affinity chromatography column is filled with protein A, eluting by using mobile phase A and mobile phase B, and both mobile phase A and mobile phase B contain arginine. Further, mobile phase A comprises 45-55mM Tris-HAc, 140-160 mM NaCl and 0.45-0.55M arginine, preferably mobile phase B comprises 110-130 mM HAc and 0.45-0.55M arginine. Further, mobile phase A included 50mM Tris-HAc, 150mM NaCl and 0.5M arginine. Further, mobile phase B included 120 mM HAc and 0.5M arginine. Further, the pH value of the mobile phase A is 6.8-7.2. Further, the pH of mobile phase B is 2.9-3.3. Further, the elution comprises the steps of balancing by using a mobile phase A when eluting for 0-1min, eluting by using a mobile phase B when eluting for 1-3min, and eluting by using the mobile phase A when eluting for 3-6 min. Further, loading the standard substance on the affinity chromatographic column comprises the step of carrying out gradient concentration dilution on the standard substance protein by utilizing a buffer solution to obtain 7-8 standard substance proteins with different concentrations, and respectively loading the standard substance proteins on the affinity chromatographic column for elution and ultraviolet detection. Further, the concentration range of the standard protein obtained by dilution is 0.1-10 g/L. Further, before loading or eluting, the standard protein, the sample to be tested, the mobile phase A and the mobile phase B were subjected to filtration treatment using a 0.22 μm filter, respectively. By applying the technical scheme of the invention, the mobile phase containing arginine is used for eluting the protein A affinity chromatography, the arginine can influence