CN114656560A

CN114656560A - Method for preparing latex enhanced immunoturbidimetric reagent by using recombinant antibody

Info

Publication number: CN114656560A
Application number: CN202210475505.8A
Authority: CN
Inventors: 王亭芳
Original assignee: Wuhan New Biocalysis Solution Co ltd
Current assignee: Wuhan New Biocalysis Solution Co ltd
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2022-06-24
Anticipated expiration: 2042-04-29
Also published as: CN114656560B

Abstract

The invention discloses a method for preparing a latex-enhanced immunoturbidimetric reagent by using a recombinant antibody, belonging to the field of in-vitro diagnostic reagents. According to the invention, an amino acid sequence is inserted between CH2 and CH3 at the Fc end of the antibody to obtain the recombinant antibody, so that the length of the Fc end is increased, and the hydrophobicity of the recombinant antibody is increased, so that the recombinant antibody can be more easily coupled with latex microspheres. The recombinant antibody is used for preparing a reaction reagent R2 for a latex enhanced immunoturbidimetry, so that the problems of unstable batch-to-batch difference, large labor input and difficult improvement of antibody yield caused by adopting common polyclonal antibodies and monoclonal antibodies can be solved.

Description

Method for preparing latex enhanced immunoturbidimetric reagent by using recombinant antibody

Technical Field

The invention relates to a method for preparing a latex-enhanced immunoturbidimetry reagent by using a recombinant antibody, in particular to a method for preparing a latex-enhanced immunoturbidimetry kit by using an Fc end modified recombinant antibody, belonging to the field of in-vitro diagnostic reagents.

Background

The latex enhanced immunoturbidimetry (PETIA) is a relatively stable and accurate method for detecting the humoral protein homogeneous phase immunoturbidimetry. The PETIA process is broadly divided into two categories: the scattering turbidimetry detection method and the transmission turbidimetry detection method. The two basic principles are very similar, and both are that the antibody is coupled on the surface of the polymer latex microsphere, and when the microsphere coupled with the antibody is combined with the antigen, the antibody is rapidly gathered together in a short time, so that the light scattering performance or the light transmittance performance of the reaction solution is changed. Moreover, the change of the light-scattering performance or the light-transmitting performance of the reaction solution has strong correlation with the concentration of the detected antigen, and the reaction solution can reflect the concentration of the detected antigen within a certain range.

Latex-enhanced immunoturbidimetry generally employs two reagents, namely a reagent (R1) and a reagent (R2), wherein R1 is a buffer solution with a coagulant at a certain concentration, and R2 is a latex microsphere solution of a conjugated antibody. R2 is the core part of latex immunoturbidimetry, and the high-quality and cheap antibody plays a decisive role in the performance and market price of latex immunoturbidimetry reagents. At present, most of latex microsphere particles adopted by PETIA detection reagents are inert microspheres and carboxyl microspheres. The former combines the antibody on the surface of the microsphere through physical adsorption, and the latter covalently combines with the amino terminal of the antibody through carboxyl to form a chemical cross-linking arm between the microsphere and the antibody, so that the steric effect is reduced, and the combination rate of the antibody is improved.

At present, most antibodies adopted by PETIA detection reagents are goat polyclonal antibodies, rabbit polyclonal antibodies or mouse monoclonal antibodies, which are all animal sources, have certain biological safety problems, and have huge animal feeding, feeding fields and manual investment. Moreover, the production consistency of the polyclonal antibody is low, and the yield is unstable; although monoclonal antibodies are produced consistently, with high specificity and sensitivity, hybridoma cell lines drift over time, resulting in changes in the antibodies produced, and over time, reduced antibody yields and poor quality may occur. Therefore, as a core raw material of a diagnostic reagent, on one hand, both a polyclonal antibody and a monoclonal antibody cannot well ensure the production quality of the PETIA detection reagent and the subsequent continuous stable production. On the other hand, the structural and functional formulas of the commonly used IGG mabs also limit further improvements of the microsphere and antibody coupling process as one of the key steps in reagent development.

Disclosure of Invention

[ problem ] to

The invention aims to provide a method for preparing a latex enhanced immunoturbidimetry reagent by using an Fc modified recombinant antibody, so that the problems of unstable multi-antibody and monoclonal antibody batch differences, large labor input, difficult improvement of antibody yield and the like are solved, the coupling efficiency of a solid phase and an antibody in the reagent preparation process is effectively improved, the cost of a PETIA detection reagent is further reduced, and a favorable support is formed for improving the quality.

[ solution ]

The invention provides a method for modifying a recombinant antibody for preparing a latex-enhanced immunoturbidimetric reaction reagent R2, which is characterized in that a plurality of amino acids are inserted between the CH2 and CH3 regions at the Fc end of the antibody so as to increase the length and hydrophobicity of the Fc end of the antibody. The number of inserted amino acids does not affect the function of the whole antibody, and the length and hydrophobicity of the Fc end of the antibody can be increased.

In certain embodiments of the invention, 10 amino acids are inserted between the CH2 and CH3 regions of the Fc terminus of an antibody, said amino acids being glycine or lysine.

In certain embodiments of the invention, 10 glycines or 10 lysines are inserted between the CH2 and CH3 regions of the Fc-terminus of an antibody against a recombinant antibody having a C-reactive protein as the antigen, the murine anti-Fab terminus and the human IgG 1-type Fc terminus.

The invention provides an Fc-terminal-modified recombinant antibody, wherein the light chain amino acid sequence of the Fc-terminal-modified recombinant antibody is shown as SEQ ID NO: 1, and the heavy chain amino acid sequence is shown as SEQ ID NO: 3 or SEQ ID NO: 4, respectively. The recombinant antibody modified by the Fc end can be used for preparing a reaction reagent R2 required by detecting C-reactive protein by using a latex enhanced immunoturbidimetry method.

The invention provides a preparation (modification) method of the Fc end modified recombinant antibody, which comprises the following steps:

(1) the Fc terminal sequence of human IgG1 antibody is edited to insert 10 glycines or 10 lysines between CH2 and CH3 regions;

(2) editing a mouse anti-Fab end and the Fc end of the human IgG1 antibody modified in the step (1) at a gene level, and obtaining base sequences for encoding heavy chains and light chains through gene synthesis;

(3) introducing the nucleotide sequence obtained in the step (2) into mammalian Cells (CHOS) by using a plasmid vector pCDNA3, and screening to obtain a recombinant cell strain with Puro and G4218 resistance;

(4) and (4) culturing the recombinant cell strain obtained in the step (3) in vitro to obtain the recombinant antibody modified by the Fc end.

The invention provides a reagent taking the Fc-terminal-modified recombinant antibody as a raw material, wherein the reagent is a reaction reagent R2 required by detecting C-reactive protein by using a latex enhanced immunoturbidimetry method, and the reagent is formed by connecting the Fc-terminal-modified recombinant antibody with a latex microsphere through a covalent bond.

The invention provides a preparation method of the reagent, which comprises the following steps:

(1) activating the latex microspheres by using ECD, centrifuging, discarding supernatant, and collecting precipitate, wherein the precipitate is the activated latex microspheres;

(2) ultrasonically resuspending the collected precipitate in a buffer system, adding a recombinant antibody to couple the activated latex microspheres and the secondary antibody, centrifuging, discarding the supernatant, and collecting the precipitate;

(3) carrying out ultrasonic re-suspension on the precipitate collected in the step (2) in a closed buffer solution, then centrifuging, removing the supernatant, and collecting the precipitate;

(4) carrying out ultrasonic re-suspension on the precipitate collected in the step (3) in a dilution buffer solution to prepare a latex solution;

(5) the latex solution was diluted in a predetermined ratio with a dilution buffer to prepare a reaction reagent R2.

The invention also provides application of the reagent R2 in detection of C-reactive protein by using a latex enhanced immunoturbidimetry method.

[ advantageous effects ]

According to the invention, an amino acid sequence is inserted between CH2 and CH3 at the Fc end, so that the length of the Fc end is increased, the hydrophobicity of the Fc end is increased, the Fc end is more easily coupled with latex microspheres, the coupling efficiency of a solid phase and an antibody in the preparation process of the reagent is effectively improved, the cost of the PETIA detection reagent is further reduced, and a favorable support is formed for improving the quality.

The invention takes a recombinant antibody modified by Fc as a raw material to prepare a reaction reagent R2 for latex enhanced immunoturbidimetry. The Fc modified recombinant antibody can be produced quickly and stably in cells, has the advantage of serving as a core raw material compared with the conventional antibody, and avoids the problems of poor and unstable polyclonal antibody and monoclonal antibody batches, huge manpower investment, difficulty in improving the antibody yield and the like.

Drawings

FIG. 1: elisa potency assay for different antibodies in example 1, FIG. 1, wherein the abscissa is lg antibody concentration and the ordinate is OD₄₅₀。

FIG. 2 is a schematic diagram: calibration curves for CRP assay reagents prepared with different antibodies.

Detailed Description

The terms:

antibodies, also called immunoglobulins (Ig), are immunoglobulins which are produced by the body from plasma cells differentiated from B cells under the stimulation of antigenic substances and which are specifically binding reactive with the corresponding antigen. Antibodies exist as one or more wye-shaped monomers, each of which is a symmetrical structure having 4 polypeptide chains, of which 2 are the longer, relatively higher molecular weight identical heavy chains (H chains) and 2 are the shorter, relatively lower molecular weight identical light chains (L chains). In the same Ig molecule, the amino acid compositions of the two H chains are identical, and the amino acid compositions of the two L chains are identical. According to the function, the Fab segment of the antibody, namely the antigen binding fragment (Fab), is composed of a complete light chain and VH and CHl structural domains of heavy chains, and has antigen binding activity; the Fc fragment, i.e., the crystallizable (Fc), consists of the CH2 and CH3 domains of the heavy chain, and has no antigen binding activity.

C-reactive protein (CRP) is a protein (acute protein) which rises sharply in plasma when a body is infected or tissues are damaged, activates complement and strengthens phagocytosis of phagocytes to play an opsonizing role, and eliminates pathogenic microorganisms invading the body and damaged, necrotic and apoptotic tissue cells.

The emulsion microsphere is prepared with styrene monomer as basic material and through emulsion polymerization, and through unique surface modification process, the microsphere has controllable functional groups. The amido on the surface of the antibody molecule and the carboxyl latex microsphere can form amido bond, so that the antibody and the latex microsphere are coupled.

EXAMPLE 1 modification and expression of recombinant antibodies

(1) Modification strategies for recombinant antibodies

The recombinant antibody is a recombinant product of a mouse anti-Fab end and a human IgG1 type Fc end, and a short peptide of 10 amino acids is inserted between the CH2 and CH3 regions of the Fc end of the antibody: GGG GGGGGGG or KKK KKKKKKK or FFFF FFFFFF.

Before modification, the light chain amino acid sequence of the antibody is shown in SEQ ID NO: 1, the heavy chain amino acid sequence is shown in SEQ ID NO: 2; after insertion of 10 amino acid modifications between the CH2 and CH3 regions at the Fc end of the antibody, the amino acid sequences of the heavy chains are as set forth in SEQ ID NOs: 3. SEQ ID NO: 4. SEQ ID NO: 5, respectively.

(2) Editing the mouse anti-Fab end and the Fc end of the human IGG1 antibody modified in the step (1) at the gene level, and obtaining the base sequences for encoding the heavy chain and the light chain through gene synthesis.

(3) The nucleotide sequence obtained in step (2) was introduced into mammalian cells (CHOS, Chinese hamster ovary adherent cells) using a plasmid vector pCDNA3, and recombinant cell lines resistant to Puro and G4218 were selected.

(4) Culturing the recombinant cell strain obtained in the step (3) in vitro to obtain a recombinant antibody 1, a recombinant antibody 2, a recombinant antibody 3 and a recombinant antibody 4; wherein, the light chain amino acid sequence of the recombinant antibody 1 is shown in SEQ ID NO: 1. the heavy chain amino acid sequence is shown in SEQ ID NO: 2, the light chain amino acid sequence of recombinant antibody 2 is shown in SEQ ID NO: 1. the heavy chain amino acid sequence is shown in SEQ ID NO: 3, the light chain amino acid sequence of recombinant antibody 3 is shown in SEQ ID NO: 1. the heavy chain amino acid sequence is shown in SEQ ID NO: 4, the light chain amino acid sequence of recombinant antibody 4 is shown in SEQ ID NO: 1. the heavy chain amino acid sequence is shown in SEQ ID NO: 5.

2. recombinant antibodies were assayed using enzyme-linked immunosorbent assay (Elisa)

(1) Native C-reactive protein (CRP) was diluted to 0.5 μ g/mL with carbonate buffer pH 9.7, added to 96-well plates every 100 μ L, and incubated at 37 ℃ for 2 hours;

(2) discarding the liquid in the wells, washing with PBST buffer (phosphate buffer containing Tween-20) at 300. mu.L per well for 3 times;

(3) discarding the liquid in the wells, adding 200 μ L of PBST blocking solution containing 1% bovine serum albumin in each well, and incubating at 37 ℃ for 2 hours;

(4) discarding the liquid in the wells, washing with PBST (PBST), 300 μ L per well, 3 times;

(5) adjusting the concentration of the recombinant antibody 1-4 obtained in the step 1 to 5mg/mL by using PBS buffer solution respectively, then carrying out double dilution to obtain 23 concentration gradients, adding one PBS buffer solution as a blank control respectively, adding 24 obtained samples into an ELISA plate, wherein each well is 100 mu L, each sample is provided with a plurality of wells, and incubating the ELISA plate at 37 ℃ for 0.5 hour;

(6) discarding liquid in the wells, washing with PBST buffer solution, washing with 300 μ L per well for 3 times;

(7) diluting rabbit anti-human IgG enzyme-labeled secondary antibody with 1% bovine serum albumin-containing PBST buffer solution according to the proportion of 1+2000, adding the diluted secondary antibody into each hole according to the proportion of 100 mu L per hole, and incubating for 30min at 37 ℃;

(8) discarding liquid in the wells, washing with PBST buffer solution, washing with 300 μ L per well for 3 times;

(9) adding 100 mu L of TMB substrate color development liquid into each hole, and reacting for 10min at room temperature in a dark place;

(10) after 50 mu L of 2M sulfuric acid stop solution is added into each hole, the enzyme label plate is placed on an enzyme label instrument, and the absorbance of each hole is recorded under the wavelength of 450 nm. As shown in table 1 and fig. 1, the results show that the four antibodies have small difference in binding titer with the antigen when detected at the Elisa level, and have good recognition effect on the target antigen.

TABLE 1 results of Elisa potency assay of different antibodies

Example 2 method for preparing latex-enhanced immunoturbidimetric reagent R2 using the recombinant antibody prepared in example 1

(1) Diluting 1mL of carboxyl latex microspheres (50nm-400nm) by using 4mL of 20-50mM Good' S buffer solution until the mass concentration of the carboxyl latex microspheres is 1%, and uniformly mixing at 25-37 ℃.

(2) 0.01g of EDC & HCl (C) was weighed₈H₁₇N₃HCl), adding into the latex microsphere solution obtained in the step (1), and rapidly stirring at 25-37 ℃ to activate carboxyl. After reaction for 10-30 min, centrifugation (30000g, 10-60min) was carried out to remove the supernatant and collect the precipitate for further use.

(3) The above pellets were resuspended in 5mL of 20-50mM Good' S buffer and dispersed by sonication (40KHz, 5-15 min).

(4) And (4) respectively adding 2mg of the recombinant antibody prepared in the example 1 into the latex microsphere solution rapidly stirred at 25-37 ℃ and subjected to ultrasonic dispersion in the step (3) to perform coupling reaction of the latex microspheres and the antibody. After reacting for 1-2 hours, centrifuging (30000g, 10-60min), removing supernatant, and collecting precipitate for later use.

(5) The above pellet was resuspended in 5mL of 20-50mM Good' S buffer and dispersed by sonication (40KHz, 5-15 min).

(6) And (4) adding 1g of bovine serum albumin into the coupled antibody latex microsphere solution which is rapidly stirred at the temperature of 25-37 ℃ and is subjected to ultrasonic dispersion in the step (5) for blocking. After reacting for 1-2 hours, centrifuging (30000g, 10-60min), removing supernatant, and collecting precipitate for later use.

(7) The above pellet was resuspended in 5mL of 20-50mM Good' S buffer, and dispersed by sonication (40KHz, 5-15min) to give R2 concentrate.

(8) The reagent R2 was prepared by diluting the above R2 concentrated solution 5 to 200 times with a latex diluent containing 20 to 50mM Good' S buffer.

Example 3 method for detecting protein C reactive protein using latex enhanced turbidimetric immunoassay prepared in example 2

(1) The instrument comprises the following steps: hitachi 7180 full-automatic biochemical analyzer

(2) Parameters are as follows:

sample size: 2 mu L of the mixture is prepared into a solution,

reagent R1: the volume of the solution is 100 mu L,

reagent R2: the volume of the solution is 100 mu L,

the fitting mode is as follows: the amount of the spline is that of the spline,

dominant wavelength: the particle diameter of the nano-particles is 570nm,

sub-wavelength: none.

(3) After 4 reagents R2 prepared in example 2 were diluted to 1+4, standard curves were prepared on a fully automatic biochemical analyzer according to the above parameters, and the standard curves are shown in fig. 2.

Then, 20 random serum samples and 10 serum positive samples were tested on a full-automatic biochemical analyzer using the 4 reagents R2 prepared in example 2, and the reproducibility of the reagent R2 was measured at a CRP concentration of 5 mg/L. The results of the experiments are shown in tables 2 to 4. In Table 2, the background is the absorbance value obtained by using water as a control at a wavelength of 570 nm; the calibration results (absorbance data) at different calibration concentrations refer to the difference in absorbance between the reagent R2 and CRP before and after the reaction for a period of time at different CRP concentrations, and the difference is linearly related to the calibration concentration within a certain range. As can be seen from table 2, different antibodies after coating showed differences in sensitivity, with antibody 1 as the control, antibody 3 having the highest sensitivity and antibody 4 having the lowest sensitivity.

In combination with the data in tables 2-4, it can be seen that the antibody exhibits the following properties in the terminal reagent when the Fc-terminus of the antibody is modified with different amino acid sequences: the antibody specificity is not affected, the coating efficiency is obviously improved by using the antibody 3, the sensitivity of the reagent R2 is improved by about one time, and the repeatability of the measurement value of the reagent R2 at a low value end is obviously improved and is optimized to 1.2% from 3.8% of the contrast.

TABLE 2 comparison of sensitivity in CRP detection with reagent R2 coated with different antibodies

TABLE 3 comparison of the accuracy of the reagent R2 obtained by coating different antibodies in the detection of serum samples

TABLE 4 repeatability data for the measurement of CRP samples at a concentration of 5mg/L with reagent R2 obtained after coating with different antibodies

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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<120> a method for preparing a latex-enhanced immunoturbidimetric reagent using a recombinant antibody

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Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Phe Phe Phe Phe Phe Phe Phe

370 375 380

Phe Phe Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

385 390 395 400

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

405 410 415

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

420 425 430

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

435 440 445

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455 460

Claims

An Fc-terminal modified recombinant antibody, wherein the light chain amino acid sequence is as set forth in SEQ ID NO: 1, and the heavy chain amino acid sequence is shown as SEQ ID NO: 3 or SEQ ID NO: 4, respectively.
2. Use of the Fc-terminal modified recombinant antibody of claim 1 for preparing a reagent R2 for detecting C-reactive protein by latex enhanced immunoturbidimetry.
3. A method for modifying a recombinant antibody used for preparing a latex-enhanced immunoturbidimetric reaction reagent R2, wherein the modification is the insertion of amino acids between the CH2 and CH3 regions of the Fc terminus of the recombinant antibody to increase the length and hydrophobicity of the Fc terminus of the antibody.
4. The method of claim 3, wherein 10 amino acids are inserted between the CH2 and CH3 regions of the Fc terminus of the antibody, said amino acids being glycine or lysine.
5. The method of claim 3 or 4, wherein the recombinant antibody is a recombinant antibody having a murine anti-Fab end and a human IgG1 type Fc end, in which the C-reactive protein is an antigen.
6. The method of claim 3, wherein the recombinant antibody is a recombinant antibody having a murine anti-Fab end and a human IgG1 type Fc end, and the recombinant antibody has a C-reactive protein as an antigen, and comprises the following steps:

(1) the Fc terminal sequence of human IgG1 antibody was re-edited by inserting 10 glycines or 10 lysines between the CH2 and CH3 regions;

(2) editing a mouse anti-Fab end and the Fc end of the human IgG1 antibody modified in the step (1) at a gene level, and obtaining base sequences for encoding heavy chains and light chains through gene synthesis;

(3) introducing the nucleotide sequence obtained in the step (2) into mammalian Cells (CHOS) by using a plasmid vector pCDNA3, and screening to obtain a recombinant cell strain with Puro and G4218 resistance;

(4) and (4) culturing the recombinant cell strain obtained in the step (3) in vitro to obtain the recombinant antibody modified by the Fc end.
7. A reagent prepared from the Fc-terminal-modified recombinant antibody according to claim 1, wherein the reagent is R2, which is a reagent required for detecting C-reactive protein by latex-enhanced immunoturbidimetry, and the reagent is obtained by linking the Fc-terminal-modified recombinant antibody to latex microspheres through covalent bonds.
8. A method for preparing the reagent according to claim 7, comprising the steps of:

(1) activating the latex microspheres by using ECD, centrifuging, discarding supernatant, and collecting precipitate, wherein the precipitate is the activated latex microspheres;

(2) ultrasonically resuspending the collected precipitate in a buffer system, adding a recombinant antibody to couple the activated latex microspheres and the secondary antibody, centrifuging, discarding the supernatant, and collecting the precipitate;

(3) carrying out ultrasonic resuspension on the precipitate collected in the step (2) in a closed buffer solution, then centrifuging, discarding the supernatant, and collecting the precipitate;

(4) carrying out ultrasonic resuspension on the precipitate collected in the step (3) in a dilution buffer solution to prepare a latex solution;

(5) the latex solution was diluted in a predetermined ratio with a dilution buffer to prepare a reaction reagent R2.
9. Use of the reagent of claim 7 for the detection of C-reactive protein by latex-enhanced immunoturbidimetry.
Recombinant antibody with modified Fc-terminus, characterized in that several amino acids are inserted between the CH2 and CH3 regions of the recombinant anti-Fc terminus, said amino acids being capable of increasing the hydrophobicity of the antibody.