Improved statin B detection kit and monoclonal antibody preparation method

Abstract

The invention discloses an improved statin B detection kit and a monoclonal antibody preparation method, and the kit comprises a first reagent and a second reagent, the first reagent comprises antibody-coupled toluenesulfonyl magnetic beads, and the second reagent comprises an antibody-coupled luminous marker; the antibody is an inhb specific monoclonal antibody, and the inhb specific monoclonal antibody is a monoclonal antibody produced by cells obtained by immune screening of a bablc mouse with an inhb gene knocked out. Compared with a commercially available Beckmann statin B kit, the kit provided by the invention realizes faster, more sensitive and simpler detection.

Description

Technical Field

[0001] The present invention relates to the field of in vitro detection technology, and in particular to an improved inhibin B detection kit and a monoclonal antibody preparation method. Background Art

[0002] Inhibin is a multifunctional growth and differentiation factor of the transforming growth factor β (TGFβ) superfamily. It is a dimeric glycoprotein hormone composed of two subunits, α and β. One α subunit and one of the two β subunits form INHA (αβA) and INHB (αβB) respectively through disulfide bonds, of which the relative molecular mass of INHB is about 32KD. INHB has the same β subunit as activins (ACT). Activins are homologous or heterologous dimers composed of two identical or different subunits connected by disulfide bonds. The expression of four activin isomers was found in the ovary, namely activin A (composed of two βA subunits), activin B (composed of two βB subunits), activin AB (composed of βA and βB subunits), and activin AC (composed of βA and βC subunits).

[0003] Inhibin B is the main marker of ovarian reserve function and testicular seminiferous tubule function. It can be used to detect female infertility caused by ovarian factors and male infertility caused by seminiferous tubule dysfunction. In the male body, inhibin B is a testicular-derived glycoprotein hormone. The serum inhibin B level in adult males is significantly negatively correlated with FSH, and has a negative feedback effect on FSH. In the female body, inhibin B is mainly produced by the granulosa cells of the medium and small antral follicles of the ovary, which can feedback inhibit the release of follicle-stimulating hormone (FSH) from the anterior pituitary and regulate the formation of follicles. INHB levels reflect ovarian reserve earlier and more directly than FSH.

[0004] Inhibin B has become an important marker analyte for studying male and female reproductive function in clinical diagnostic analysis. Some of the many applications include: Down syndrome screening (inhibin A), male infertility testing (inhibin B), ovarian reserve / menopausal onset (inhibin B), and ovarian cancer (inhibin αC subunit and inhibin B).

[0005] Two commercial inhibin B immunoassay kits and their monoclonal antibody raw materials are currently available from DSL and OBI (both Beckman Coulter companies). Both tests use the same pair of monoclonal antibodies that Groome and colleagues developed in this laboratory more than 20 years ago for synthetic peptides, where the capture antibody (C5) is generated from a peptide of the inhibin βB subunit and the R1 detection antibody is generated from a peptide of the inhibin α subunit. Both tests require a methionine oxidation step with hydrogen peroxide or heat denaturation followed by the addition of SDS, because the two methionines of the βB subunit peptide spontaneously oxidize to sulfoxides during the monoclonal antibody immunoassay, while the methionines in natural samples are not oxidized. Therefore, in order to obtain full immunoreactivity of the antibody in the kit test and improve the sensitivity of the kit, it is necessary to add an oxidant to the kit to oxidize the sample in advance, and in the ELISA kit, the assay needs to be incubated overnight with the sample to obtain sufficient sensitivity.

[0006] Manufacturers such as Yahuilong in the market all use the above-mentioned methods and raw materials. Although the reaction time is shortened to a certain extent on the magnetic particle chemiluminescence platform and no overnight reaction is required, sample processing is still required and the reaction time takes more than 45 minutes to complete. In addition, the current inhibin B kit has a cross-reaction of about 0.5% with inhibin A.

[0007] Therefore, for inhibin B detection kits, how to omit sample pretreatment, improve sensitivity, and shorten reaction time has not yet been fully resolved. Summary of the invention

[0008] In order to solve one or more problems existing in the prior art, the present invention provides an improved inhibin B detection kit and a method for preparing a monoclonal antibody. The technical solution adopted by the present invention to solve the above problems is: an improved inhibin B detection kit, the kit comprising: a first reagent and a second reagent; the first reagent comprises an antibody coupled to tosyl magnetic beads, and the second reagent comprises an antibody coupled to a luminescent marker; The antibody is an inhb-specific monoclonal antibody, which is a monoclonal antibody produced by cells obtained by immune screening of bablc mice with inhb gene knocked out.

[0009] In some embodiments, it also includes: a third reagent, the third reagent includes: a first luminescent reagent and a second luminescent reagent, the first luminescent reagent is 0.1 mol / L hydrochloric acid and 0.3% hydrogen peroxide, and the second luminescent reagent is 0.15 mol / L sodium hydroxide and 0.05% Triton 100.

[0010] In some embodiments, for the first reagent, the reaction buffer for the coupling reaction between the antibody and the tosyl magnetic beads is CBS, and the coupling catalyst is a coupling buffer containing 3M ammonium sulfate.

[0011] Furthermore, for the coupling reaction, 1 mg of the tosyl magnetic beads was added to every 20 ug of the antibody, the reaction volume of the magnetic beads was 10 mg / ml, and the volume ratio of the reaction buffer to the coupling catalyst was 2:1.

[0012] Furthermore, the uncoupled magnetic beads were blocked by adding 5% casein, 5% BSA in PBS, pH 7.4 blocking solution after the coupling reaction.

[0013] Furthermore, before performing the blocking, uncoupled antibody was washed using PBST.

[0014] Furthermore, after the blocking is completed, 50 mM TBS, 5% BSA, 0.02% Tween 20, and 0.05% Proclin 300 magnetic bead diluent are added to dilute the magnetic bead concentration to 0.15-0.3 mg / ml.

[0015] In some embodiments, the second reagent is obtained by diluting a labeled antibody solution with an acridine diluent; NHS-SA-acridine is added to the antibody and incubated and purified to obtain the labeled antibody solution.

[0016] Furthermore, the acridine diluent consists of 50 mM MES, 0.15 M NaCl, 0.5% BSA, 0.05% Tween 20, and 0.5% casein.

[0017] And a method for preparing the above-mentioned inhb-specific monoclonal antibody, which comprises: S10, breeding homozygous mice with wild-type mice, performing PCR identification on the bred mice using specific primers, and screening to obtain subtype homozygous mice, wherein the homozygous mice are mice with completely excised inhb genes, and the wild-type mice are mice with inhb genes not excised; S20, immunizing homozygous mice of the subtype using a multi-site repeated immunization method, wherein the protein used in the multi-site repeated immunization method is the expressed and purified ACTBB protein; S021, after performing multiple immunizations, taking spleen cells and osteoma cells from homozygous mice of the subtype and fusing them to obtain hybridoma cells; S022, screening the hybridoma cells to obtain inhb-specific monoclonal cells, using the inhb-specific monoclonal cells to produce ascites to obtain the inhb-specific monoclonal antibodies.

[0018] Since the homology of the βB subunit gene of inhibin B in humans and mice reaches 99%, immunizing bablc mice with inhibin B protein by conventional methods basically does not produce immunogenicity, so it is difficult to develop improved antibodies against other epitopes for the βB subunit. However, this application uses the inhibin B gene of balb / c mice to knock out the inhibin B gene, repeatedly immunizes mice with the expressed recombinant ACTBB at multiple sites, and screens monoclonal antibodies with the recombinant expressed inhb, thus producing a highly specific monoclonal inhibin βB (clone number 5G1 / 8A4).

[0019] The inhb-specific monoclonal antibody (antibody 5G1 / 8A4) was shown to be highly specific in the inhibin B detection kit without any sample pretreatment steps and was compared with a commercially available inhibin B assay, with significant correlation, ultimately making the detection using the kit using antibody 5G1 / 8A4 faster, more sensitive and simpler. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the PCR identification results of gene knockout mice; Figure 2 This is a schematic diagram of the titer results of four immune sera in Example 2; Figure 3 This is a table comparing the clinical relevance of the kit of the present application in Example 5 and the commercially available Beckman Inhibin B kit; Figure 4 is a linear regression model diagram of the clinical correlation comparison table of Example 5; Figure 5 The sensitivity test table of the kit of the present application in Example 6; Figure 6 This is a cross-reaction interference experiment table of the kit of the present application in Example 7. DETAILED DESCRIPTION

[0021] In order to make the above-mentioned objects, features and advantages of the present invention more understandable, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from this description, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0022] The preparation process of the monoclonal antibody is as follows: S10, breeding homozygous mice with wild-type mice, identifying the bred mice by PCR using specific primers, and screening to obtain subtype homozygous mice (obtaining complete INHB immunoreactivity), wherein the homozygous mice are mice with complete excision of the inhb gene, and the wild-type mice are mice with inhb gene not excised; S20, immunizing homozygous mice of the subtype using a multi-site repeated immunization method, wherein the protein used in the multi-site repeated immunization method is the expressed and purified ACTBB protein; S021, after performing multiple immunizations, taking spleen cells and osteoma cells from homozygous mice of the subtype and fusing them to obtain hybridoma cells; S022, screening the hybridoma cells to obtain inhb-specific monoclonal cells, using the inhb-specific monoclonal cells to produce ascites to obtain the inhb-specific monoclonal antibodies.

[0023] Example 1, preparation and identification of genetically modified mice, wherein the genetically modified bablc mice are from Saiye Biotechnology; the genetically modified mice are named C57BL / 6N-Inhbb, the sequence name is KOCMP-16324-Inhbb, the knockout gene name is Inhbb, the NCBI ID is 16324, and the chain length region C57BL / 6N is removed; First, homozygous mice (complete removal of the inhb gene) were bred with wild-type mice (non-removal of the inhb gene), and the resulting mice were subjected to PCR using specific primers to identify whether the subtype mice had the inhb gene removed. The identification process of gene-removed mice is as follows: 0.2 cm of the mouse tail is taken, the mouse gene is released using a one-step mouse gene extraction kit, and the mouse full-sequence gene is obtained after heating to destroy the protease activity. Specific primers are set by the gene-knocked mouse gene and the gene of the mouse that has not been knocked out as follows: PCR Primer 1-Gene excision success specific primer (Annealing Temperature 60.0 ºC): F1:5'-GTCCCTTGGAACTCCTTAGAAACA-3' R1: '-AATTAAGACCCTTCTCCTCCCTC-3' Product size: 575 bp PCR Primer 2 - Gene-specific primer (Annealing Temperature 60.0 ºC): F2: 5'-TTCTCCCGCTTTGTAGAAAACCA-3' R1: 5'-AATTAAGACCCTTCTTTCCTCCCTC-3' Product size: 634 bp Homozygous: one band with 575 bp Heterozygous: two bands with 575 bp and 634 bp Wild type: one band with 634 bp The results of PCR identification of gene knockout mice are as follows Figure 1 As shown, mice 6 and 7 are homozygous, with only one band of 575 bp; mice 4, 8, and 12 are wild-type, with only one band of 634; mice 2, 3, 9, 10, and 13 are heterozygous, with two bands of 575 / 634 bp. Mice 6 and 7 (with complete INHB immunoreactivity) were selected for the next step of immunization.

[0024] Example 2, INHB protein immunization, a multi-site repeated immunization method was used to immunize mice No. 6 and No. 7 (to obtain complete INHB immune reactivity), and the protein used in the multi-site repeated immunization method was the expressed and purified ACTBB protein; wherein, in the first immunization, the protein dosage was 60-100ug / mouse, and a second immunization was performed two weeks later, and the protein dosage for the second immunization was 30-60ug / mouse. Blood was collected from the tail vein 3 days after the second immunization to test the mouse serum titer. The results showed that the mouse serum titer was high and the immunization effect was good; the third and fourth immunizations were performed in the same manner as the second immunization, and the serum titer results of the fourth immunization were as follows: Figure 2 As shown; 3 days after the four immunizations, the mouse tail vein blood was continued to be collected to test the serum titer. The serum titer results showed that the titer was greater than 400,000. The higher the titer, the better the immune effect. 60-100ug / ampoule shock immunization can be arranged; spleen cells were taken and fused with myeloma cells, and the inhb-specific monoclonal cell line was obtained through subgram screening, and then ascites production was carried out to mass-produce inhb-specific monoclonal antibodies.

[0025] Example 3, preparation of the kit, preparation of the first reagent, coupling the obtained inhb-specific monoclonal antibody with tosyl magnetic beads (manufacturer is JSR or Thermo), the reaction buffer of the coupling reaction is CBS, its pH is 9.6, and the coupling catalyst is a coupling buffer solution containing 3M ammonium sulfate; 1mg of the tosyl magnetic beads is added to every 20ug of the antibody, the magnetic bead reaction volume is 10mg / ml, the reaction buffer is 2 / 3 of the reaction buffer, 1 / 3 of the coupling catalyst, and the reaction is carried out at 37°C for 16-24h; then the excess uncoupled antibody is washed with PBST; then 5% casein, 5% BSA PBS, PH7.4 blocking solution is added to block the uncoupled magnetic beads, and the blocking is carried out for 12-24h. After the blocking is completed, 50mM TBS, 5% BSA, 0.02% Tween 20, 0.05% Proclin300 magnetic bead diluent are added to dilute the magnetic bead concentration to 0.15-0.3mg / ml to obtain the first reagent.

[0026] Preparation of the second reagent: add 30ug of NHS-SA-acridine to every 1mg of antibody (INHB-specific monoclonal antibody), incubate at room temperature for 2h, purify with molecular sieve to obtain labeled antibody solution, and dilute with acridine diluent to 0.1-0.4ug / ml according to the antibody concentration to obtain the second reagent. The acridine diluent is 50mM MES, 0.15M NaCl, 0.5% BSA, 0.05% Tween 20, and 0.5% casein.

[0027] And a third reagent, the third reagent includes: a first luminescent reagent and a second luminescent reagent, the first luminescent reagent is 0.1 mol / L hydrochloric acid and 0.3% hydrogen peroxide, and the second luminescent reagent is 0.15 mol / L sodium hydroxide and 0.05% Triton 100.

[0028] Example 4, use of the kit, kit reaction process: 50ul of sample, 50ul of the first reagent, 50ul of the second reagent, incubate for 20min, magnetic separation and wash 3 times, add 100ul of the first luminescent reagent, 100ul of the second luminescent reagent, and then read the luminescent signal value; the entire reaction process takes 25min to complete.

[0029] Example 5, clinical relevance, the kit obtained in Example 3 of the present application was compared with the commercially available Beckman inhibin B kit for clinical relevance, wherein a total of 34 clinical samples, the measured values ​​of the two kits are as follows Figure 3 As shown, based on Figure 3 A linear regression model was established for analysis, and we obtained Figure 4 The linear regression model diagram shown, Figure 4The horizontal axis X is the clinical measurement value of the Beckmanstatin B kit, and the vertical axis Y is the clinical measurement value of the kit of this application. The linear regression equation is y = 1.0243x - 6.821 ( Figure 4 ), Figure 4 The discrete points in the table are the clinical values ​​measured by the Beckman Statins B Kit; Figure 3 , Figure 4 , the correlation R-square is calculated to be 0.9855, and the slope K of the linear regression equation is 1.0243, and it is concluded that the kit of the present application has a good correlation.

[0030] Example 6, kit sensitivity, analytical sensitivity is defined as the photon number measurement of the zero value calibrator test 20 times, taking the average value plus twice the standard deviation, and substituting the obtained photon number into the standard curve to obtain the sensitivity, such as Figure 5 As shown, the sensitivity of the inhibin B chemiluminescent immunoassay kit was calculated to be 2.14 pg / mL. It can be seen that the kit of the present application has a higher sensitivity than the commercially available kit (11.98 pg / ml).

[0031] Example 7, cross-reaction rate, pure serum was added with analogs for cross-reaction interference experiment, the cross-reaction substances included: inhibin A-inha, activin AB-ACTAB, activin BB, ACTBB, and the ratio of the concentration of mixed serum and pure serum with added interfering substances to the concentration of added interfering substances was measured respectively to calculate the cross-reaction rate of the kit, such as Figure 6 As shown, the cross-reaction rate is less than 0.05%, which is 10 times lower than the 0.5% cross-reaction on the market.

[0032] Since the homology of the βB subunit gene of inhibin B in humans and mice reaches 99%, immunizing bablc mice with inhibin B protein by conventional methods basically does not produce immunogenicity, so it is difficult to develop improved antibodies against other epitopes for the βB subunit. However, this application uses the inhibin B gene of balb / c mice to knock out the inhibin B gene, repeatedly immunizes mice with the expressed recombinant ACTBB at multiple sites, and screens monoclonal antibodies with the recombinant expressed inhb, thus producing a highly specific monoclonal inhibin βB (clone number 5G1 / 8A4).

[0033] The inhb-specific monoclonal antibody (antibody 5G1 / 8A4) was shown to be highly specific in the inhibin B detection kit without any sample pretreatment steps and was compared with a commercially available inhibin B assay, with significant correlation, ultimately making the detection using the kit using antibody 5G1 / 8A4 faster, more sensitive and simpler.

[0034] The above-described embodiments only express one or more implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. An improved inhibin B detection kit, characterized in that: The kit comprises: a first reagent and a second reagent; the first reagent comprises an antibody coupled to tosyl magnetic beads, and the second reagent comprises an antibody coupled to a luminescent marker; The antibody is an inhb-specific monoclonal antibody, which is a monoclonal antibody produced by cells obtained by immune screening of bablc mice with inhb gene knocked out.

2. The improved inhibin B detection kit according to claim 1, characterized in that: Also includes: The third reagent includes: a first luminescent reagent and a second luminescent reagent, the first luminescent reagent is 0.1 mol / L hydrochloric acid and 0.3% hydrogen peroxide, and the second luminescent reagent is 0.15 mol / L sodium hydroxide and 0.05% Triton 100.

3. The improved inhibin B detection kit according to claim 1, characterized in that: For the first reagent, the reaction buffer for the coupling reaction between the antibody and the tosyl magnetic beads is CBS, and the coupling catalyst is a coupling buffer containing 3M ammonium sulfate.

4. The improved inhibin B detection kit according to claim 3, characterized in that: For the coupling reaction, 1 mg of the tosyl magnetic beads was added to every 20 ug of the antibody, the reaction volume of the magnetic beads was 10 mg / ml, and the volume ratio of the reaction buffer to the coupling catalyst was 2:

1.

5. The improved inhibin B detection kit according to claim 3, characterized in that: Block the uncoupled beads by adding 5% casein, 5% BSA in PBS, pH 7.4, after the coupling reaction.

6. The improved inhibin B detection kit according to claim 5, characterized in that: Before performing the blocking, washing of unconjugated antibodies was performed using PBST.

7. The improved inhibin B detection kit according to claim 5, characterized in that: After the blocking step, 50 mM TBS, 5% BSA, 0.02% Tween 20, and 0.05% Proclin 300 magnetic bead diluent were added to dilute the magnetic bead concentration to 0.15-0.3 mg / ml.

8. The improved inhibin B detection kit according to claim 1, characterized in that: The second reagent is obtained by diluting the labeled antibody solution with an acridine diluent; NHS-SA-acridine is added to the antibody and incubated and purified to obtain the labeled antibody solution.

9. The improved inhibin B detection kit according to claim 8, characterized in that: The acridine diluent consists of 50 mM MES, 0.15 M NaCl, 0.5% BSA, 0.05% Tween 20, and 0.5% casein.

10. A method for preparing a monoclonal antibody, wherein the monoclonal antibody is the inhb-specific monoclonal antibody of claim 1, characterized in that: S10, breeding the homozygous mice with wild-type mice, performing PCR identification on the bred mice using specific primers, and screening to obtain subtype homozygous mice, wherein the homozygous mice are mice with complete excision of the inhb gene, and the wild-type mice are mice with no excision of the inhb gene; S20, immunizing homozygous mice of the subtype using a multi-site repeated immunization method, wherein the protein used in the multi-site repeated immunization method is the expressed and purified ACTBB protein; S021, after performing multiple immunizations, taking spleen cells and osteoma cells from homozygous mice of the subtype and fusing them to obtain hybridoma cells; S022, screening the hybridoma cells to obtain inhb-specific monoclonal cells, using the inhb-specific monoclonal cells to produce ascites to obtain the inhb-specific monoclonal antibodies.

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