An improved inhibin B detection kit and monoclonal antibody preparation method

Abstract

The present invention discloses an improved inhibin B detection kit and monoclonal antibody preparation method. 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 inhibin B-specific monoclonal antibody produced by immune screening of cells obtained from BabLc mice with inhibin B gene knockout. Compared to the commercially available Beckman Inhibin B kit, the kit achieves 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] Inhibins are multifunctional growth and differentiation factors that belong to the transforming growth factor β (TGFβ) superfamily. They are dimeric glycoprotein hormones 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. INHB has a molecular weight of approximately 32 kDa. INHB shares the same β subunit with activins (ACTs). Activins are homodimers or heterodimers composed of two identical or different subunits linked by disulfide bonds. Four activin isoforms have been found in the ovary: 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 a primary marker of ovarian reserve and testicular seminiferous tubule function, and can be used to detect female infertility caused by ovarian factors and male infertility caused by seminiferous tubule dysfunction. In men, inhibin B is a testicular-derived glycoprotein hormone. Serum inhibin B levels in adult males are significantly negatively correlated with FSH, exerting a negative feedback effect on FSH. In women, inhibin B is primarily produced by the granulosa cells of small and medium-sized antral follicles of the ovaries, where it can feedback-inhibit the release of follicle-stimulating hormone (FSH) from the anterior pituitary, regulating follicle formation. INHB levels reflect ovarian reserve earlier and more directly than FSH.

[0004] Inhibin B has become an important biomarker in clinical diagnostic analysis for the study of male and female reproductive function. Some of its 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 corresponding monoclonal antibody raw materials are currently available from DSL and OBI (both Beckman Coulter companies). Both assays utilize the same pair of monoclonal antibodies developed by Groome and colleagues in their laboratory over 20 years ago against synthetic peptides. The capture antibody (C5) is generated from a peptide derived from the βB subunit of inhibin, and the detection antibody R1 is generated from a peptide derived from the α subunit of inhibin. Both assays require a methionine oxidation step using hydrogen peroxide or heat denaturation followed by the addition of SDS. This is because the two methionine amino acids in the βB subunit peptide spontaneously oxidize to sulfoxides during the monoclonal antibody immunoassay, whereas methionines in natural samples are not oxidized. Therefore, to obtain full immunoreactivity of the antibodies and improve kit sensitivity, an oxidizing agent must be added to the kit to pre-oxidize the sample. Furthermore, for the enzyme immunoassay kit, adequate sensitivity requires an overnight incubation with the sample.

[0006] Manufacturers such as Yahuilong use the aforementioned method and raw materials. While this approach, using a magnetic microparticle chemiluminescence platform, shortens the reaction time somewhat, eliminating the need for overnight reactions, sample processing is still required, and the reaction time can exceed 45 minutes. Furthermore, the current inhibin B kit exhibits approximately 0.5% cross-reactivity with inhibin A.

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

[0008] To address 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 present invention employs the following technical solutions to address the aforementioned problems: an improved inhibin B detection kit comprising: a first reagent and a second reagent; the first reagent comprising an antibody coupled to tosyl magnetic beads, the second reagent comprising an antibody coupled to a luminescent marker;

[0009] 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 knockout.

[0010] In some embodiments, it also includes: a third reagent, the third reagent including: 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.

[0011] 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.

[0012] Furthermore, for the coupling reaction, 1 mg of the tosyl magnetic beads was added to every 20 ug of the antibody, the magnetic bead reaction volume was 10 mg / ml, and the volume ratio of the reaction buffer to the coupling catalyst was 2:1.

[0013] Furthermore, the uncoupled magnetic beads were blocked by adding 5% casein, 5% BSA in PBS, pH 7.4 blocking solution after the coupling reaction.

[0014] Furthermore, before performing the blocking step, uncoupled antibodies were washed using PBST.

[0015] Furthermore, 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.

[0016] 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.

[0017] Furthermore, the acridine diluent consists of 50 mM MES 0.15 M NaCl, 0.5% BSA, 0.05% Tween 20, and 0.5% casein.

[0018] And a method for preparing the above-mentioned inhb-specific monoclonal antibody, comprising: S10, breeding homozygous mice with wild-type mice, identifying the bred mice by PCR 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 non-excision of the inhb gene;

[0019] 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;

[0020] S021, after multiple immunizations, taking spleen cells and osteoblasts from homozygous mice of the subtype and fusing them to obtain hybridoma cells;

[0021] S022, screening the hybridoma cells to obtain inhb-specific monoclonal cells, using the obtained inhb-specific monoclonal cells to produce ascites to obtain the inhb-specific monoclonal antibody.

[0022] Because the βB subunit gene of inhibin B shares 99% homology between humans and mice, conventional immunization of bab / c mice with inhibin B protein produces little immunogenicity. Therefore, developing antibodies targeting other epitopes of the βB subunit is difficult. However, this application, by knocking out the inhibin B gene in bab / c mice, repeatedly immunizing mice at multiple sites with recombinant ACTBB, and screening monoclonal antibodies using recombinantly expressed inhb, has generated a highly specific monoclonal antibody against inhibin βB (clone number 5G1 / 8A4).

[0023] The inhb-specific monoclonal antibody (antibody 5G1 / 8A4) was shown to be highly specific in the inhibin B detection kit without the need for any sample pretreatment steps, and was compared with a commercially available inhibin B assay, showing significant correlation. Ultimately, the detection using the kit using antibody 5G1 / 8A4 can be faster, more sensitive, and simpler. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the PCR identification results of gene knockout mice;

[0025] Figure 2 This is a schematic diagram of the titer results of the four immune sera in Example 2;

[0026] 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;

[0027] Figure 4 This is a linear regression model diagram of the clinical correlation comparison table in Example 5;

[0028] Figure 5 This is a sensitivity test table of the kit of the present application in Example 6;

[0029] Figure 6 This is a table showing the cross-reaction interference experiment of the kit of the present application in Example 7. DETAILED DESCRIPTION

[0030] To make the above-mentioned objects, features, and advantages of the present invention more readily understood, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following description sets forth many specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0031] The monoclonal antibody preparation process is as follows: S10, breeding homozygous mice with wild-type mice, identifying the resulting mice by PCR using specific primers, and screening to obtain subtype homozygous mice (obtaining complete INHB immunoreactivity), wherein the homozygous mice are mice in which the inhb gene is completely removed, and the wild-type mice are mice in which the inhb gene is not removed;

[0032] 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;

[0033] S021, after multiple immunizations, taking spleen cells and osteoblasts from homozygous mice of the subtype and fusing them to obtain hybridoma cells;

[0034] S022, screening the hybridoma cells to obtain inhb-specific monoclonal cells, using the obtained inhb-specific monoclonal cells to produce ascites to obtain the inhb-specific monoclonal antibody.

[0035] Example 1, preparation and identification of genetically modified mice, wherein the genetically modified bablc mice were obtained from Saiye Bio; the genetically modified mice were named C57BL / 6N-Inhbb, the sequence name was KOCMP-16324-Inhbb, the knockout gene was named Inhbb, the NCBI ID was 16324, and the chain length region C57BL / 6N was excised;

[0036] First, homozygous mice (completely ablated inhb gene) were bred with wild-type mice (non-ablated inhb gene). PCR was then performed using specific primers to identify the subtype of mice with inhb gene ablation.

[0037] The identification process for gene-ablated mice is as follows: 0.2 cm of the mouse tail is taken, and the mouse gene is released using a one-step mouse gene extraction kit. After heating to destroy the protease activity, the full sequence of the mouse gene is obtained. Specific primers are set based on the gene of the gene-knocked mouse and the gene of the mouse that has not been gene-knocked as follows:

[0038] PCR Primer 1 - Gene excision specific primer (Annealing Temperature 60.0 ºC):

[0039] F1:5'-GTCCCTTGGAACTCCTTAGAAACA-3'

[0040] R1: '-AATTAAGACCCTTCTCCTCCCTC-3'

[0041] Product size: 575 bp

[0042] PCR Primer 2 - Gene-specific primer (Annealing Temperature 60.0 ºC):

[0043] F2: 5'-TTCTCCCGCTTTGTAGAAAACCA-3'

[0044] R1: 5'-AATTAAGACCCTTCTTTCCTCCCTC-3'

[0045] Product size: 634 bp

[0046] Homozygous: one band with 575 bp

[0047] Heterozygote: two bands with 575 bp and 634 bp

[0048] Wild type: one band with 634 bp

[0049] The results of PCR identification of gene knockout mice are as follows Figure 1 As shown, mice 6 and 7 are homozygous, with only a single band at 575 bp; mice 4, 8, and 12 are wild-type, with only a single band at 634 bp; and mice 2, 3, 9, 10, and 13 are heterozygous, with two bands at 575 and 634 bp. Mice 6 and 7 (which exhibited complete INHB immunoreactivity) were selected for the next immunization step.

[0050] 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 the second immunization was performed two weeks later, and the protein dosage for the second immunization was 30-60ug / mouse. The tail vein blood was collected 3 days after the second immunization to test the mouse serum titer. The results showed that the mouse serum titer was high and the immune effect was good; the third and fourth immunizations were performed in the same way 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, venous blood was collected from the mouse tail 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. A 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.

[0051] Example 3, kit preparation, preparation of the first reagent: The obtained inhb-specific monoclonal antibody was coupled to tosyl magnetic beads (manufactured by JSR or Thermo). The reaction buffer for the coupling reaction was CBS, pH 9.6, and the coupling catalyst was a coupling buffer solution containing 3M ammonium sulfate. 1mg of the tosyl magnetic beads was added for every 20ug of the antibody, the magnetic bead reaction volume was 10mg / ml, and the reaction buffer was 2 / 3 reaction buffer and 1 / 3 coupling catalyst. The reaction was incubated at 37°C for 16-24 hours. Excess uncoupled antibody was then washed with PBST. Uncoupled magnetic beads were then blocked with 5% casein, 5% BSA in PBS, pH 7.4 blocking buffer for 12-24 hours. After blocking, 50mM TBS, 5% BSA, 0.02% Tween 20, and 0.05% Proclin 300 magnetic bead diluent was added to dilute the magnetic beads to a concentration of 0.15-0.3mg / ml to obtain the first reagent.

[0052] To prepare the second reagent, add 30 μg of NHS-SA-acridine per 1 mg of antibody (inhb-specific monoclonal antibody) and incubate at room temperature for 2 hours. Purify the labeled antibody solution through molecular sieves and dilute it to 0.1-0.4 μg / ml with acridine diluent, which contains 50 mM MES, 0.15 M NaCl, 0.5% BSA, 0.05% Tween 20, and 0.5% casein.

[0053] And a third reagent, the third reagent includes: a first luminescent reagent and a second luminescent reagent, the first luminescent reagent is 0.1mol / L hydrochloric acid and 0.3% hydrogen peroxide, and the second luminescent reagent is 0.15mol / L sodium hydroxide and 0.05% Triton 100.

[0054] 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 washing 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.

[0055] Example 5, clinical relevance, the kit obtained in Example 3 of this 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 graph shown, Figure 4 The 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 punctuation points in the table are the clinical values ​​measured by the Beckman Inhibin 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. It is concluded that the kit of the present application has a good correlation.

[0056] Example 6, kit sensitivity, analytical sensitivity is defined as the average value plus twice the standard deviation of the photon number measured 20 times for the zero value calibrator test, and the obtained photon number is put 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).

[0057] Example 7, cross-reaction rate, pure serum was added with analogs for cross-reaction interference experiment, cross-reaction substances included: inhibin A-inha, activin AB-ACTAB, activin BB, ACTBB, respectively, the concentration of mixed serum and pure serum with added interfering substances was measured to calculate the cross-reaction rate of the kit, such as Figure 6As shown, the cross-reaction rate is less than 0.05%, which is 10 times lower than the 0.5% cross-reaction on the market.

[0058] Because the βB subunit gene of inhibin B shares 99% homology between humans and mice, conventional immunization of bab / c mice with inhibin B protein produces little immunogenicity. Therefore, developing antibodies targeting other epitopes of the βB subunit is difficult. However, this application, by knocking out the inhibin B gene in bab / c mice, repeatedly immunizing mice at multiple sites with recombinant ACTBB, and screening monoclonal antibodies using recombinantly expressed inhb, has generated a highly specific monoclonal antibody against inhibin βB (clone number 5G1 / 8A4).

[0059] The inhb-specific monoclonal antibody (antibody 5G1 / 8A4) was shown to be highly specific in the inhibin B detection kit without the need for any sample pretreatment steps, and was compared with a commercially available inhibin B assay, showing significant correlation. Ultimately, the detection using the kit using antibody 5G1 / 8A4 can be faster, more sensitive, and simpler.

[0060] The embodiments described above merely represent one or more embodiments of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the scope of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended 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 knockout. The method for preparing the inhb-specific monoclonal antibody comprises: S10, breeding homozygous mice with wild-type mice, identifying the bred mice by PCR 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 inhb gene not excision; S20, immunizing the subtype homozygous mice 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; 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; For the coupling reaction, 1 mg of the tosyl magnetic beads was added to every 20 μg of the antibody, the magnetic bead reaction volume was 10 mg / ml, and the volume ratio of the reaction buffer to the coupling catalyst was 2:1; Block the uncoupled beads by adding 5% casein, 5% BSA in PBS, pH 7.4 blocking buffer after the coupling reaction.

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 Before performing the blocking, washing of unconjugated antibodies was performed using PBST.

4. The improved inhibin B detection kit according to claim 1, 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.

5. 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 acridine diluent; NHS-SA-acridine is added to the antibody and incubated and purified to obtain the labeled antibody solution.

6. The improved inhibin B detection kit according to claim 5, 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.

7. The improved inhibin B detection kit according to claim 1, characterized in that The method for preparing the inhb-specific monoclonal antibody further comprises: S021, after multiple immunizations, taking spleen cells and osteoblasts 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 obtained inhb-specific monoclonal cells to produce ascites to obtain the inhb-specific monoclonal antibody.

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