LU502312B1

LU502312B1 - Process for coating keyhole limpet hemocyanin (klh) and kit for quantitative detection of mouse klh-specific igg antibodies

Info

Publication number: LU502312B1
Application number: LU502312A
Authority: LU
Inventors: Wenhua Zhong; Hui Huang; Zhaoxin Yang
Original assignee: Hainan Medical Univ
Priority date: 2022-06-20
Filing date: 2022-06-20
Publication date: 2022-12-20

Abstract

The present invention discloses a kit for quantitative detection of mouse KLH-specific IgG antibodies, which includes KLH-specific IgG antibody-captured microspheres, sample pretreatment lgM captured microspheres, standard substances, FITC labeled detection fluorescent antibodies, a sample diluent, and a washing buffer; and preparation of the KLH-specific IgG antibody-captured microspheres includes the following steps: (1) dissolving KLH proteins in a carbonate buffer containing 0.1% dithiothreitol (DTT); (2) activating microspheres; and (3) adding the activated microspheres, and after the reaction is completed, performing blocking. In the present invention, by adjusting a coating solution, KLH proteins are depolymerized and effectively ligated to polystyrene carboxyl microspheres, thereby improving efficiency of coupling between the KLH proteins and the microspheres; and in addition, by performing pretreatment on a to-be-tested sample, interference of lgM antibodies in immune response is reduced, and sensitivity and accuracy of quantitative detection of lgG antibodies in immune serum are improved.

Description

PROCESS FOR COATING KEYHOLE LIMPET HEMOCYANIN (KLH) AND KIT FOR QUANTITATIVE DETECTION OF MOUSE KLH-SPECIFIC IGG ANTIBODIES FIELD OF THE INVENTION

[0001] The present invention relates to the field of detection kits, and in particular, to a process for coating keyhole limpet hemocyanin (KLH) and a kit for quantitative detection of mouse KLH-specific IgG antibodies.

BACKGROUND OF THE INVENTION

[0002] In recent years, TDAR test is considered to be a functional test for detecting potential immunotoxicity of drugs and having good predictability. It can reflect the influence of a to-be-tested drug on an entire immune system by artificially importing foreign antigens, predict a change in an immune function, and predict characteristics of drug immune regulation and immunotoxicity. Therefore, the method has gradually been widely used in drug immunotoxicology research. However, the conventional TDAR test has the problems of poor specificity of the imported antigens, complex operation, large variability, and the like, which lead to great defects in the specificity, sensitivity, precision, repeatability, operability, and the like of the test. In recent years, many scholars at home and abroad have studied keyhole limpet hemocyanin (KLH) as an imported foreign antigen. During detection of KLH-specific IgG antibodies in peripheral blood of experimental animals such as rats and pigs by using the ELISA method, it is found that compared with other antigens, the KLH, as a foreign antigen, has the advantages of less variability, stable results, and the like, and is more suitable as a T cell-dependent antigen.

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[0003] Although the TDAR test method using the KLH as a specific antigen has many advantages, there are still some problems:

[0004] (1) The KLH is a soluble protein extracted from marine organisms, and for mammals, the KLH is a foreign protein with high immunogenicity. The KLH, as a natural polymer protein, has a molecular weight of 8000-9000 KDa, and has two subunits, KLH; (400 KDa) and KLH, (345 KDa). For microspheres with a diameter of 1-10 um, the KLH protein having a molecular weight more than 8000 KDa is not suitable as a coupling protein, and therefore it needs to be dissociated into monomers. Usually, when the pH of a coating solution is 9.6, the KLH polymer protein dissociates, but the dissociated KLH subunits are not stable and may again form polymer, and therefore coupling efficiency of the KLH protein and the microspheres 1s low, resulting in a low detection level.

[0005] (2) Different immune doses, immune pathways, and cycles may produce different immune effects. In an immune response process, lgG and 1gM antibodies may be produced. Therefore, there is interference of an IgM detection matrix in serum quantification, resulting in reduction in accuracy of KLH-IgG antibody detection.

[0006] In addition, most of the TDAR test detection methods use an indirect ELISA method, and experimental results are mainly qualitative. Therefore, it is impossible to fully reflect immunotoxicity for different immunosuppressive drugs or stimulating drugs. Besides, some known immunosuppressants, such as chemotherapy drugs, have immunosuppression themselves, for which, what needs to be investigated 1s a degree of immunosuppression, and qualitative analysis cannot meet test needs.

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BRIEF SUMMARY OF THE INVENTION

[0007] To solve the problems existing in the existing technology, the present invention provides a process for coating keyhole limpet hemocyanin (KLH) and a kit for quantitative detection of mouse KLH-specific IgG antibodies. By adjusting a coating solution, KLH proteins are depolymerized and effectively ligated to polystyrene carboxyl microspheres, thereby improving efficiency of coupling between the KLH proteins and the microspheres; and in addition, by performing pretreatment on a to-be-tested sample, interference of 1gM antibodies in immune response is reduced, and sensitivity and accuracy of quantitative detection of 1gG antibodies in immune serum are improved.

[0008] The present invention is implemented by the following technical solutions:

[0009] A process for coating keyhole limpet hemocyanin (KLH) includes the following steps: (1) dissolving KLH proteins in a carbonate buffer containing 0.1% dithiothreitol (DTT), to obtain a coating protein solution; (2) activating carboxyls on surfaces of microspheres by activators to obtain activated microspheres; and (3) adding the activated microspheres after activation to the coating protein solution in step (1) for coupling, and after the reaction is completed, performing blocking to obtain KLH-specific IgG antibody-captured microspheres.

[0010] As a preferred solution of the present invention, a conventional microsphere coating solution is generally a carbonate coating solution, and the coating solution used in the present invention is obtained by adding

0.1% dithiothreitol (DTT) to a conventional carbonate coating solution, which can effectively combine KLH monomers with polystyrene carboxyl microspheres, thereby improving chemical coupling efficiency.

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[0011] As a preferred solution of the present invention, a composition proportion of the coating protein solution in step (1) is as follows: a concentration of KLH proteins is 80-160 g/L, a concentration of dithiothreitol is 1 g/L, and a pH value of the carbonate buffer is 9.6.

[0012] As a preferred solution of the present invention, the activators in step (2) are 1-ethyl-3-[3-dimethylaminopropyl] carbonimine (EDC) and acetyl-glycine N-hydroxysuccinimide ester (S-NHS), and the activation time 1s 20 min; and the microspheres are polystyrene carboxyl microspheres.

[0013] As a preferred solution of the present invention, a ratio of a quantity of the activated microspheres to a volume of the coating solution in step (3) is 500/uL.

[0014] As a preferred solution of the present invention, the coupling reaction in step (3) lasts 2 hours, and a blocking agent is a 2% BSA solution.

[0015] A kit for quantitative detection of mouse KLH-specific IgG antibodies includes KLH-specific IgG antibody-captured microspheres, sample pretreatment lgM captured microspheres, standard substances, FITC labeled detection fluorescent antibodies, a sample diluent, and a washing buffer.

[0016] The content proportion of the reagents in the kit is as follows:

[0017] sample pretreatment IgM captured microspheres: 1.5 mL (1.25x10")/tube, with a microsphere dosage of 3 uL/test, which can be used for 500 experiments, and used for removing interference of lgM from serum;

[0018] KLH-specific IgG antibody-captured microspheres: 1.5 mL 4

(1.25x107)/tube, with a microsphere dosage of 6 uL/test, which can be used for 250 experiments;

[0019] standard substances: at a concentration of 0.4 mg/mL, each tube of standard substances being diluted with 200 uL of sample diluent as a highest concentration of the standard substances;

[0020] FITC labeled detection fluorescent antibodies: 2 mL, at a concentration of 0.06 mg/mL, and with a dosage of 100 uL/test;

[0021] sample diluent: 25 mL of PBS phosphate buffer with a concentration of 0.01 mol/L and the pH of 7.2-7.4 and containing 1% BSA, used for diluting the standard substances or a to-be-tested sample; and washing buffer:

[0022] 500 mL of PBS phosphate buffer with a concentration of 0.01 mol/L and the pH of 7.2-7.4 and containing 0.05% tween-20, used for cleaning steps and resuspending washed microspheres.

[0023] A method for preparing the sample pretreatment 1gM captured microspheres includes: coupling activated microspheres with anti-mouse IgM-specific antibodies for 2 hours, and after the reaction is completed, performing blocking to obtain IgM antibody-captured microspheres.

[0024] As a preferred solution of the present invention, the anti-mouse IgM-specific antibodies are anti-mouse IgM pu chain-specific IgG antibodies.

[0025] A method for use and operation of the kit includes the following steps: (1) taking sample pretreatment lgM captured microspheres to add to a to-be-tested serum sample for pretreatment for 1 h, to remove KLH-specific IgM antibodies from the to-be-tested serum sample; (2) adding the KLH-specific IgG antibody-captured microspheres that have undergone the pretreatment to the serum sample; and (3) adding FITC labeled detection fluorescent antibodies and then measuring a fluorescence intensity MFI value of an FITC channel.

[0026] The kit of the present invention includes sample pretreatment 1gM captured microspheres, and before IgM antibodies are detected, a serum sample is pre-treated, which can remove KLH-specific IgM antibodies from the serum sample, thereby reducing interference of the KLH-specific IgM antibodies in the to-be-tested serum sample on quantitative detection of IgG antibodies; in addition, by using the KLH-specific IgG antibody-captured microspheres prepared by the coating process of the present invention, lgG antibodies in the immune serum can be better quantitatively analyzed.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. In the process for coating keyhole limpet hemocyanin (KLH) of the present invention, by improving the coating solution, KLH proteins can be depolymerized and effectively ligated to polystyrene carboxyl microspheres, thereby improving efficiency of coupling.

[0029] 2. In the kit for quantitative detection of mouse KLH-specific IgG antibodies of the present invention, by pre-treating a to-be-tested sample, interference of 1gM antibodies in immune response is reduced, so that IgG antibodies in the immune serum can be better quantitatively analyzed.

[0030] 3. The kit for quantitative detection of mouse KLH-specific IgG antibodies of the present invention requires a small amount of samples, has a high detection sensitivity, is convenient to operate, has high practicability, and can be directly used to perform quantitative analysis of a TDAR 6 experiment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The drawings illustrated herein are used to provide a further understanding of the embodiments of the present invention, and constitute a part of the present application, and do not constitute a limitation to the embodiments of the present invention. In the drawings:

[0032] FIG. 1 is a diagram of the linearity curve of a standard substance solution according to an embodiment of the present invention;

[0033] FIG. 2 is the diagram of the detection concentration values of mouse KLH-specific IgG antibodies in a to-be-tested sample according to an embodiment of the present invention;

[0034] FIG. 3 is a diagram of the flow analyzer of the serum sample according to an embodiment of the present invention;

[0035] FIG. 4 is a comparative diagram of MFI values of the to-be-tested sample before and after pre-treatment according to an embodiment of the present invention; and

[0036] FIG. 5 is a comparative diagram of MFI values of antibody captured microspheres respectively prepared by using a coating solution of the present invention and an existing coating solution according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below with reference to embodiments and drawings. The schematic implementations of the present invention and descriptions thereof are only used to explain the present invention, and are not intended to limit the 7 present invention.

[0038] Embodiment 1

[0039] A process for coating KLH proteins specifically includes the following steps:

[0040] (1) KLH proteins were dissolved in a carbonate buffer containing

0.1% dithiothreitol (DTT) with the pH of 9.6, to obtain a coating protein solution;

[0041] 2) carboxyls on surfaces of polystyrene carboxyl microspheres were activated by activators EDC and S-NHS for 20 min, to obtain activated microspheres;

[0042] 3) the polystyrene carboxyl microspheres after activation were added to the coating protein solution for coupling for 2 h; and

[0043] 4) after the reaction was completed, 2% bovine serum albumin was added to the solution for blocking for 30 min, to obtain KLH-specific IgG antibody-captured microspheres; then the captured microspheres were added to a PBS phosphate buffer storage solution containing 0.05% sodium azide with the pH of 7.2-7.4 for storage.

[0044] In the embodiment, an addition amount of the microspheres, and types and addition amounts of the blocking agent and the storage solution are the same as those in the existing technology, and therefore the addition amounts and addition types of the substances are not described one by one.

[0045] In the present invention, by using the improved coating solution, that is, the carbonate buffer with the pH of 9.6 and containing 0.1% dithiothreitol (DTT), the KLH proteins can be depolymerized, thereby improving coupling efficiency between the KLH proteins and the microspheres.

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[0046] Comparative Embodiment 1

[0047] A process for coating KLH proteins differs from embodiment 1 in that: a conventional carbonate buffer free of 0.1% dithiothreitol (DTT) was used in step (1).

[0048] The KLH-specific IgG antibody-captured microspheres prepared in Example 1 and the microspheres prepared in comparative embodiment 1 were taken to measure fluorescence intensity MFI values. Results are shown in FIG 5. It can be clearly learned from the figure that the MFI values of this embodiment are significantly higher than those of comparative embodiment 1, which indicates that the improved coating solution of the present invention can improve coupling between the KLH proteins and the microspheres.

[0049] Embodiment 2

[0050] A kit for quantitative detection of mouse KLH-specific IgG antibodi es was provided and the reagent proportion was as follows:

[0051] sample pretreatment IgM captured microspheres: 1.5 mL (1.25x107) /tube, with a microsphere dosage of 3 uL/test, which can be used for 500 ex periments, and used for removing the interference of IgM from serum;

[0052] KLH-specific IgG antibody-captured microspheres: 1.5 mL (1.25x1 07)/tube, with a microsphere dosage of 6 uL/test, which can be used for 25 0 experiments;

[0053] standard substances: at a concentration of 0.4 mg/mL, each tube of s tandard substances being diluted with 200 uL of sample diluent as a highest concentration of the standard substances;

[0054] FITC labeled detection fluorescent antibodies: 2 mL, at a concentrat 9 ion of 0.06 mg/mL, and with a dosage of 100 uL/test;

[0055] sample diluent: 25 mL of PBS phosphate buffer with a concentratio n of 0.01 mol/L and the pH of 7.2-7.4 and containing 1% BSA, used for dil uting the standard substances or a to-be-tested sample; and

[0056] washing buffer: 500 mL of PBS phosphate buffer with a concentrati on of 0.01 mol/L and the pH of 7.2-7.4 and containing 0.05% tween-20, use d for cleaning steps and resuspending washed microspheres.

[0057] The specific test steps of the kit are in the following:

[0058] 1. Preparation of a microsphere standard solution

[0059] 1.1 KLH-specific IgG antibody-captured microspheres were taken a nd diluted by 1.2 times ;

[0060] 1.2 100 uL of a standard substance solution was taken and diluted b y 500000, 250000, 125000, 62500, 31250, 15625, 7812.5, and 3906.25 (pg/ mL) by using a 1% BSA sample diluent;

[0061] 1.3 50 uL of standard substances and 50 pL. of resuspended KLH-sp ecific IgG antibody-captured microspheres were added to each sample tube , oscillated for 30s, and incubated at room temperature for 2 h;

[0062] 1.4 1:25 diluted FITC labeled detection fluorescent antibodies were added to each sample tube and incubated at room temperature for 1 h;

[0063] 1.5 500 uL of washing buffer was added for washing, and centrifuge d at 14000 g for 4 min, to remove a supernatant; and

[0064] 1.6 200 uL of washing buffer was added, and uniform vortex mixing was performed.

[0065] 2. Draw of a standard curve

[0066] Through detection and data analysis, a detection microsphere gate w as circled in an FSC/SSC scatter diagram, to obtain 3500 microspheres, and fluorescence intensity MFI values in an FITC channel were detected. The standard curve was drawn with a dilution concentration of the standard substances as the abscissa (X) and a logarithm of the mean fluorescence intensity (MFI) as the ordinate (Y). As shown in FIG. 1, measured results were fitted by using linear regression, to obtain equations: y = (A-D)/[1+(</C)"B]"E+D, R? = 0.9993, À = 10.090, B = 14.122, C = 21.873, D = 15.805, and E = -11.800, indicating that linearity of the method is good.

[0067] 3. Pre-treatment of the to-be-tested sample

[0068] 3.1 Serum was diluted to 1:100, and 100 pL of diluted serum was added to each sample tube;

[0069] 3.2 50 pL of resuspended sample pretreatment 1gM captured microspheres were again added and incubated at room temperature for 1 h; and

[0070] 3.3 centrifugation at 14000 g was performed for 4 min, to extract 50 pL of supernatant.

[0071] 4. Quantitative detection of IgG antibodies in the to-be-tested samp le

[0072] 4.1 50 uL of resuspended KLH-specific IgG antibody-captured mi crospheres were added and incubated at room temperature for 2 h;

[0073] 4.2 100 uL of 1:25 diluted FITC labeled detection antibodies were added and incubated at room temperature for 2 h;

[0074] 4.3 500 uL of washing buffer was added for washing, and centrifug ed at 14000 g for 4 min, to remove a supernatant; and

[0075] 4.4 200 uL of washing buffer was added for washing, and uniform 11 vortex mixing was performed.

[0076] 5. Detection of the to-be-tested sample

[0077] FIG 2 illustrates the concentration values (std1-std8) of mouse KLH-specific IgG antibodies in standard substances and the concentration value result (Testl) of antibodies in the to-be-tested serum sample is calculated according to the standard curve.

[0078] A detection microsphere gate was circled in an FSC/SSC scatter diagram, to obtain 3500 microspheres, and fluorescence intensity MFI values in an FITC channel were detected. As shown in FIG. 3, the left side was the KLH polyclonal antibody microsphere flow analyzer FSC/SSC diagram for the to-be-tested sample; and the middle and the right sides were diagrams of the results obtained by using the fluorescent microsphere flow cytometer for the to-be-tested sample obtained by regulating the FSC and the SSC.

[0079] Comparative Embodiment 2

[0080] A kit for quantitative detection of mouse KLH-specific IgG antibo dies differs from embodiment 2 in that comparative embodiment 2 does not include sample pretreatment IgM captured microspheres.

[0081] The specific detection operation steps of the kit differ from those o f Example 2 in that step 3 of pre-treatment of the to-be-tested sample is not included.

[0082] FIG 4 illustrates MFI values of a to-be-tested sample in embodiment 2 after pretreatment and MFI values of the to-be-tested sample in comparative embodiment 2 without pretreatment. As can be learned from the figure, the MFI values of embodiment 2 are significantly higher than those of comparative embodiment 2, indicating that KLH-specific IgM antibodies in the sample are removed from the to-be-tested sample through the pretreatment, thereby reducing interference thereof on the detection.

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[0083] The specific implementations described above further describe the mt objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the foregoing descriptions are only specific implementations of the present invention and are not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, and the like made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

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Claims

WHAT IS CLAIMED IS:

1. A process for coating keyhole limpet hemocyanin (KLH), comprising the following steps: (1) dissolving KLH proteins in a carbonate buffer containing 0.1% dithiothreitol (DTT), to obtain a coating protein solution; (2) activating carboxyls on surfaces of microspheres by activators to obtain activated microspheres; and (3) adding the activated microspheres after activation to the coating protein solution in step (1) for coupling, and after the reaction is completed, performing blocking to obtain KLH-specific IgG antibody-captured microspheres.

2. The process for coating keyhole limpet hemocyanin (KLH) according to claim 1, wherein a composition proportion of the coating protein solution in step (1) is as follows: a concentration of KLH proteins is 80-160 g/L, a concentration of dithiothreitol is 1 g/L, and a pH value of the carbonate buffer is 9.6.

3. The process for coating keyhole limpet hemocyanin (KLH) according to claim 1, wherein the activators in step (2) are EDC and S-NHS.

4. The process for coating keyhole limpet hemocyanin (KLH) according to claim 1, wherein a ratio of a quantity of the activated microspheres to a volume of the coating solution in step (3) 1s 500/uL.

5. The process for coating keyhole limpet hemocyanin (KLH) according to claim 1, wherein the coupling reaction in step (3) lasts 2 hours, and a blocking agent is a 2% BSA solution.

6. A kit for quantitative detection of mouse KLH-specific IgG antibodies, comprising KLH-specific IgG antibody-captured microspheres that is prepared by the process according to any one of claims 1 to 5, sample pretreatment 1gM captured microspheres, standard substances, FITC labeled detection fluorescent antibodies, a sample diluent, and a washing 14 buffer.

7. The kit for quantitative detection of mouse KLH-specific IgG antibodies according to claim 6, wherein a method for preparing the sample pretreatment lgM captured microspheres comprises: coupling activated microspheres with anti-mouse IgM-specific antibodies, and after the reaction 1s completed, performing blocking to obtain IgM antibody-captured microspheres.

8. The kit for quantitative detection of mouse KLH-specific IgG antibodies according to claim 7, wherein the anti-mouse IgM-specific antibodies are anti-mouse IgM u chain-specific IgG antibodies.

9. The kit for quantitative detection of mouse KLH-specific IgG antibodies according to claim 6, wherein a method for use and operation of the kit comprises the following steps: (1) taking sample pretreatment 1gM captured microspheres to add a to-be-tested serum sample for pretreatment, to remove KLH-specific [gM antibodies from the to-be-tested serum sample; (2) adding the KLH-specific IgG antibody-captured microspheres that have undergone the pretreatment to the serum sample; and (3) adding FITC labeled detection fluorescent antibodies and then measuring a fluorescence intensity MFI value of an FITC channel.