Reagent, kit and detection method for free light chain detection
By using a solid-phase carrier labeled with F(ab')2 antibody fragments and a composite blocking agent, and by enzymatically digesting the antibody fragments to address the non-specific reaction of the antibody Fc segment, the non-specific reaction of the antibody Fc segment is solved, thus resolving the technical problems existing in the prior art and achieving a highly efficient and specific reaction. This significantly improves the specificity and accuracy of free light chain detection and achieves new technical effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING BIOSTEC BIOTECH
- Filing Date
- 2026-03-20
- Publication Date
- 2026-05-12
AI Technical Summary
Among existing methods for detecting free light chains, latex-enhanced immunoturbidimetry suffers from non-specific reactions caused by antibody Fc fragments and endogenous interfering substances, resulting in abnormal detection linearity and low specificity.
The solid-phase carrier was labeled with F(ab')2 antibody fragment and combined with a composite blocking agent. The Fc fragment was removed by enzymatic hydrolysis of the antibody to reduce non-specific reactions. The blocking agent was added to the liquid-phase reaction solution to shield interfering substances and improve detection specificity and linearity.
It significantly improves the specificity and accuracy of free light chain detection, solves the problem of non-linearity, and meets the precision needs of clinical testing.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of detection kit technology, specifically to a reagent, kit, and detection method for detecting free light chains. Background Technology
[0002] Immunoglobulin (Ig) monomers are composed of two identical heavy chains (H chains) and two identical light chains (L chains) linked by disulfide bonds. Light chains are classified into two types: Kappa chains (κ chains) and Lambda chains (λ chains). Due to their smaller molecular weight, light chains are synthesized faster than heavy chains. After binding with heavy chains, approximately 40% of light chains remain free in the serum, known as free light chains (FLCs). In normal individuals, serum FLC levels are maintained within a certain range. However, abnormal plasma cells secrete significantly more monoclonal light chains than heavy chains, resulting in excessive FLCs in the serum, as seen in conditions such as multiple myeloma and systemic amyloidosis. Clinically, various immune system diseases exhibit abnormalities in immunoglobulins and light chains. In autoimmune diseases, autoantigens are complex and diverse. B cells, stimulated by different antigens, form diverse plasma cell clones, resulting in not only increased quantities of synthesized immunoglobulins but also polyclonal synthesis. Free light chain detection reagents have wide clinical applications.
[0003] Traditional methods for analyzing free light chains include serum protein electrophoresis and immunofixation electrophoresis, but their low sensitivity and inability to quantify them fail to meet clinical needs. Immunoturbidimetric analysis effectively solves this problem, and most commercially available free light chain reagents currently use this method. The biggest advantage of this method is the design of antibodies that specifically recognize and bind to antigenic epitopes at hidden sites on Ig molecules. This allows for highly efficient and accurate detection and analysis of free light chains through changes in the turbidity of the antigen-antibody complex.
[0004] However, common problems exist in actual clinical applications, with some samples exhibiting disproportionate linearity. The core issue is the occurrence of nonspecific reactions. Latex-enhanced immunoturbidimetry often uses intact antibody-labeled solid-phase microspheres. The Fc fragment of the antibody contains antigen-binding sites, complement-binding sites, etc., and the binding reaction of the Fc fragment reduces specificity. In addition, endogenous interfering substances in the sample, such as heterophile antibodies, human anti-animal antibodies, and rheumatoid factor, can nonspecifically bind to the antibody. All of these factors reduce specificity, further leading to disproportionate linearity.
[0005] In existing technologies, screening for highly specific antibodies reduces the occurrence of side effects, but it cannot completely eliminate them. Most commercial antibodies contain Fc fragments, and some antibodies do not meet the standards for anti-interference performance, resulting in poor clinical specificity and linearity.
[0006] Based on this, this application is hereby submitted. Summary of the Invention
[0007] The technical problem this invention aims to solve is that the latex-enhanced immunoturbidimetric assay for free light chains suffers from non-specific reactions caused by antibody Fc fragments and endogenous interfering substances, which cannot be eradicated by existing methods, resulting in abnormal detection linearity and low specificity. The objective is to provide a reagent, kit, and detection method for the detection of free light chains.
[0008] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a free light chain detection kit, comprising the following components: A solid-phase support with F(ab')2 antibody fragments labeled on its surface; Liquid-phase reaction solution, comprising a composite blocking agent for inhibiting non-specific reactions of interfering substances; The F(ab')2 antibody fragment therein works in conjunction with the composite blocking agent to improve the specificity and linearity of the detection.
[0009] As one of the preferred embodiments, the F(ab')2 antibody fragment is prepared by the following steps: using rabbit anti-human free light chain polyclonal antibody as raw material, after digestion with pepsin, the Fc fragment and the undigested intact antibody are removed by Protein A chromatography column, thereby obtaining the purified F(ab')2 antibody fragment.
[0010] As one of the preferred embodiments, the composite blocking agent includes 0.1 mg / mL to 0.5 mg / mL rheumatoid factor antibody, 0.1 mg / mL to 0.5 mg / mL rabbit IgG, 0.1 mg / mL to 0.5 mg / mL rabbit IgM, and 0.1 mg / mL to 0.5 mg / mL heterophile antibody blocking agent.
[0011] As one of the preferred embodiments, the working concentration of the composite blocking agent in the liquid-phase reaction solution is 0.1 mg / mL-0.5 mg / mL.
[0012] As one of the preferred embodiments, the solid support is latex microspheres with a particle size of 60-200 nm.
[0013] Furthermore, the latex microspheres, with a wavelength of 60–200 nm, possess a moderate and sufficiently large specific surface area, enabling efficient loading of F(ab')2 antibody fragments. This increases antigen-antibody binding sites and significantly enhances the light scattering signal after immune complex formation, thereby improving the detection sensitivity for free light chains and meeting the accurate detection needs of low-concentration clinical samples. It also ensures reagent dispersion stability and reduces non-specific interference and background turbidity. Synergistically, it works with the F(ab')2 antibody fragments and composite blocking agents to significantly improve the specificity, linear range, and accuracy of free light chain immunoturbidimetric detection. As a preferred embodiment, the F(ab')2 antibody fragments are covalently coupled to the surface of the latex microspheres.
[0014] As one of the preferred embodiments, the liquid-phase reaction solution further includes 100 mM / L HEPES buffer, sodium chloride, polyethylene glycol, and surfactant.
[0015] Secondly, the present invention provides a kit for detecting free light chains, comprising: R1 reagent: the above liquid-phase reaction solution; R2 reagent: the solid-phase carrier and latex preservation solution of the above-mentioned conjugated F(ab')2 antibody fragment.
[0016] As one of the preferred embodiments, the latex preservation solution comprises 100 mM / L HEPES buffer, 2%–15% sucrose, 0.1%–1% BSA, and 0.01%–0.1% ProClin300.
[0017] Thirdly, a method for detecting the content of free light chains in a sample includes the following steps: Mix the sample to be tested with reagent R1 of the above kit and pre-incubate; Add reagent R2 from the kit to the above mixture to initiate the immune reaction; Monitor the turbidity changes in the reaction system and calculate the concentration of free light chains in the sample based on the standard curve.
[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The reagent for detecting free light chains of the present invention uses a solid-phase carrier labeled with F(ab')2 antibody fragments and a composite blocking agent. The two work synergistically to form a closed-loop protection from two dimensions: "eliminating antibody self-interference" and "shielding sample exogenous interference". This completely avoids non-specific side reactions that cannot be eradicated by a single method and synergistically improves the binding targeting and detection specificity of the target analyte.
[0019] 2. The reagent for free light chain detection of the present invention effectively eliminates non-specific binding caused by the Fc segment of intact antibodies, and can also specifically shield the effects of endogenous interfering substances such as heterophilic antibodies, human anti-animal antibodies, and rheumatoid factor in the sample. It completely avoids various side reactions caused by the antibody itself and exogenous interfering substances from two dimensions; it greatly improves the specificity and binding targeting of the detection for free light chain target analytes, successfully solves the common problem of sample linearity disproportion in clinical testing, significantly optimizes the accuracy, stability and reliability of the detection results, and meets the actual clinical needs for accurate detection of free light chains. Detailed Implementation
[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are only used to explain this invention and are not intended to limit this invention.
[0021] Throughout this specification, references to “an embodiment,” “an example,” or “an example” mean that a particular feature, structure, or characteristic described in connection with that embodiment or example is included in at least one embodiment of the invention. Therefore, the phrases “an embodiment,” “an example,” “an example,” or “an example” appearing in various places throughout the specification do not necessarily refer to the same embodiment or example. Furthermore, specific features, structures, or characteristics can be combined in one or more embodiments or examples in any suitable combination and / or sub-combination. The term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.
[0022] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60–120 and 80–110 are listed for a specific parameter, it is understood that ranges of 60–110 and 80–120 are also expected. Furthermore, if minimum range values of 1 and 2 are listed, and if maximum range values of 3, 4, and 5 are listed, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0" and "5" have been listed in this document; "0-5" is merely a shortened representation of these numerical combinations. Furthermore, when a parameter is described as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc. Unless otherwise specified, all steps in this application can be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the method may also include step (c), indicating that step (c) can be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.
[0023] In existing technologies, latex-enhanced immunoturbidimetry for the detection and analysis of free light chains typically uses solid-phase microspheres labeled with full-length antibodies. Full-length antibodies contain an antigen-binding Fab region and a multifunctional Fc region. Because the Fc region contains antigen-binding sites, complement-binding sites, etc., the binding reaction of the Fc region reduces specificity, resulting in non-specific reactions when this fragment binds to interfering substances. Furthermore, endogenous interfering substances in the sample, such as heterophilic antibodies, human anti-animal antibodies, and rheumatoid factor, can cause non-specific reactions. All of these factors reduce specificity, further leading to disproportionate linearity.
[0024] Immunoturbidimetry is based on the specific reaction of antigen and antibody to form a large molecular complex, which produces turbidity in the solution. When light passes through the solution, scattering and transmission occur, weakening the light intensity. In the case of antibody excess, the light intensity is directly proportional to the analyte concentration, thus allowing for quantitative calculation of the analyte concentration. However, under certain conditions, the results can be interfered with, such as by heterophile antibodies, autoantibodies, and radioactive ionomers (RF). These factors can lead to false positives or false negatives. Therefore, improving the specificity of the reaction is crucial for accurate quantitative analysis.
[0025] To address the aforementioned issues, research has revealed that antibodies can be enzymatically hydrolyzed into monovalent Fab-binding and divalent F(ab')2 fragments, as well as Fc fragments, under enzymatic catalysis. Enzymatic treatment of the antibody and removal of the Fc fragment reduces non-specific reactions caused by the Fc fragment. Labeling microspheres with the free light chain F(ab')2 antibody fragment and adding a composite blocking agent to the R1 reaction solution reduces side reactions caused by the antibody itself and interfering agents, improves specificity for the target analyte, and resolves the linearity disproportion problem.
[0026] Specifically, the preparation steps for the free light chain F(ab')2 antibody fragment are as follows: (1) Dialyze the rabbit anti-human free light chain polyclonal antibody in citrate buffer, and dissolve the pepsin in citrate buffer.
[0027] (2) Rabbit anti-human free light chain polyclonal antibody and pepsin were mixed at a ratio of (1-5):25 and incubated in a 37°C water bath for 2 hours.
[0028] (3) Add sodium hydroxide to adjust the pH to about 7 to terminate the reaction.
[0029] (4) The product was purified using a protein A chromatography column.
[0030] Furthermore, the preparation steps of the antibody microspheres are as follows: Add latex microspheres (concentration 10% (w / v), particle size 60-200 nm) to morpholine ethanesulfonic acid buffer (pH 6.0), along with 50 mg / ml EDC and 50 mg / ml NHS activator, mix well, and activate at 37 degrees for 30 min.
[0031] Centrifuge (10000-20000 rpm) for 30 min and remove the supernatant.
[0032] Resuspended in hydroxyethylpiperazine ethanesulfonic acid buffer (pH 7.5).
[0033] Add the F(ab')2 antibody fragment and conjugate at 37 degrees for 2 hours.
[0034] Add blocking agent (0.5% (w / v) BSA solution) and block at 37 degrees for 30 min.
[0035] Add latex preservation solution (100 mM / L HEPES buffer, pH 7.5, 2% (w / v) to 15% (w / v) sucrose, 0.1% (w / v) to 1% (w / v) BSA and 0.01% (w / v) to 0.1% (w / v) ProClin300). Example 1
[0036] A reagent kit for the detection of free light chains using immunoturbidimetry, the components and amounts of each reagent are as follows: R1 reagent: 100 mM / L HEPES (pH 7.5), 4% sodium chloride, 3% polyethylene glycol 6000, 0.1% Triton X-100, 0.2 mg / ml blocking agents (rheumatoid factor antibody, rabbit IgG, rabbit IgM, and heterophile antibody blocking agents), and 0.05% ProClin 300. R2 reagent: 60nm latex microspheres conjugated with 100mM / L HEPES (pH 7.5), 2% sucrose, 0.75% BSA, 0.05% ProClin300, and F(ab')2 antibody fragment. Example 2
[0037] A reagent kit for the detection of free light chains using immunoturbidimetry, the components and amounts of each reagent are as follows: R1 reagent: 100 mM / L HEPES (pH 7.5), 3.5% sodium chloride, 2.5% polyethylene glycol 6000, 0.1% Triton X-100, 0.2 mg / ml blocking agents (rheumatoid factor antibody, rabbit IgG, rabbit IgM and heterophile antibody blocking agents) and 0.05% ProClin300.
[0038] R2 reagent: 80nm latex microspheres conjugated with 100mM / L HEPES (pH 7.5), 7.5% sucrose, 0.5% BSA, 0.05% ProClin300 and F(ab')2 antibody fragment. Example 3
[0039] A reagent kit for the detection of free light chains using immunoturbidimetry, the components and amounts of each reagent are as follows: R1 reagent: 100 mM / L HEPES (pH 7.5), 3% sodium chloride, 1% polyethylene glycol 6000, 0.1% Triton X-100, 0.2 mg / ml blocking agents (rheumatoid factor antibody, rabbit IgG, rabbit IgM and heterophile antibody blocking agents) and 0.05% ProClin 300.
[0040] R2 reagent: 130 nm latex microspheres conjugated with 100 mM / L HEPES (pH 7.5), 10% sucrose, 0.25% BSA, 0.05% ProClin300, and F(ab')2 antibody fragment.
[0041] Comparative Example 1 Based on Example 1, the difference between this comparative example and Example 1 is that there is no composite blocking agent in reagent R1.
[0042] Comparative Example 2 Commercially available reagents were selected, and their composition was as follows: Reagent R1: Tris(hydroxymethyl)aminomethane buffer and polyethylene glycol; Reagent R2: Glycine buffer and antibody latex microspheres; Calibrator (recombinant protein); Quality control (recombinant protein).
[0043] Test 1: Linearity determination: The reagent was diluted with deionized water to six gradient concentrations. Each gradient concentration was measured three times, and the average value was calculated. The relative deviation between the measured average value and the expected value was calculated.
[0044] After aspirating reagent R1 and the sample, incubate at 37°C for 5 minutes. Then add reagent R2, and after reacting for 18 seconds, read the absorbance A1. After 5 minutes, read the absorbance A2. The sample concentration was calculated based on the instrument calibration curve, as shown in Tables 1 and 2.
[0045]
[0046]
[0047] Conclusion: In Comparative Example 2, at high concentration (dilution ratio 1), the κ chain deviation was 2.09% and the λ chain deviation was 1.9%, with the deviation showing a significant upward trend as the concentration increased; moreover, the concentration at each dilution point showed a gradual upward trend as the dilution ratio increased, highlighting the problem of non-linearity.
[0048] At the same high concentration, in Examples 1-3, the highest deviation of the κ chain was only 0.22%, and the highest deviation of the λ chain was only 0.30%. Moreover, the deviation remained below 6% across the entire concentration range, and there was no phenomenon of the deviation increasing dramatically with increasing concentration. The concentrations at each dilution point were proportional.
[0049] For free κ light chains: In Examples 1-3, the deviation range of the reagents of the present invention is 0.21% to 3.82%, while the deviation range of Comparative Example 2 is 2.09% to 25.02%.
[0050] For free λ light chains: In Examples 1-3, the deviation range of the reagents of the present invention is 0.18% to 5.30%, while the deviation range of Comparative Example 2 is 1.99% to 26.98%.
[0051] For free κ light chains / free λ light chains: In Comparative Example 1, in the group without the addition of the composite blocker, the concentrations at each dilution point were higher than those in the other examples; and the concentrations at each dilution point gradually increased with increasing dilution ratio.
[0052] In summary, through the synergistic effect of antibody tablet F(ab')2 and the composite blocking agent, the reagent of this invention greatly improves the problem of non-linearity of free light chains, and further enhances the specificity and accuracy of this reagent.
[0053] In clinical applications, commercially available reagent kits exhibit common problems: significant differences in test results between different kits; and disproportionate linearity in some samples. This may be due to the properties of the antibodies themselves, such as low antibody specificity and weak anti-interference ability. Latex-enhanced immunoturbidimetry typically uses full-length antibodies labeled with solid-phase microspheres. Full-length antibodies contain an antigen-binding Fab region and a multifunctional Fc region. Under enzymatic catalysis, antibodies can be enzymatically cleaved into monovalent Fab fragments, divalent F(ab')2 fragments, and the Fc fragment. Because the antibody Fc fragment contains antigen-binding sites, complement-binding sites, etc., this fragment produces a non-specific reaction after binding to interfering substances.
[0054] This invention reduces non-specific reactions caused by the Fc fragment by enzymatically treating the antibody and removing the Fc fragment. Furthermore, endogenous interfering substances in the sample, such as heterophilic antibodies, human anti-animal antibodies, and rheumatoid factor, can cause non-specific reactions. As shown in Tables 1 and 2, this invention significantly improves the problem of disproportionate linearity of free light chains.
[0055] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A reagent for the detection of free light chains using immunoturbidimetric assay, characterized in that, It includes the following components: A solid-phase support with F(ab')2 antibody fragments labeled on its surface; Liquid-phase reaction solution, comprising a composite blocking agent for inhibiting non-specific reactions of interfering substances; The F(ab')2 antibody fragment therein works in conjunction with the composite blocking agent to improve the specificity and linearity of the detection.
2. The reagent for the detection of free light chains by immunoturbidimetric assay according to claim 1, characterized in that, The F(ab')2 antibody fragment was prepared by the following steps: using rabbit anti-human free light chain polyclonal antibody as raw material, after digestion with pepsin, the Fc fragment and undigested intact antibody were removed by Protein A chromatography column, thereby obtaining the purified F(ab')2 antibody fragment.
3. The reagent for the detection of free light chains by immunoturbidimetric assay according to claim 1, characterized in that, The composite blocking agent includes 0.1 mg / mL to 0.5 mg / mL rheumatoid factor antibody, 0.1 mg / mL to 0.5 mg / mL rabbit IgG, 0.1 mg / mL to 0.5 mg / mL rabbit IgM, and 0.1 mg / mL to 0.5 mg / mL heterophile antibody blocking agent.
4. The reagent for the detection of free light chains by immunoturbidimetric assay according to claim 3, characterized in that, The working concentration of the composite blocking agent in the liquid reaction solution is 0.1 mg / mL-0.5 mg / mL.
5. The reagent for the detection of free light chains by immunoturbidimetric assay according to claim 1, characterized in that, The solid support is latex microspheres with a particle size of 60-200 nm.
6. The reagent for the detection of free light chains by immunoturbidimetric assay according to claim 5, characterized in that, The F(ab')2 antibody fragment is covalently coupled to the surface of the latex microspheres.
7. The reagent for the detection of free light chains by immunoturbidimetric assay according to claim 1, characterized in that, The liquid reaction solution also includes 100 mM / L HEPES buffer, sodium chloride, polyethylene glycol, and surfactant.
8. A reagent kit for detecting free light chains, characterized in that, include: R1 reagent: the liquid-phase reaction solution according to any one of claims 1-7; R2 reagent: The solid-phase carrier and latex preservation solution for conjugating the F(ab')2 antibody fragment as described in any one of claims 1-7.
9. The kit for detecting free light chains according to claim 8, characterized in that, The latex preservation solution comprises 100 mM / L HEPES buffer, 2%–15% sucrose, 0.1%–1% BSA, and 0.01%–0.1% ProClin300.
10. A method for detecting the content of free light chains in a sample, characterized in that, Includes the following steps: The sample to be tested is mixed with reagent R1 of the kit as described in claim 8 or 9 and pre-incubated; Add reagent R2 from the kit to the above mixture to initiate the immune reaction; Monitor the turbidity changes in the reaction system and calculate the concentration of free light chains in the sample based on the standard curve.