Kappa light chain detection kit and preparation method thereof

Abstract

The invention discloses a kappa light chain detection kit and a preparation method, and relates to the technical field of clinical in-vitro detection reagents, the kit is in a double-reagent form, the double reagents comprise a reagent R1 and a reagent R2, the reagent R2 comprises a capture antibody and a detection antibody which can be specifically combined with different epitopes of a kappa light chain, and the detection antibody comprises a reagent R1 and a reagent R2 which can be specifically combined with different epitopes of the kappa light chain. A capture antibody-antigen-detection antibody sandwich structure is formed to improve the hook effect in detection. The kappa light chain detection reagent provided by the invention solves the problems that the existing kappa light chain detection reagent mostly uses a single antibody and a hook effect is easy to occur due to reduction of a HOOK point, a capture antibody-antigen-detection antibody sandwich structure is formed by two antibodies (capture antibody and detection antibody) aiming at different epitopes of an antigen, and the core is a specific binding mode, so that the recognition precision of the antigen can be improved, and the detection accuracy of the kappa light chain detection reagent is improved. And non-specific binding is reduced, the detection sensitivity and HOOK point are greatly improved, and further popularization and application in the market are facilitated.

Description

Technical Field

[0001] This invention relates to the field of clinical in vitro diagnostic reagent technology, specifically to a κ light chain detection kit and its preparation method. 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 bound to the heavy chains are called bound light chains, while those not bound to the heavy chains are called free light chains. The sum of bound and free light chains is the total light chain. Light chains are classified into two types based on their structure and antigenicity: κ (kappa) and λ (lambda). Under normal circumstances, light chains can be detected in both serum and urine. Total light chains are consistently detectable in the serum of healthy individuals, while only a small amount are present in urine. When kidney damage or multiple myeloma occurs, abnormal secretion by plasma cells leads to abnormal levels of immunoglobulins and light chains in the serum, resulting in significant differences in serum and urine light chain levels compared to normal individuals, and a noticeable change in the κ / λ ratio.

[0003] Currently, there are many methods available for measuring the κ light chain in blood and urine, such as gel isoelectric aggregation, Western blotting, immunoturbidimetry, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), immunofixation electrophoresis, high-performance liquid chromatography (HPLC), and chemiluminescent immunoassay. While ELISA offers advantages such as high sensitivity, high accuracy, rapid reaction, and strong specificity, it requires a dedicated ELISA instrument and has relatively high reagent costs. In contrast, immunoturbidimetry is simple to operate, highly automated, fast, and offers high sensitivity and precision. It is suitable for ordinary automated biochemical analyzers and spectrophotometers and is widely used.

[0004] Immunoturbidimetry is currently the most commonly used clinical method for detecting total light chains. It utilizes the reaction between anti-κ light chain antibodies and antigens in the sample to form antigen-antibody complexes, leading to an increase in solution turbidity. The light chain content can be calculated by detecting changes in turbidity (scattered or transmitted light intensity). This method requires the addition of an appropriate proportion of antigen to the quantitative antibody solution to ensure a rapid reaction and the formation of the largest possible number of immune complexes (ICs), resulting in the strongest light signal and ensuring accurate results.

[0005] However, in actual testing, an imbalance in the proportion of antigen added to the sample can lead to unrealized test results. In particular, when there is an excess of antigen, the immune complexes formed by the binding of antigen and antibody are reduced, which will directly cause deviations in the test results of specific proteins, i.e., the hook effect. This can easily cause strongly positive samples to be mistested as weakly positive or even false negative results, directly masking the true pathological condition.

[0006] Total Light Chains (TLC) is the sum of bound light chains and free light chains. The detection sites for free light chains are hidden sites inside the light chains, while the detection sites for traditional κ light chain reagents are exposed sites on the outside where light chains bind to heavy chains.

[0007] Current immunoturbidimetric assays for κ light chain detection use a single antibody. When there is an excess of antigen, a large amount of antigen will occupy antibody sites in a monovalent binding form, leading to a reduction in the complex and potentially causing false positives or false negatives. In complex samples, extraneous proteins may bind nonspecifically to the single antibody, indirectly consuming the antibody and reducing the actual concentration of antibody that can bind to the target antigen, thus lowering the hook point.

[0008] Therefore, this application is hereby submitted. Summary of the Invention

[0009] The technical problem to be solved by this invention is that most existing κ light chain detection reagents use a single antibody, and the hook point is prone to hook effect when lowered. The purpose is to provide a κ light chain detection kit and its preparation method.

[0010] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a κ light chain detection kit, the kit being in the form of a dual reagent, the dual reagents being reagent R1 and reagent R2, wherein reagent R2 includes a capture antibody and a detection antibody capable of specifically binding to different epitopes of the κ light chain, forming a sandwich structure of capture antibody-antigen-detection antibody to improve the hook effect in detection.

[0011] As one of the optional implementation methods, the capture antibody is a rabbit anti-human κ light chain antibody, and the detection antibody is a rabbit anti-human κ free light chain antibody.

[0012] The reason for choosing rabbit anti-human κ light chain antibody and rabbit anti-human κ free light chain antibody in this protocol is as follows: The rabbit's immune system exhibits strong immunogenicity to human proteins (such as the κ light chain), producing high-titer, high-affinity polyclonal antibodies with a broad epitope recognition range, covering multiple regions of the κ light chain. This makes them suitable for epitope differentiation design using a double-antibody sandwich assay. Furthermore, rabbit-derived antibodies show high specificity for the human κ light chain and low cross-reactivity with other species or other human immunoglobulins (such as the λ light chain and heavy chain), reducing non-specific interference in detection.

[0013] As one of the optional embodiments, in reagent R2, the concentration of the rabbit anti-human κ light chain antibody is 0.3-0.9 g / L, and the concentration of the rabbit anti-human κ free light chain antibody is 0.3-0.9 g / L.

[0014] As one of the optional implementation methods, the reagent R2 further includes sodium chloride at a concentration of 2-10 g / L and sodium azide at a concentration of 0.05-0.2%.

[0015] As one of the optional embodiments, the reagent R1 comprises the following components: 4-hydroxyethylpiperazine ethanesulfonic acid buffer at a concentration of 0.15 mol / L, sodium chloride at a concentration of 4.8 g / L, polyethylene glycol 20000 at a concentration of 30-50 g / L, and Tween 80 at a concentration of 0.1%.

[0016] Secondly, the present invention provides a method for preparing the above-mentioned reagent kit, including the preparation step of reagent R2: Provide antibody diluent; The capture antibody and the detection antibody were diluted with the antibody diluent described above to obtain solution A and solution B, respectively. Mix solution A and solution B in a certain proportion.

[0017] As one optional implementation, the antibody diluent comprises 50 mM phosphate buffer at pH 7.4 and 0.1% preservative.

[0018] In this protocol, sodium azide is used as the preservative. It can inhibit the growth and reproduction of microorganisms such as bacteria and fungi, preventing the buffer solution from deteriorating due to microbial contamination. At the same time, it will not react with the core components of the buffer system, nor will it significantly interfere with most biological experiments (such as protein electrophoresis, immunoassay, cell washing, etc.).

[0019] Thirdly, a method for detecting the content of κ light chains in a sample, using the above-mentioned kit, includes the following steps: The sample to be tested is mixed with reagent R1 and pre-incubated; Add the reagent R2 to initiate the reaction; Measure the signal and calculate the concentration.

[0020] As one of the optional implementation methods, the pre-incubation time is 5 minutes and the temperature is 37°C.

[0021] As one of the optional implementation methods, the sample is a blood sample or a urine sample.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. By using two antibodies (capture antibody and detection antibody) targeting different epitopes of the antigen, a sandwich structure of capture antibody-antigen-detection antibody is formed. The core is the specific binding mode, which can improve the recognition accuracy of antigen, reduce non-specific binding, and greatly improve the sensitivity and hook point of detection, which is conducive to further promotion and use in the market.

[0023] 2. By complementing the epitopes of the capture antibody and the detection antibody, the problem of competitive inhibition of high-concentration samples is solved at the molecular level, rather than relying on traditional reagent formulation optimization.

[0024] 3. The combination of the broad spectrum of capture antibodies and the specificity of detection antibodies ensures both detection sensitivity and avoids the hook effect, achieving the dual advantages of high sensitivity and wide linear range. Detailed Implementation

[0025] 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.

[0026] 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.

[0027] 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. Example 1

[0028] This embodiment provides a κ light chain detection kit, which is a two-reagent kit including reagent R1 and reagent R2. Preparation of reagent R1: 4-Hydroxyethylpiperazine ethanesulfonic acid (HEPES) 0.15 mol / L Sodium chloride (NaCl) 4.8 g / L Polyethylene glycol 20000 (PEG20000) 45g / L Tween 80 0.1% Sodium azide (NaN3) 0.1% Preparation of reagent R2: Rabbit anti-human κ light chain antibody 0.8 g / L Rabbit anti-human κ free light chain antibody 0.4 g / L Sodium chloride (NaCl) 5g / L Sodium azide (NaN3) 0.1% Specifically, the preparation steps for reagent R2 are as follows: Prepare antibody dilution buffer (50mM pH7.4 buffer + 0.1% preservative). Rabbit anti-human κ light chain antibody and rabbit anti-human κ free light chain antibody were diluted with the above antibody diluent to obtain solution A and solution B, respectively. Mix solution A and solution B in a ratio of 1:3 to 3:1.

[0029] The role of rabbit anti-human κ light chain antibody is to broadly capture all κ light chains (including bound and free forms) in the sample, ensuring that no target is missed and guaranteeing the sensitivity of the detection.

[0030] The role of rabbit anti-human κ free light chain antibody is to recognize only the free specific epitopes of free κ light chain (which are masked by the heavy chain in bound κ light chain and are only exposed when free), avoiding interference from bound κ light chain. Example 2

[0031] This embodiment provides a κ light chain detection kit, which is a two-reagent kit including reagent R1 and reagent R2. Preparation of reagent R1: 4-Hydroxyethylpiperazine ethanesulfonic acid (HEPES) 0.15 mol / L Sodium chloride (NaCl) 4.8 g / L Polyethylene glycol 20000 (PEG20000) 45g / L Tween 80 0.1% Sodium azide (NaN3) 0.1% Preparation of reagent R2: Rabbit anti-human κ light chain antibody 0.43 g / L Rabbit anti-human κ free light chain antibody 0.9 g / L Sodium chloride (NaCl) 5g / L Sodium azide (NaN3) 0.1% Example 3

[0032] This embodiment provides a κ light chain detection kit, which is a two-reagent kit including reagent R1 and reagent R2. Preparation of reagent R1: 4-Hydroxyethylpiperazine ethanesulfonic acid (HEPES) 0.15 mol / L Sodium chloride (NaCl) 4.8 g / L Polyethylene glycol 20000 (PEG20000) 45g / L Tween 80 0.1% Sodium azide (NaN3) 0.1% Preparation of reagent R2: Rabbit anti-human κ light chain antibody 0.6 g / L Rabbit anti-human κ free light chain antibody 0.6 g / L Sodium chloride (NaCl) 5g / L Sodium azide (NaN3) 0.1% Specifically, this invention discloses a serum and urine κ light chain detection kit (immunoturbidimetric method) for use in a fully automated specific protein analyzer. It combines rabbit anti-human κ light chain antibody and rabbit anti-human free κ light chain antibody, forming a sandwich structure of capture antibody-antigen-detection antibody using two antibodies targeting different epitopes of the antigen (capture antibody and detection antibody). The core feature is a specific binding mode, which improves the accuracy of antigen recognition, reduces non-specific binding, and greatly enhances detection sensitivity and hook point, facilitating further market adoption. Furthermore, this detection system is operated using a fully automated specific protein analyzer, ensuring accurate results while offering greater convenience and speed. Large-scale direct operation is possible in routine laboratories.

[0033] The detection methods and protocols are provided in the operation manuals or instrument software for the BioSystems BA400, Chongqing Bosideng BA200, Chongqing Bosideng BST300, Chongqing Bosideng BST320, and Chongqing Bosideng BA400 fully automated specific protein analyzers. The operation is as follows: Open the κ light chain detection reagent bottle cap and place it in the corresponding reagent slot of the instrument. Shake gently before first use to avoid air bubbles and potential sampling errors. Take a sample with an extremely high value and place it in the corresponding sample slot for testing. When testing, please select the correct sample type; otherwise, incorrect results will occur. Click "Start" on the instrument. The sampling needle will draw R1 into the reaction dish; then draw the sample again, mix well, incubate at 37°C for 5 minutes, add R2, and the instrument will automatically detect. The instrument's detection system will automatically calculate the results. Comparative Example 1

[0034] This comparative example provides a κ light chain detection kit, which is a two-reagent kit including reagent R1 and reagent R2. Preparation of reagent R1: 4-Hydroxyethylpiperazine ethanesulfonic acid (HEPES) 0.15 mol / L Sodium chloride (NaCl) 4.8 g / L Polyethylene glycol 20000 (PEG20000) 45g / L Tween 80 0.1% Sodium azide (NaN3) 0.1% Preparation of reagent R2: Rabbit anti-human κ light chain antibody 0.8 g / L Sodium chloride (NaCl) 5g / L Sodium azide (NaN3) 0.1% Comparative Example 2

[0035] This comparative example provides a κ light chain detection kit, which is a two-reagent kit including reagent R1 and reagent R2. Preparation of reagent R1: 4-Hydroxyethylpiperazine ethanesulfonic acid (HEPES) 0.15 mol / L Sodium chloride (NaCl) 4.8 g / L Polyethylene glycol 20000 (PEG20000) 45g / L Tween 80 0.1% Sodium azide (NaN3) 0.1% Preparation of reagent R2: Rabbit anti-human κ free light chain antibody 0.8 g / L Sodium chloride (NaCl) 5g / L Sodium azide (NaN3) 0.1% Comparative Example 3

[0036] Commercially available κ light chain reagent from manufacturer A was selected as a control reagent, and the following experiments were conducted: Take the same sample containing the κ light chain and perform serial dilutions at ratios of 1 (undiluted), 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, and 1 / 256 to prepare a series of samples with different concentrations.

[0037] Each dilution sample was added to the reaction system of a commercially available comparative reagent and the reagent of the present invention, and the κ light chain was detected under the same conditions (temperature, incubation time, etc.). The absorbance value corresponding to each dilution was recorded and the concentration was calculated.

[0038] The concentration measured at each dilution was multiplied by the corresponding dilution factor to obtain the inverse multiplier concentration, which represents the theoretical concentration of the original sample and is used to evaluate the consistency of results at different dilutions. The final experimental data are shown in Table 1.

[0039]

[0040]

[0041] in conclusion: In Example 1, the absorbance gradually increased from 1 to 1 / 4 (1.2522 to 1.3634), and then steadily decreased (1.2965 to 0.4389), conforming to the normal pattern of "concentration decreases → absorbance decreases," without signal reversal. On the other hand, the absorbance at undiluted was as high as 1.2522, much higher than the 0.1822 of the control reagent, indicating that the signal was not severely suppressed at high concentrations. Finally, the inverse product concentration from high to low dilution (8304.85 to 32.12) showed a stable multiple relationship without serious deviation, indicating a significant increase in the HOOK point.

[0042] In Example 2, the absorbance gradually increased from 1 to 1 / 4 (1.2321 to 1.3532), and then steadily decreased (1.2763 to 0.4176), conforming to the normal pattern of "concentration decreases → absorbance decreases," without signal reversal. On the other hand, the absorbance at undiluted was as high as 1.2321, much higher than the 0.1822 of the control reagent, indicating that the signal was not severely suppressed at high concentrations. Finally, the inverse product concentration from high to low dilution (8392.75 to 33.17) showed a stable multiple relationship without serious deviation, indicating a significant increase in the HOOK point.

[0043] In Example 3, the absorbance gradually increased from 1 to 1 / 4 (1.2331 to 1.3839), and then steadily decreased (1.2657 to 0.4238), conforming to the normal pattern of "concentration decreases → absorbance decreases," without signal reversal. On the other hand, the absorbance at undiluted was as high as 1.2331, much higher than the 0.1822 of the control reagent, indicating that the signal was not severely suppressed at high concentrations. Finally, the inverse product concentration from high to low dilution (8320.21 to 31.89) showed a stable multiple relationship without serious deviation, indicating a significant increase in the HOOK point.

[0044] In Comparative Example 1, as the concentration increased from 1 / 16 to 1, the absorbance gradually decreased, and a concentration value appeared, indicating that the signal was suppressed at high concentrations, exhibiting the hook effect. Furthermore, the absorbance at undiluted concentration was only 0.3421, far lower than the 0.5782 at 1 / 16, demonstrating the signal inversion characteristic of the hook effect. Additionally, the high-concentration region (1~1 / 16) was initially judged as "Out," but inverse concentration multiplication showed the presence of a large number of κ light chains in the sample, indicating a false negative, a direct result of the hook effect.

[0045] In Comparative Example 2, as the concentration increased from 1 / 16 to 1, the absorbance gradually decreased, and a concentration value appeared, indicating that the signal was suppressed at high concentrations, exhibiting the hook effect. Furthermore, the absorbance at undiluted concentration was only 0.3072, far lower than the 0.4231 at 1 / 16, demonstrating the signal inversion characteristic of the hook effect. Additionally, the high-concentration region (1~1 / 16) was initially judged as "Out," but inverse concentration multiplication showed the presence of a large number of κ light chains in the sample, indicating a false negative, a direct result of the hook effect.

[0046] In Comparative Example 3, with the existing reagents, the absorbance gradually decreased as the concentration increased from 1 / 16 to 1, and a concentration value appeared, indicating that the signal was suppressed at high concentrations, exhibiting the HOOK effect. Furthermore, the absorbance at undiluted concentration was only 0.1822, far lower than the 0.571 at 1 / 16, a signal inversion characteristic of the HOOK effect. Additionally, the high concentration region (1~1 / 16) was judged as "Out," but the inverse concentration multiplication showed the presence of a large number of κ light chains in the sample, indicating a false negative, a direct result of the HOOK effect.

[0047] It is known that in clinical applications, commercially available reagent kits have relatively low hook points, causing strongly positive samples to be misdetected as weakly positive or even false negative results. The hook point depends on the maximum ability of the antibody to bind to the antigen in the detection system. When the antigen concentration exceeds this ability, a hook effect is triggered. Currently, immunoturbidimetric assays mostly use single antibodies. However, total light chains contain bound light chains and free light chains. The detection site for free light chains is a hidden site inside the light chain, while the detection site for traditional κ light chain reagents is an exposed site on the outside where light chains bind to heavy chains. We combine rabbit anti-human κ light chain antibody and rabbit anti-human κ free light chain antibody. Through two antibodies targeting different epitopes of the antigen (capture antibody and detection antibody), a sandwich structure of "capture antibody-antigen-detection antibody" is formed. The core is a specific binding mode, which can improve the recognition accuracy of antigens, reduce non-specific binding, and greatly improve the sensitivity and hook point of the detection.

[0048] 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 κ light chain detection kit, characterized in that, The kit is in the form of a dual reagent, namely reagent R1 and reagent R2. Reagent R2 includes a capture antibody and a detection antibody that can specifically bind to different epitopes of the κ light chain, forming a sandwich structure of capture antibody-antigen-detection antibody to improve the hook effect in detection.

2. The κ light chain detection kit according to claim 1, characterized in that, The capture antibody is a rabbit anti-human κ light chain antibody, and the detection antibody is a rabbit anti-human κ free light chain antibody.

3. The κ light chain detection kit according to claim 1, characterized in that, In reagent R2, the concentration of the rabbit anti-human κ light chain antibody is 0.3-0.9 g / L, and the concentration of the rabbit anti-human κ free light chain antibody is 0.3-0.9 g / L.

4. The κ light chain detection kit according to claim 1, characterized in that, The reagent R2 also includes sodium chloride at a concentration of 2-10 g / L and sodium azide at a concentration of 0.05-0.2%.

5. The κ light chain detection kit according to claim 4, characterized in that, The reagent R1 comprises the following components: 0.15 mol / L of 4-hydroxyethylpiperazine ethanesulfonic acid buffer, 4.8 g / L of sodium chloride, 30-50 g / L of polyethylene glycol 20000, and 0.1% of Tween 80.

6. A method for preparing the kit according to any one of claims 1-5, characterized in that, Including the preparation steps of reagent R2: Provide antibody diluent; The capture antibody and the detection antibody were diluted with the antibody diluent described above to obtain solution A and solution B, respectively. Mix solution A and solution B in a certain proportion.

7. The method for preparing the reagent kit according to claim 6, characterized in that, The antibody diluent comprises 50 mM phosphate buffer at pH 7.4 and 0.1% preservative.

8. A method for detecting the content of κ light chains in a sample, characterized in that, Using the kit as described in any one of claims 1-7, the following steps are included: The sample to be tested is mixed with reagent R1 and pre-incubated; Add the reagent R2 to initiate the reaction; Measure the corresponding signal and calculate the concentration.

9. The method for detecting the content of κ light chains in a sample according to claim 8, characterized in that, The pre-incubation time is 5 minutes and the temperature is 37℃.

10. The method for detecting the content of κ light chains in a sample according to claim 8, characterized in that, The sample is either a blood sample or a urine sample.

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