A serum free light chain detection reagent and detection method

Abstract

The present invention provides a serum free light chain detection reagent and detection method, comprising: a first reagent comprising a reaction buffer and an anti-human IgG-Fab polyclonal antibody; and a second reagent comprising a nanosphere-labeled anti-human FLC polyclonal antibody. The anti-human IgG-Fab polyclonal antibody is used to block cross-reactions between bound light chains of immunoglobulins in the sample and the anti-human FLC polyclonal antibody. By adding the anti-human IgG-Fab polyclonal antibody to the reaction buffer, the present invention effectively addresses the cross-reaction problem between the anti-FLC polyclonal antibody and light chains bound to immunoglobulins, while ensuring the long-term stability of the reagent.

Description

Technical Field

[0001] The present invention relates to the field of medical immunology, and in particular to a serum free light chain detection reagent and a detection method. Background Art

[0002] Serum free light chains (FLCs) are immunoglobulin light chains produced by B cells. They are classified into two types: kappa (κ) and lambda (λ), with molecular weights of 22 kDa (monomer kappa) and 45 kDa (dimer lambda), respectively. FLCs have multiple biological functions, including antiviral replication, inhibition of neutrophil apoptosis, activation of the complement system, and regulation of inflammatory responses.

[0003] FLC detection methods include electrophoresis, immunofixation electrophoresis, and latex immunoturbidimetry. Latex immunoturbidimetry is widely used due to its high sensitivity and ease of detection. The latex immunoturbidimetry method is based on the specific binding reaction between antigen and antibody, quantitatively analyzing FLC concentration by measuring the light scattering intensity or light absorption of the antigen-antibody complex.

[0004] To address the cross-reactivity issue between anti-FLC polyclonal antibodies and light chains bound to immunoglobulins (Igs), patent applications CN116930479A and CN116953216A propose a blocking agent for reducing cross-reactivity in polyclonal antibody-based free light chain detection reagents and their applications. These patents utilize a peptide composition to pre-shield the exposed surface of the FLC polyclonal antibody cross-linked to polystyrene microspheres in the R2 component of a latex-enhanced immunoturbidimetric reagent based on anti-FLC polyclonal antibodies, thereby preventing cross-reactivity between the antibody and light chains bound to immunoglobulins (Igs). However, these peptide compositions all consist of light chain constant region sequences. Due to the diversity of antigenic sites recognized by polyclonal antibodies, these peptides, each several dozen amino acids in length, potentially harbor multiple antibody binding sites. Consequently, the peptides, linked to two antibodies, can cause aggregation of adjacent polystyrene microspheres, compromising the stability of the reagent. Summary of the Invention

[0005] The invention not only solves the cross-reaction problem by adding anti-human IgG-Fab antibodies to the reaction buffer, but also eliminates the instability problem caused by adding light chain polypeptide fragments to a latex solution sensitized with free light chain antibodies.

[0006] The present invention provides a serum free light chain detection reagent, comprising:

[0007] A first reagent, comprising a reaction buffer and an anti-human IgG-Fab polyclonal antibody;

[0008] A second reagent includes a nanosphere-labeled anti-human FLC polyclonal antibody;

[0009] Among them, anti-human IgG-Fab polyclonal antibody is used to block the cross reaction between the binding light chain of immunoglobulin in the sample and the anti-human FLC polyclonal antibody.

[0010] The present invention technically adopts the design of a double antibody reagent, in which the first antibody reagent uses anti-human IgG-Fab polyclonal antibody to block the binding light chain in the immunoglobulin, preventing it from non-specific binding with the anti-FLC polyclonal antibody in the second antibody reagent, thereby ensuring the accuracy of the detection.

[0011] Furthermore, the anti-human IgG-Fab polyclonal antibody is at least one of a goat anti-human IgG-Fab antibody and a rabbit anti-human IgG-Fab antibody.

[0012] Anti-human IgG-Fab polyclonal antibodies are antibodies that can specifically bind to the Fab segment of immunoglobulins. Specifically, this can be achieved using polyclonal antibodies obtained by immunizing sheep or rabbits. They block cross-reactions by blocking the antigen-binding sites that bind to the light chain in immunoglobulins, and can effectively cover interference from immunoglobulins from different species.

[0013] Furthermore, the anti-human FLC polyclonal antibody is a sheep polyclonal antibody or a rabbit polyclonal antibody prepared from FLC immunogen.

[0014] By using FLC as an immunogen, highly specific polyclonal antibodies against FLC can be prepared. These antibodies can efficiently bind to free light chains without cross-reacting with light chains bound to immunoglobulins.

[0015] Furthermore, the nanoparticles include polystyrene microspheres, colloidal gold microspheres, and magnetic beads.

[0016] After the nanosphere-labeled antibody binds to the free light chain, the concentration of the free light chain in the serum is quantitatively analyzed by optical methods such as changes in scattered light intensity or transmittance.

[0017] Furthermore, the first reagent also includes: a macromolecular polymer, a salt, and a preservative.

[0018] The buffer and macromolecular polymer in the reaction system work synergistically to improve the efficiency of antigen-antibody binding, while preservatives and salts ensure the long-term storage stability of the reagent. This achieves a dual improvement in detection specificity and reagent stability without the introduction of additional blocking agents.

[0019] Furthermore, the second reagent also includes a buffer, a protective protein, and a preservative.

[0020] The addition of a buffer maintains the acid-base balance and ionic strength of the reaction system, ensuring uniform surface charge distribution on the nanospheres. The protective protein forms a physical barrier layer on the surface of the microspheres, effectively blocking nonspecific aggregation caused by van der Waals forces between the microspheres. The preservative continuously inhibits microbial proliferation during long-term storage, preventing elevated background signals due to reagent contamination. The synergistic effect of the components ensures that the antibody-labeled nanospheres remain monodisperse when stored at 4°C-8°C, maintaining antibody activity at over 95% of its initial value throughout their shelf life.

[0021] The present invention also provides a method for detecting serum free light chains, comprising the following steps:

[0022] S10, mixing the sample or calibrator with the first reagent to allow the anti-human IgG-Fab polyclonal antibody to react with the bound light chain of Igs;

[0023] S20, adding a second reagent to allow the anti-human FLC polyclonal antibody labeled with the nanospheres to specifically bind to the FLC in the sample to form a complex;

[0024] S30. Detect the light scattering signal or absorbance of the complex and quantitatively analyze the FLC concentration.

[0025] In the first stage, after the sample is mixed with the primary reagent, the anti-human IgG-Fab polyclonal antibody preferentially binds to the bound light chains of immunoglobulins, creating a steric hindrance that prevents subsequent detection antibodies from cross-reacting with the bound light chains. In the second stage, nanoparticles labeled with anti-human FLC polyclonal antibodies are added. These antibodies specifically bind only to unbound free light chains, forming a complex. Due to the large surface area of ​​the nanoparticles and their optical signal amplification effect, the complex produces significant light scattering or absorbance changes in the solution. Finally, the detection system measures the signal intensity at a specific wavelength, establishing a standard curve for quantitative analysis.

[0026] Furthermore, in S30 , the detected wavelength is 570 nm and the sub-wavelength is 800 nm.

[0027] By calculating the difference between the main wavelength signal and the sub-wavelength signal, the influence of nonspecific light scattering can be eliminated, so that the detection results only reflect the concentration change of the target complex.

[0028] After adopting the technical solution of the present invention, the following technical effects can be achieved:

[0029] The present invention provides a serum free light chain detection reagent and detection method. The first reagent uses anti-human IgG-Fab polyclonal antibodies to block the cross-reaction between immunoglobulin-bound light chains in the sample and anti-human FLC polyclonal antibodies. A second reagent labeled with nanoparticles is combined to achieve specific detection, effectively avoiding the cross-reaction problem of traditional polyclonal antibodies and bound light chains, and improving the accuracy of detection. DETAILED DESCRIPTION

[0030] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort shall fall within the scope of protection of the present invention.

[0031] In existing technologies, when latex immunoturbidimetry is used to detect serum free light chains, anti-FLC polyclonal antibodies easily cross-react with immunoglobulin-bound light chains, resulting in high test results. Existing solutions use peptide blocking agents to prevent cross-reactions, but peptide fragments easily bind to labeled antibodies, causing microsphere aggregation and reducing reagent stability.

[0032] To address this issue, researchers discovered that the Fab fragments of immunoglobulin light chains are exposed on the surface of the antibody molecule, while the Fab regions of free light chains remain free. Based on this discovery, they considered using anti-human IgG-Fab polyclonal antibodies to specifically bind to the Fab fragments of immunoglobulins in the sample, pre-blocking the epitopes that bind to the light chains and thus preventing nonspecific binding to the detection antibody. Furthermore, by placing the blocking and detection antibodies in separate reagents, direct contact between the blocking agent and the labeled antibody is avoided, fundamentally addressing the reagent stability issue.

[0033] This application proposes a serum free light chain detection reagent, comprising a first reagent and a second reagent. The first reagent comprises a reaction buffer and an anti-human IgG-Fab polyclonal antibody, and the second reagent comprises a nanosphere-labeled anti-human FLC polyclonal antibody. The anti-human IgG-Fab polyclonal antibody is used to block cross-reaction between bound light chains in immunoglobulins in the sample and the anti-human FLC polyclonal antibody.

[0034] The technical solution of this embodiment technically adopts the design of a double antibody reagent, in which the first antibody reagent uses an anti-human IgG-Fab polyclonal antibody to block the binding light chain in the immunoglobulin, preventing it from nonspecifically binding with the anti-FLC polyclonal antibody in the second antibody reagent, thereby ensuring the accuracy of the detection.

[0035] Anti-human IgG-Fab polyclonal antibodies can specifically recognize and bind to the Fab region of IgG, thereby blocking or masking epitopes on immunoglobulins that may cross-react with anti-FLC antibodies. By pre-blocking these sites, cross-reactions can be effectively avoided. The technology in this example significantly reduces cross-reactions between anti-FLC polyclonal antibodies and bound light chains, improving the specificity and accuracy of detection.

[0036] Specifically, nanosphere-labeled anti-human FLC polyclonal antibodies are complexes formed by immobilizing anti-human FLC polyclonal antibodies on the surface of nanospheres, which can be implemented using polystyrene microspheres, colloidal gold microspheres, or magnetic beads. The nanospheres amplify the optical signal of the antigen-antibody complex, improving detection sensitivity.

[0037] Specifically, the anti-human IgG-Fab polyclonal antibody in the first reagent, after mixing with the sample in reaction buffer, preferentially binds to the Fab fragment of the immunoglobulin, covering the antigenic epitope bound to the light chain. In the second reagent, the nanosphere-labeled anti-human FLC polyclonal antibody specifically binds only to unblocked free light chains, forming a stable antigen-antibody complex. This staged reaction design blocks interference from bound light chains while avoiding direct contact between the blocking agent and the labeled antibody.

[0038] The present application places the blocking antibody independently in the first reagent and realizes the steric hindrance effect through step-by-step reaction, which not only effectively blocks the cross-reaction, but also avoids the latex aggregation caused by the interaction between the blocking agent and the labeled antibody, thereby maintaining the colloidal stability of the reagent.

[0039] In some embodiments of the present application, the anti-human IgG-Fab polyclonal antibody is at least one of a goat anti-human IgG-Fab antibody and a rabbit anti-human IgG-Fab antibody.

[0040] Specifically, by selecting at least one of a goat anti-human IgG-Fab antibody and a rabbit anti-human IgG-Fab antibody, interference from immunoglobulins from different species can be effectively mitigated. As immune animals, sheep and rabbits recognize a wide range of antigenic epitopes, and the polyclonal antibodies they produce can recognize conformational epitopes within the Fab segment, thereby blocking multiple potential binding sites for the light chain.

[0041] Anti-human IgG-Fab polyclonal antibodies derived from sheep and rabbit have broad cross-reactivity and can effectively block the Fab region on immunoglobulins, avoiding cross-reaction with the anti-FLC polyclonal antibody in the secondary antibody reagent.

[0042] The immune systems of sheep and rabbits are capable of producing multiple antibodies targeting the human IgG-Fab region. These polyclonal antibodies can cover more antigenic epitopes, thereby improving the blocking effect. The technology in this example significantly enhances the blocking effect on the binding light chain of immunoglobulins by using polyclonal antibodies derived from sheep or rabbits, further improving the specificity and accuracy of the test.

[0043] In some embodiments of the present application, the anti-human FLC polyclonal antibody is a sheep polyclonal antibody or a rabbit polyclonal antibody prepared from FLC immunogen.

[0044] By using FLC as an immunogen, highly specific polyclonal antibodies against FLC can be prepared. These antibodies can efficiently bind to free light chains without cross-reacting with light chains bound to immunoglobulins.

[0045] By selecting polyclonal antibodies from specific sources, cross-reactivity with bound light chains is eliminated during the antibody preparation stage, eliminating the need for the addition of blocking peptides and fundamentally avoiding aggregation issues caused by the introduction of exogenous peptides. This solves the cross-reactivity issue between anti-FLC polyclonal antibodies and immunoglobulin-bound light chains, ensuring detection specificity. It also avoids latex aggregation caused by the interaction between blocking agents and labeled antibodies, improving the long-term stability of the reagent system.

[0046] In some embodiments of the present application, the nanospheres include polystyrene microspheres, colloidal gold microspheres, and magnetic beads.

[0047] Specifically, when the antibody combines with the free light chains in the serum to form an antigen-antibody complex, the complex formed will also be larger because the volume of the nanospheres is much larger than that of a single antibody molecule, which increases the scattering or absorption of light by the complex, thereby improving the sensitivity of detection.

[0048] Specifically, fixing antibodies that specifically recognize free light chains in serum on the surface of nanoparticles can improve their stability and avoid degradation or inactivation of free antibodies in solution. In addition, chemical modification of the microsphere surface can provide a stable and suitable environment for the antibodies to maintain their activity and extend the shelf life of the reagents. The immunoturbidimetric method using nanoparticles can achieve automated detection. Simply add the sample and reagents, and the instrument will automatically complete the subsequent reaction and signal reading, greatly simplifying the operation process and improving the convenience of detection.

[0049] In the serum free light chain detection reagent, after the nanosphere-labeled antibody binds to the free light chain, the concentration of free light chains in the serum is quantitatively analyzed by optical methods such as changes in scattered light intensity or transmittance.

[0050] In some embodiments of the present application, the first reagent further includes a macromolecular polymer, a salt, and a preservative.

[0051] Specifically, the buffer maintains the pH of the reaction system at 7.4 to ensure sufficient binding of the anti-human IgG-Fab polyclonal antibody to the bound light chain. Preferably, 100 mmol / L HEPES is used. By optimizing the concentration and pH of the HEPES buffer, the accuracy and stability of the assay are improved, and interfering factors in the sample are reduced.

[0052] Specifically, the macromolecular polymer accelerates the formation of the antigen-antibody complex and improves the detection sensitivity by forming a steric hindrance effect. Preferably, polyethylene glycol 6000 is used.

[0053] Specifically, salt substances maintain the spatial conformation and biological activity of antibodies by adjusting the ionic strength of the solution, thereby facilitating antibody binding, while reducing nonspecific binding through salting-out. Preferably, sodium chloride is used at a concentration of 50.0 g / L.

[0054] Specifically, the preservative effectively inhibits bacterial growth and prolongs the shelf life of the reagent, allowing the reagent to maintain stability for more than 12 months at 2° C.-8° C. Preferably, the preservative is sodium azide at a concentration of 1 g / L.

[0055] In some embodiments of the present application, the second reagent further includes a buffer, a protective protein, and a preservative.

[0056] Specifically, the buffer is preferably 20 mmol / L HEPES, pH 7.4. The protective protein is one of bovine serum albumin (BSA), casein, fish serum albumin, or fish gelatin. The preservative is a chemical substance that inhibits microbial growth, specifically sodium azide, which blocks microbial metabolic activity and ensures storage stability of the reagent.

[0057] By adding a buffer, the acid-base balance and ionic strength of the reaction system are maintained, ensuring uniform charge distribution on the surface of the nanospheres. The protective protein forms a physical barrier layer on the surface of the microspheres, effectively blocking nonspecific aggregation caused by van der Waals forces between the microspheres, reducing signal loss during the detection process, and improving detection accuracy. The preservative continuously inhibits microbial proliferation during long-term storage, avoiding increased detection background signals due to reagent contamination. The synergistic effect of these four components allows the antibody-labeled nanospheres to remain monodisperse when stored at 4°C-8°C, and the antibody activity remains above 95% of the initial value during the shelf life.

[0058] The present application further proposes a method for detecting serum free light chains, which uses a detection reagent including a first reagent and a second reagent, including the following steps: mixing a sample or a calibrator with the first reagent to allow the anti-human IgG-Fab polyclonal antibody to react with the Igs-bound light chain; adding the second reagent to allow the nanosphere-labeled anti-human FLC polyclonal antibody to specifically bind to the FLC in the sample to form a complex; detecting the light scattering signal or absorbance of the complex to quantitatively analyze the FLC concentration.

[0059] This example achieves quantitative analysis of kappa (Kappa) light chain or lambda (Lambda) light chain through the binding of specific antibodies.

[0060] Among them, anti-human IgG-Fab polyclonal antibodies refer to polyclonal antibodies produced against the Fab fragment of human immunoglobulin G, which can be specifically achieved using goat anti-human IgG-Fab antibodies or rabbit anti-human IgG-Fab antibodies. This antibody specifically binds to the bound light chain of immunoglobulin, blocking its cross-reaction with subsequent detection antibodies. Nanosphere-labeled anti-human FLC polyclonal antibodies refer to polyclonal antibodies that recognize free light chains that are fixed to the surface of nanospheres. Specifically, polystyrene microspheres, colloidal gold microspheres, or magnetic beads can be used as carriers. Nanospheres enhance the intensity of the light signal by increasing the volume of the complex, thereby improving detection sensitivity.

[0061] Specifically, this method achieves specific detection through a step-by-step reaction mechanism. In the first stage, after the sample is mixed with the first reagent, the anti-human IgG-Fab polyclonal antibody preferentially binds to the bound light chain of the immunoglobulin, forming a steric hindrance effect, preventing the subsequent detection antibody from cross-reacting with the bound light chain. In the second stage, nanoparticles labeled with anti-human FLC polyclonal antibodies are added. These antibodies specifically bind only to unbound free light chains to form a complex. Due to the large surface area and optical signal amplification effect of the nanoparticles, the complex produces significant light scattering or absorbance changes in the solution. The amount of the complex is positively correlated with the FLC concentration level in the sample or calibration material. By detecting the light scattering or transmittance of the complex, the FLC in the sample can be quantitatively analyzed. Finally, the signal intensity at a specific wavelength is measured by the detection system, and a standard curve is established to complete the quantitative analysis.

[0062] This assay effectively addresses the cross-reactivity issue between anti-FLC polyclonal antibodies and immunoglobulin-binding light chains, while maintaining the stability of the nanosphere labeling system through a step-by-step reaction design. This method enhances the specificity of serum free light chain detection while ensuring consistent performance during storage and use.

[0063] In some embodiments of the present application, when detecting the light scattering signal or absorbance of the complex and quantitatively analyzing the FLC concentration, the detection wavelength is 570 nm and the sub-wavelength is 800 nm.

[0064] Specifically, the primary detection wavelength of 570nm is used to capture the specific wavelength band of light scattering or absorption characteristics of the antigen-antibody complex. This wavelength is selected as the characteristic absorption peak of the antigen-antibody complex and can be achieved using a spectrophotometer or light scattering detector. The secondary wavelength of 800nm ​​is used to subtract the near-infrared band of background interference signals. Specifically, it can be achieved using a dual-wavelength differential detection system. This wavelength can eliminate interference from nonspecific scattering such as lipemia and hemolysis in the sample.

[0065] In addition, the scattering characteristics of the near-infrared band are more suitable for the particle size distribution range of colloidal gold or polystyrene microspheres, which can avoid signal distortion caused by microsphere aggregation.

[0066] This example provides specific wavelengths to optimize detection performance and enhance sensitivity and specificity. By setting wavelengths of 570 nm and 800 nm and calculating the difference between the primary and secondary wavelength signals, the effects of nonspecific light scattering can be eliminated, allowing the detection results to reflect only changes in the concentration of the target complex, thereby improving detection sensitivity and specificity. The technology in this example significantly enhances detection sensitivity and specificity, reduces background interference in the sample, and improves the signal-to-noise ratio of the detection signal.

[0067] Comparative Example 1

[0068] A serum free light chain detection reagent, the raw material components and dosages of the reagent are as follows:

[0069] First reagent: 100 mmol / L HEPES (pH 7.4), 2.0 g / L polyethylene glycol 6000, 2.0 g / L BSA, 50.0 g / L sodium chloride, 1 g / L sodium azide;

[0070] Secondary reagent: 20 mmol / L HEPES (pH 7.4), 2.5 g / L polystyrene microsphere-labeled anti-human FLC-κ antibody or human FLC-λ antibody, 2.0 g / L BSA, and 1 g / L sodium azide.

[0071] Example 1

[0072] A serum free light chain detection reagent, the raw material components and dosages of the reagent are as follows:

[0073] The first reagent: 100 mmol / L HEPES (pH 7.4), 2.0 g / L polyethylene glycol 6000, 2.0 g / L BSA, 50.0 g / L sodium chloride, 1 g / L sodium azide, 10 g / L rabbit anti-human IgG (H+L) polyclonal antibody;

[0074] Secondary reagent: 20 mmol / L HEPES (pH 7.4), 2.5 g / L polystyrene microsphere-labeled anti-human FLC-κ antibody or human FLC-λ antibody, 2.0 g / L BSA, and 1 g / L sodium azide.

[0075] Example 2

[0076] A serum free light chain detection reagent, the raw material components and dosages of the reagent are as follows:

[0077] The first reagent: 100 mmol / L HEPES (pH 7.4), 2.0 g / L polyethylene glycol 6000, 2.0 g / L BSA, 50.0 g / L sodium chloride, 1 g / L sodium azide, 10 g / L rabbit anti-human IgG-F(ab)2 polyclonal antibody;

[0078] Secondary reagent: 20 mmol / L HEPES (pH 7.4), 2.5 g / L polystyrene microsphere-labeled anti-human FLC-κ antibody or human FLC-λ antibody, 2.0 g / L BSA, and 1 g / L sodium azide.

[0079] Example 3

[0080] A serum free light chain detection reagent, the raw material components and dosages of the reagent are as follows:

[0081] The first reagent: 100 mmol / L HEPES (pH 7.4), 2.0 g / L polyethylene glycol 6000, 2.0 g / L BSA, 50.0 g / L sodium chloride, 1 g / L sodium azide, 10 g / L mouse anti-human IgG-Fab monoclonal antibody;

[0082] Secondary reagent: 20 mmol / L HEPES (pH 7.4), 2.5 g / L polystyrene microsphere-labeled anti-human FLC-κ antibody or human FLC-λ antibody, 2.0 g / L BSA, and 1 g / L sodium azide.

[0083] Example 4

[0084] A serum free light chain detection reagent, the raw material components and dosages of the reagent are as follows:

[0085] The first reagent: 100 mmol / L HEPES (pH 7.4), 2.0 g / L polyethylene glycol 6000, 2.0 g / L BSA, 50.0 g / L sodium chloride, 1 g / L sodium azide, 10 g / L goat anti-human IgG-Fab polyclonal antibody;

[0086] Secondary reagent: 20 mmol / L HEPES (pH 7.4), 2.5 g / L polystyrene microsphere-labeled anti-human FLC-κ antibody or human FLC-λ antibody, 2.0 g / L BSA, and 1 g / L sodium azide.

[0087] Example 5

[0088] A serum free light chain detection reagent, the raw material components and dosages of the reagent are as follows:

[0089] The first reagent: 100 mmol / L HEPES (pH 7.4), 2.0 g / L polyethylene glycol 6000, 2.0 g / L BSA, 50.0 g / L sodium chloride, 1 g / L sodium azide, 10 g / L rabbit anti-human IgG-Fab polyclonal antibody;

[0090] Secondary reagent: 20 mmol / L HEPES (pH 7.4), 2.5 g / L polystyrene microsphere-labeled anti-human FLC-κ antibody or human FLC-λ antibody, 2.0 g / L BSA, and 1 g / L sodium azide.

[0091] Determination of cross-reactivity of serum free light chain detection reagent to human IgG:

[0092] Comparative Example 1 and Examples 1-5 were used to measure samples of varying concentrations prepared with human IgG. The instrument used was a Hitachi 7180 automated analyzer with the following parameters: 570 nm wavelength, 800 nm sub-wavelength, 5.6 μl sample or calibrator, 210 μl R1, 70 μl R2, and reading points 18-34.

[0093] For IgG samples of different concentrations, the lower the measured value of the comparative example and the example, the lower the cross reaction is considered to be.

[0094] As shown in Tables 1 and 2, the comparative example reagent 1, which did not contain anti-cross antibodies, had the highest cross-reaction, while Examples 1-3 had some cross-reaction inhibition effect, but it was not very obvious. However, Examples 4 and 5, which respectively contained goat anti-human IgG-Fab polyclonal antibody and rabbit anti-human IgG-Fab polyclonal antibody in reagent 1, had the strongest cross-reaction inhibition ability.

[0095] Table 1: FLC-κ reagent anti-human IgG cross-reactivity results (mg / L)

[0096] Table 2: FLC-λ reagent anti-human IgG cross-reactivity results (mg / L)

[0097] Serum free light chain detection reagent stability determination:

[0098] Examples 4 and 5 were stored at 2°C-8°C, and three levels of quality control samples were tested at 0, 3, 6, 9, and 12 months of storage. A relative deviation of less than 5% compared to the value measured at 0 month was considered to indicate better stability. The instrument used was a Hitachi 7180 automated analyzer with the following parameters: 570 nm wavelength, 800 nm sub-wavelength, 5.6 μl sample or calibrator, 210 μl R1, 70 μl R2, and reading points 18-34.

[0099] The results are shown in Table 3. The FLC-κ reagent and FLC-λ reagent prepared according to Example 4 and Example 5 can be stable at 2°C-8°C for 1 year.

[0100] Table 3: Reagent stability verification results

[0101]

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A serum free light chain detection reagent, characterized in that: The detection reagent includes: A first reagent, comprising a reaction buffer and an anti-human IgG-Fab polyclonal antibody; A second reagent, comprising an anti-human FLC-κ antibody or an anti-human FLC-λ antibody labeled with nanospheres; The anti-human IgG-Fab polyclonal antibody is used to block the cross-reaction between the binding light chain in the immunoglobulin in the sample and the anti-human FLC-κ antibody or the anti-human FLC-λ antibody.

2. The detection reagent according to claim 1, characterized in that The anti-human IgG-Fab polyclonal antibody is at least one of a goat anti-human IgG-Fab antibody and a rabbit anti-human IgG-Fab antibody.

3. The detection reagent according to claim 1, characterized in that The nanometer microspheres include polystyrene microspheres, colloidal gold microspheres and magnetic beads.

4. The detection reagent according to claim 1, characterized in that The first reagent also includes: a macromolecular polymer, a salt, and a preservative.

5. The detection reagent according to claim 1, characterized in that The second reagent also includes a buffer, a protective protein, and a preservative.

6. A method for detecting serum free light chains, characterized in that: Using the detection reagent according to any one of claims 1 to 5, comprising the following steps: S10, mixing the sample or calibrator with the first reagent to allow the anti-human IgG-Fab polyclonal antibody to react with the bound light chain of Igs; S20, adding the second reagent to allow the anti-human FLC-κ antibody or anti-human FLC-λ antibody labeled with the nanospheres to specifically bind to the FLC-κ or FLC-λ in the sample to form a complex; S30. Detect the light scattering signal or absorbance of the complex and quantitatively analyze the concentration of FLC-κ or FLC-λ.

7. The detection method according to claim 6, characterized in that In S30 , the detected wavelength is 570 nm and the sub-wavelength is 800 nm.

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