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CN-122017255-A - Reagent, kit and detection method for free light chain detection

CN122017255ACN 122017255 ACN122017255 ACN 122017255ACN-122017255-A

Abstract

The invention discloses a reagent, a kit and a detection method for detecting a free light chain, and relates to the technical field of detection kits, comprising the following components, wherein the surface of a solid phase carrier is marked with a F (ab ') 2 antibody fragment; the liquid phase reaction solution comprises a composite blocking agent for inhibiting nonspecific reaction of endogenous interferents, wherein the F (ab') 2 antibody fragment and the composite blocking agent are combined to improve the specificity and the reaction linearity of detection. The invention solves the problems that the existing means can not eradicate the free light chain due to nonspecific reaction caused by an Fc segment of an antibody and an endogenous interference object, and the detection linearity is abnormal and the specificity is low, and the solid phase carrier marked with the F (ab') 2 antibody fragment and the composite blocker are used for synergism, so that the double-dimensional closed loop protection is formed by eliminating the self interference of the antibody and shielding the exogenous interference of a sample, and compared with the single use of the antibody optimization or interference shielding means, the combination targeting property and the detection specificity of a target analyte are synergistically improved.

Inventors

  • LIN YUAN
  • LIU ZHEN
  • LI XUERUI
  • ZHANG NANA

Assignees

  • 重庆博士泰生物技术有限公司

Dates

Publication Date
20260512
Application Date
20260320

Claims (10)

  1. 1. A reagent for free light chain immunoturbidimetry detection, comprising the following components: A solid phase carrier, the surface of which is marked with F (ab') 2 antibody fragment; A liquid phase reaction liquid including a complex blocker for inhibiting a non-specific reaction of an interfering substance; Wherein the F (ab') 2 antibody fragment interacts with the complex blocker to improve specificity and linearity of the assay.
  2. 2. The reagent for detecting free light chain immunoturbidimetry according to claim 1, wherein the F (ab ') 2 antibody fragment is prepared by using rabbit anti-human free light chain polyclonal antibody as a raw material, performing pepsin enzymolysis, and removing the Fc fragment and the intact antibodies which are not subjected to enzymolysis by using a Protein A chromatographic column, thereby obtaining a purified F (ab') 2 antibody fragment.
  3. 3. The reagent for free light chain immunoturbidimetry detection of claim 1, wherein the composite blocker comprises 0.1 mg/mL to 0.5mg/mL rheumatoid factor antibody, 0.1 mg/mL to 0.5mg/mL rabbit IgG, 0.1 mg/mL to 0.5mg/mL rabbit IgM, and 0.1 mg/mL to 0.5mg/mL amphotropic antibody blocker.
  4. 4. The reagent for detecting free light chain immunoturbidimetry according to claim 3, wherein the working concentration of the complex blocker in the liquid phase reaction solution is 0.1 mg/mL to 0.5mg/mL.
  5. 5. The reagent for detecting free light chain immunoturbidimetry according to claim 1, wherein the solid phase carrier is latex microsphere with a particle size of 60-200nm.
  6. 6. The reagent for free light chain immunoturbidimetry detection of claim 5, wherein the F (ab') 2 antibody fragment is coupled to the surface of the latex microsphere by a covalent bond.
  7. 7. The reagent for detecting free light chain immunoturbidimetry according to claim 1, wherein the liquid phase reaction solution further comprises 100mM/LHEPES buffer, sodium chloride, polyethylene glycol and a surfactant.
  8. 8. A kit for free light chain detection comprising: R1 reagent is the liquid phase reaction liquid according to any one of claims 1 to 7; R2 reagent the solid phase carrier of the conjugated F (ab') 2 antibody fragment of any one of claims 1-7 and a latex preservative solution.
  9. 9. The kit for free light chain detection of claim 8, wherein the latex preservative solution comprises 100mM/L HEPES buffer, 2% -15% sucrose, 0.1% -1% bsa, and 0.01% -0.1% proclin300.
  10. 10. A method for detecting the content of free light chains in a sample, comprising the steps of: mixing a sample to be tested with the reagent of the kit R1 as claimed in claim 8 or 9, and pre-incubating; Adding an R2 reagent of the kit into the mixed system, and starting an immune reaction; The turbidity change of the reaction system is monitored, and the concentration of the free light chain in the sample is calculated according to a standard curve.

Description

Reagent, kit and detection method for free light chain detection Technical Field The invention relates to the technical field of detection kits, in particular to a reagent, a kit and a detection method for detecting a free light chain. Background Immunoglobulin (Ig) molecular monomers are composed of 2 identical heavy chains (H chains) and 2 identical light chains (L chains) and are linked by disulfide bonds. Light chains are classified into Kappa chains (Kappa chains) and Lambda chains (Lambda chains). Because of the small molecular weight of the light chain, the synthesis rate is faster than that of the heavy chain, and about 40% of the light chain is free in serum after binding to the heavy chain. I.e., free Light Chain (FLC). Normal human serum free light chain will remain within a certain range. The monoclonal light chain secreted by abnormal plasma cells is obviously more than the heavy chain, so that excessive free light chain such as multiple myeloma, systemic amyloidosis and the like can appear in serum, and the immune system diseases clinically appear in various immune system diseases, such as the abnormality of immune globulin and light chain. In autoimmune diseases, the autoantigens are complex and diverse, and B cells are stimulated by different antigens to form various plasma cell clones, and the synthesized immunoglobulins are not only increased in quantity, but also polyclonal. Free light chain detection reagents have wide clinical applications. The traditional free light chain analysis method comprises serum protein electrophoresis, immune fixation electrophoresis and the like, but has low sensitivity, cannot be quantitatively detected, and cannot meet clinical requirements. The problem is effectively solved by the immune turbidimetry analysis, and the method is basically adopted by the free light chain reagent sold in the market at present. The method has the biggest spot that an antibody is designed to only recognize and specifically combine with the epitope of a hidden site on an Ig molecule, and the high-efficiency and accurate free light chain detection analysis is realized through the turbidity change of an antigen-antibody complex. However, there is a common problem in practical clinical applications, the linearity of a part of the sample is not proportional, and the core problem is to generate nonspecific reactions. Latex-enhanced immunonephelometry techniques often employ solid-phase microspheres labeled with intact antibodies. The Fc segment of the antibody contains an antigen binding site, a complement binding site and the like, the binding reaction of the Fc segment reduces the specificity, and in addition, endogenous interferents in a sample such as a heterophilic antibody, a human anti-animal antibody, a rheumatoid factor and the like are subjected to nonspecific binding with the antibody. All of the above factors reduce specificity, further resulting in disproportionate linearity. In the prior art, side reactions are reduced by screening high-specificity antibodies, but the side reactions cannot be completely avoided. Most commercial antibodies contain Fc fragments, and some antibodies do not reach standard in anti-interference performance, resulting in poor clinical specificity and linear results. Based on this, the present application has been proposed. Disclosure of Invention The invention aims to solve the technical problems that the free light chain is detected by a latex enhanced immunoturbidimetry method, the nonspecific reaction is caused by an Fc segment of an antibody and endogenous interferents, the existing means cannot eradicate the reaction, and the detection of linear abnormality is low in specificity. The purpose is to provide a reagent, a kit and a detection method for detecting free light chains. The invention is realized by the following technical scheme: in a first aspect, the invention provides a free light chain detection kit comprising the following components: A solid phase carrier, the surface of which is marked with F (ab') 2 antibody fragment; A liquid phase reaction liquid including a complex blocker for inhibiting a non-specific reaction of an interfering substance; Wherein the F (ab') 2 antibody fragment interacts with the complex blocker to improve specificity and linearity of the assay. As one of the preferred embodiments, the F (ab ') 2 antibody fragment is prepared by using rabbit anti-human free light chain polyclonal antibody as a raw material, performing pepsin enzymolysis, and removing the Fc fragment and the intact antibodies without enzymolysis by using a Protein A chromatographic column to obtain the purified F (ab') 2 antibody fragment. As one of the preferred embodiments, the composite blocker comprises 0.1 mg/mL-0.5 mg/mL of rheumatoid factor antibody, 0.1 mg/mL-0.5 mg/mL of rabbit IgG, 0.1 mg/mL-0.5 mg/mL of rabbit IgM and 0.1 mg/mL-0.5 mg/mL of amphotropic antibody blocker. As one of the preferred embodiments, the working concentr