A blood total light chain calibrator, a kit and its application

Abstract

The present invention discloses a blood total light chain calibrator, a kit and an application thereof. The invention belongs to the technical field of detection reagents. The invention comprises: a Lambda LC calibrator and a Kappa LC calibrator; the concentrations of the Lambda LC calibrator are 1.5 g / L, 4 g / L, 7 g / L and 11 g / L respectively; the concentrations of the Kappa LC calibrator are 2.00 g / L, 5.00 g / L, 9.00 g / L and 15.00 g / L respectively. The present invention has found that at different stages of the disease, although TLC and FLC in the blood will increase, the FLC / TLC ratio is positively correlated with the severity of the disease. By providing calibrators of different concentrations and changing the ratio of BLC and FLC, calibrators close to normal people, patients and patients of different degrees of disease are obtained. The correlation coefficients are all above 0.95, and the sample deviation is less than ±15%.

Description

Technical Field

[0001] The present invention relates to the technical field of detection reagents, and more specifically to a blood total light chain calibrator, a kit and applications. Background Art

[0002] The immunoglobulin light chains (LC) in the blood are divided into binding light chains (BLC) that are bound to the heavy chains by disulfide bonds, and free light chains (FLC) that are free. Total LC in serum, abbreviated as TLC = BLC + FLC

[0003] Serum FLC has important clinical significance in many diseases such as myeloma and light chain deposition disease

[0004] Blood TLC testing, as an auxiliary diagnosis + indicator for a variety of diseases, is currently widely carried out in the laboratory departments of large tertiary hospitals. However, they are almost all monopolized by the BNII detection system and supporting reagents of Siemens of Germany, the IM800 detection system and supporting reagents of Beckman Coulter BC of the United States, and the Cobas detection system and supporting reagents of Roche of Switzerland; among them, the LC detection reagents of Siemens BNII are the most mainstream. Only a few companies in China have related detection kits, such as Ningbo Prebac Company, Chongqing Bostai Company, etc., but their test results are quite different from Siemens LC.

[0005] For example, Figure 1 "Light Chain LC Lambda Clinical Comparison", the correlation coefficient is less than 0.6.

[0006] Existing LC patents do not mention how to solve the problem of consistency of clinical results, but only solve the problem of reagent sensitivity and detection convenience.

[0007] In addition, currently common commercial total light chain detection antibodies include Kappa&Lambda LC rabbit polyclonal antibody from Dako (under Agilent Technologies, USA), catalog numbers Q0498&Q0499, and Kappa&Lambda LC sheep polyclonal antibody from Nittobo, Japan; commercial free light chain antibodies include Kappa&Lambda FLC rabbit polyclonal antibody from Dako (under Agilent Technologies, USA), catalog numbers A0100&A0101.

[0008] TLC detection, currently all reagents use immunoturbidimetry or latex immunoturbidimetry. Antibodies for detecting LC are divided into:

[0009] Bindingsite antibody, an antibody that only recognizes the epitope of the binding surface between LC and immunoglobulin heavy chain, abbreviated as BS-Ab;

[0010] Outsite antibody, an antibody that recognizes an outer surface epitope of LC that is not bound to the immunoglobulin heavy chain, abbreviated as OS-Ab;

[0011] Among them, BS-Ab is the FLC detection antibody;

[0012] The detection antibody of total light chain is actually a mixture of BS-Ab and OS-Ab. The general ratio of BS-Ab:OS-Ab is about 1:1, such as Q0498&Q0499;

[0013] A0100&A0101 are almost all BS-Ab.

[0014] In principle, immunoturbidimetry and latex immunoturbidimetry establish a corresponding relationship (standard curve) between the turbidity signal value (absorbance for transmission detection and scattered light intensity for scattering detection) and the known concentration of the analyte (calibrator), and then test the samples of unknown concentration. The automated instrument automatically calculates the concentration of the analyte based on the standard curve using the turbidity signal value of the sample.

[0015] Detection accuracy: NMPA generally allows a detection deviation of ±15%

[0016] However, in TLC detection, approximately, one unit of BLC can only form one unit of turbidity; but one unit of monomer FLC can form 3 units of turbidity, and one unit of dimer FLC can form approximately 3 units of turbidity...; It can be understood that in different samples, if the content of BLC, FLC monomer, dimer, and polymer is inconsistent, the corresponding relationship between turbidity and concentration will be messed up, and the detection deviation will far exceed the ±15% allowed by NMPA;

[0017] TLC in the blood, the FLC content of normal people is very low, about 1-2% of TLC. Studies have found that this content will change at different stages of various FLC-related diseases, increasing from 2% to 50%;

[0018] When testing blood TLC, there is a problem of differences in the ability of BLC and FLC to form turbidity. The differences in BLC and FLC content between normal people and patients, and between different patients, lead to confusion in the corresponding relationship between concentration and turbidity, which cannot be solved.

[0019] Therefore, how to provide a blood total light chain calibrator, a kit and its application to overcome the above-mentioned technical obstacles is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0020] In view of this, the present invention provides a blood total light chain calibrator, a kit and an application.

[0021] The applicant's previous research solved the problem of clinical results of FLC detection by using high NaCL in the buffer and high surfactant TW-20 in the reagent. It is now speculated that the above extreme conditions may be used to dissociate FLC dimers and polymers in the analyte into monomers for detection, which can be directly referenced in TLC detection.

[0022] In order to achieve the above object, the present invention adopts the following technical solution:

[0023] A blood total light chain calibrator, comprising: a Lambda LC calibrator and a Kappa LC calibrator; the concentrations of the Lambda LC calibrator are 1.5 g / L, 4 g / L, 7 g / L, and 11 g / L respectively;

[0024] The concentrations of Kappa LC calibrants were 2.00 g / L, 5.00 g / L, 9.00 g / L, and 15.00 g / L;

[0025] When the Lambda LC calibrant concentration is 1.5 g / L, it includes 98% BLC and 2% FLC by mass;

[0026] When the Lambda LC calibrant concentration is 4 g / L, it includes 82% BLC and 18% FLC by mass;

[0027] When the Lambda LC calibrant concentration is 7 g / L, it includes 66% BLC and 34% FLC by mass;

[0028] When the Lambda LC calibrant concentration is 11 g / L, it includes 50% BLC and 50% FLC by mass;

[0029] When the concentration of Kappa LC calibrant is 2.00 g / L, it includes 98% BLC + 2% FLC by mass;

[0030] When the concentration of Kappa LC calibrant is 5.00 g / L, it includes 82% BLC and 18% FLC by mass;

[0031] When the concentration of Kappa LC calibrant is 9.00 g / L, it includes 66% BLC and 34% FLC by mass;

[0032] When the concentration of Kappa LC calibrator is 15 g / L, it includes 50% BLC and 50% FLC by mass.

[0033] A detection reagent comprising the above-mentioned calibrator.

[0034] A detection kit comprising the above-mentioned calibrator.

[0035] Through the above technical solutions, it can be known that compared with the prior art, the present invention discloses a blood total light chain calibrator, a kit and an application, and the technical effect achieved is: the present invention has found that in different stages of the disease, although TLC and FLC in the blood will increase, the FLC / TLC ratio is positively correlated with the severity of the disease. By providing calibrators of different concentrations and changing the ratio of BLC and FLC, calibrators close to normal people, patients, and patients of different degrees of disease are obtained. The correlation coefficients are all above 0.95, and the sample deviation is less than ±15%. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0037] Figure 1 The accompanying drawing is a clinical comparison diagram of the light chain LC Lambda of the prior art provided by the present invention.

[0038] Figure 2 The accompanying drawing is a schematic diagram of the detection principle provided by the present invention.

[0039] Figure 3 The accompanying drawing is a schematic diagram of the binding light chain reaction principle provided by the present invention.

[0040] Figure 4 The accompanying drawing is a schematic diagram of the free light chain reaction principle provided by the present invention.

[0041] Figure 5 The accompanying drawing is a comparison diagram of Lambda LC detection provided by the present invention.

[0042] Figure 6 The accompanying drawing is a comparison diagram of Kappa LC detection provided by the present invention. DETAILED DESCRIPTION

[0043] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The embodiment of the present invention discloses a blood total light chain calibrator, a kit and an application.

[0045] In the examples, the raw materials, reagents, instruments and equipment not mentioned are all commercially available or recorded in the prior art, for example:

[0046] (Reagents) R1: 4-hydroxyethylpiperazineethanesulfonic acid 100mmol / L, sodium chloride 2200mmol / L, Tween-20 1ml / L and preservative Proclin-3000.3ml / L;

[0047] (latex reagent coupled with antibody) R2

[0048] Use commercial TLC antibodies, such as Kappa&Lambda LC rabbit polyclonal antibody from Dako (Agilent Technologies, USA), catalog number Q0498&Q0499, or Kappa&Lambda LC sheep polyclonal antibody from Nittobo, Japan;

[0049] The latex microspheres used are commercial microspheres, such as latex microspheres SS (item number CM-1-1) produced by Chengdu Zhijiexin Company, activator (item number WSC0245), and reaction terminator (item number 0208E);

[0050] Specifically: 1L Kappa LC reagent R2 preparation process is: 800ml latex microspheres SS, add 20ml activator at room temperature, mix at room temperature 180rpm for 15min; add Q0498 KAPPA LC antibody 25ml, or 3 times the mass concentration of goat polyclonal antibody (the titer of goat polyclonal antibody is slightly lower, and the feed amount is 3 times that of rabbit polyclonal antibody); room temperature coupling reaction for 3 hours, add 20ml terminator, and add 160ml diluent (50mM Taps-NaOH, pH8.0, containing 0.5ml per liter PC300 preservative), add 6ml Tween-20, mix for 1 hour, and prepare KAPPA LC reagent R2.

[0051] The preparation process of 1L Lambda LC reagent R2 is as follows: 800ml latex microspheres SS, add 20ml activator at room temperature, mix at room temperature at 180rpm for 15min; add 18ml Q0499 Lambda LC antibody, or 3 times the mass concentration of goat polyclonal antibody (the titer of goat polyclonal antibody is slightly lower, and the mg feeding amount is 3 times that of rabbit polyclonal antibody); carry out coupling reaction at room temperature for 3 hours, add 20ml terminator, and add 160ml diluent (50mM Taps-NaOH, pH8.0, containing 0.5ml per liter PC300 preservative), add 6ml Tween-20, mix for 1 hour, and prepare Lambda LC reagent R2.

[0052] Detection principle: Figures 2 to 4 shown.

[0053] For blood TLC detection, TLC detection is currently mainly carried out by immunoturbidimetry, which is divided into two methods: ordinary immunoturbidimetry in which antibodies and antigens directly form turbidity, or antibodies are labeled on latex microspheres to form larger immune complexes with antigens. Both are calibrated on automated detection instruments (specific protein analyzers, biochemical analyzers) using calibrators of known concentrations, and the instrument establishes a relationship diagram (calibration curve) between turbidity signal values ​​(transmission is absorbance value, scattering is scattered light intensity value) and concentration values, and then detects the turbidity signal value of blood samples, and the instrument automatically calculates the TLC concentration of the object being measured according to the calibration curve.

[0054] Example 1

[0055] A blood total light chain calibrator: Lambda LC (linear range 0.15-11 g / L), the calibrator distribution is 1.5 g / L (mass proportion 98% BLC + 2% FLC), 4 g / L (82% BLC + 18% FLC), 7 g / L (66% BLC + 34% FLC), 11 g / L (50% BLC & FLC).

[0056] Lambda LC parameters on Hitachi 3500 were: wavelength 600 nm, sample 2 μl (automatically diluted 75 times by the instrument), R1 170 μl, R2 70 μl, two-point endpoint method, 18-34 reading points; 5-point calibration (0.00, 1.50, 4.00, 7.00, 11.00 g / L), spline curve 2 fitting calibration curve; prepared Lambda LC reagent R2 was used with universal reagent R1, and calibration products with the above ratio were used for calibration. Clinical samples were measured on Hitachi 3500, and these samples were measured on Siemens BNII on the same day. Statistical data, correlation and deviation calculated using Excel are shown in Table 1.

[0057] Table 1

[0058]

[0059]

[0060]

[0061] Example 2

[0062] A blood total light chain calibrator: Kappa LC (linear range 0.2-15 g / L) calibrator distribution is 2 g / L (98% BLC + 2% FLC), 5 g / L (82% BLC + 18% FLC), 9 g / L (66% BLC + 34% FLC), 15 g / L (50% BLC & FLC);

[0063] The parameters for Kappa LC on Hitachi 3500 were: wavelength 600nm, sample 2μl (automatically diluted 75 times by the instrument), R1170μl, R270μl, two-point endpoint method, 18-34 reading points; 5-point calibration (0.00, 2.00, 5.00, 9.00, 15.00g / L), spline curve 2 fitting calibration curve; the prepared Kappa LC reagent R2 was used with the universal reagent R1, and the calibration product with the above ratio was used for calibration, and clinical samples were measured on Hitachi 3500, and these samples were measured on the Siemens BNII on the same day, and the statistics were calculated, and the correlation and deviation were calculated using Excel.

[0064] Table 2

[0065]

[0066]

[0067]

[0068] Technical Effects

[0069] The experimental results of Examples 1 and 2 show that:

[0070] At different stages of the disease, although TLC and FLC in the blood will increase, the FLC / TLC ratio is positively correlated with the severity of the disease. The core technology is to use different concentrations of calibrators to change the ratio of BLC and FLC, so as to obtain calibrators close to normal people, patients, and patients with different degrees of disease.

[0071] The present invention obtains test results very close to those of Siemens, such as Figure 5 , 6 , the correlation coefficients all reached above 0.95, and the sample deviation was less than ±15%.

[0072] Comparative experiment

[0073] Comparative Example 1

[0074] If normal human BLC (98% by mass) and FLC (2%) are used to prepare calibrants of all concentrations, the turbidity of samples from moderate to severe patients will increase significantly due to the increase in the proportion of FLC, and the LC antigen per unit concentration will increase significantly, causing the measurement results of moderate to high-value samples (moderate to severe patients) to be significantly higher. Although this does not affect the diagnosis, it does not meet the accuracy requirements of the Food and Drug Administration.

[0075] For a patient whose real Kappa LC concentration (measured by Siemens BNII) is 14 g / L (50% BLC, 50% FLC), a normal person's BLC (98%) and FLC (2%) are used to prepare calibrants of all concentrations. During the measurement, 50% BLC forms a turbidity of about 7 g / L, but 50% FLC can form at least 3 times the turbidity of BLC per gram, resulting in a signal value of at least 21 g / L, 7+21=28 g / L, which is much greater than the real concentration of the sample of 14 g / L. Although the patient will be diagnosed with a serious disease, the deviation far exceeds the NMPA requirement of 15%.

[0076] Comparative Example 2

[0077] If the calibration material used for seriously ill patients, i.e. a 50% mix of FLC and BLC, is used for calibration, the low and medium value samples (normal subjects and mild to moderately ill patients) will have a relatively low turbidity produced by the unit concentration of LC antigen due to the high BLC content, thus resulting in low test results and an underestimated condition.

[0078] For a patient with a median LambdaLC, the true concentration (measured by Siemens BNII) is 3.0g / L (85% BLC, 15% FLC), of which (15% BLC plus 15% FLC) can measure a turbidity of 0.9g / L on the above calibration curve, but the remaining 70% BLC has only 1 / 3 of the turbidity forming ability of FLC, and the estimated measured value is 0.8g / L. The final measurement result is 0.9+0.8=1.7g / L, which is slightly more than the reference value of 2.1g / L. The patient who was originally moderately positive at 3.0g / L was measured as negative, resulting in missed diagnosis and delayed treatment.

[0079] The above lists comparative proportions of two extreme cases. Those skilled in the art can infer that the blood TLC calibration product prepared with a ratio close to the ratio discovered in the present invention can make the TLC detection accuracy basically meet the NMPA requirements.

[0080] Example 3

[0081] A method for preparing a blood total light chain calibrator:

[0082] FLC: Commercially pure products, such as Yashrj's FLC antigen pure products (DLCKHP 0003 / 15 & DLCLHP 0003 / 15), with a purity greater than 98%;

[0083] Pure BLC (greater than 99% purity): human immunoglobulin injection can be used, using 80-100kd centrifugal ultrafiltration tubes (Sartorius), to remove FLC, retain IGG (BLC), and re-dissolve for preparation.

[0084] BLC can be reconstituted using diluents sold by BioRong Diagnostics (Shanghai) Co., Ltd., such as the diluent with catalog number BYS92420.

[0085] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0086] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A blood total light chain calibrator, characterized in that: include: Lambda LC calibrators and Kappa LC calibrators; the concentrations of the Lambda LC calibrators are 1.5 g / L, 4 g / L, 7 g / L, and 11 g / L, respectively; The concentrations of the Kappa LC calibrants are 2.00 g / L, 5.00 g / L, 9.00 g / L, and 15.00 g / L; When the concentration of the Lambda LC calibrant is 1.5 g / L, it includes 98% BLC and 2% FLC by mass; When the concentration of the Lambda LC calibrant is 4 g / L, it includes 82% BLC and 18% FLC by mass; When the concentration of the Lambda LC calibrant is 7 g / L, it includes 66% BLC and 34% FLC by mass; When the concentration of the Lambda LC calibrant is 11 g / L, it includes 50% BLC and 50% FLC by mass; When the concentration of the Kappa LC calibrator is 2.00 g / L, it includes 98% BLC and 2% FLC by mass; When the concentration of the Kappa LC calibrator is 5.00 g / L, it includes 82% BLC and 18% FLC by mass; When the concentration of the Kappa LC calibrant is 9.00 g / L, it includes 66% BLC and 34% FLC by mass; When the concentration of the Kappa LC calibrator is 15 g / L, it includes 50% BLC and 50% FLC by mass.

2. A detection reagent comprising the blood total light chain calibrator according to claim 1.

3. A detection kit comprising the blood total light chain calibrator according to claim 1.

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