Latex enhanced immunoturbidimetry kit for detecting free hemoglobin

Abstract

The invention discloses a latex enhanced immunoturbidimetric kit for detecting free hemoglobin, and belongs to the technical field of medicine detection. The latex enhanced immunoturbidimetric kit for detecting the free hemoglobin comprises a reagent R1 and a reagent R2, the reagent R1 comprises 50 mM of tris (hydroxymethyl) aminomethane, 0.5 g / L to 2 g / L of a coagulant, 50 mg / L to 200 mg / L of a blocking agent, 0.5 g / L to 1.5 g / L of sodium chloride, 0.05 g / L to 0.15 g / L of bovine serum albumin, 0.25 g / L to 0.75 g / L of trehalose and 0.05 g / L to 0.5 g / L of a preservative; the reagent R2 is prepared from 50 mM of hydroxyethyl piperazine ethanesulfonic acid, 0.5 g / L to 1.5 g / L of sodium chloride, 0.05 g / L to 0.15 g / L of bovine serum albumin, 3 g / L to 7 g / L of glycerinum, 1 g / L to 3 g / L of trehalose, 0.05 g / L to 0.5 g / L of preservative and 0.05 g / L to 0.2 g / L of anti-human hemoglobin antibody sensitized latex particles. Through chemical coupling of a hemoglobin monoclonal antibody and carboxyl microspheres, an'antigen-monoclonal antibody-microsphere-monoclonal antibody-antigen 'compound detection system is constructed.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical detection, and particularly relates to a latex enhanced immunoturbidimetric kit for detecting free hemoglobin. Background Art

[0002] Normal human hemoglobin is distributed in red blood cells in the blood. After the metabolism and apoptosis of red blood cells, hemoglobin is released into the blood in a free state. Therefore, there is about 50 mg / L of free hemoglobin in the plasma of healthy people, but the content of free hemoglobin in urine is extremely low. The increase in plasma free hemoglobin is an indication of intravascular hemolysis. If the plasma free hemoglobin reaches a certain level (900 - 1000 mg / L), it can be excreted through urine, that is, accompanied by hemoglobinuria, which is seen in favism, PNH, paroxysmal cold hemoglobinuria, unstable hemoglobinopathy, cold agglutination syndrome, etc. In autoimmune hemolytic anemia, sickle cell anemia and thalassemia, plasma free hemoglobin may increase slightly or moderately. Severe transfusion reactions, especially blood type incompatibility, can occur, resulting in a large increase in plasma free hemoglobin, which may be toxic to renal tubules, cause severe kidney damage and endanger life. After extracorporeal circulation, such as the application of cardiopulmonary bypass and artificial kidney surgery, a large number of red blood cells can be mechanically damaged, which can also increase plasma free hemoglobin. When the amount of hemoglobin exceeds the binding capacity of haptoglobin, plasma free hemoglobin is filtered through the glomerulus. When it exceeds 1.00 - 1.35 g / L, hemoglobin can be excreted through urine, that is, hemoglobinuria can occur. In addition, when blood vessels are damaged or their permeability changes under certain pathological conditions, red blood cells can enter the surrounding tissues through the blood vessel wall and release hemoglobin. At this time, free hemoglobin can be detected in related specimens.

[0003] The detection of trace free hemoglobin in medical laboratories is also called occult blood test or hidden blood test, which is one of the most commonly used examination items in clinical differential diagnosis of hemorrhagic or hemolytic diseases. Existing methodologies are mostly qualitative or semi - quantitative experiments. Currently, the commonly used detection methods in laboratories mainly include colorimetry, chemical method and gold label method. The content of free hemoglobin in specimens such as normal urine, feces, gastric juice, serous cavity effusion, cerebrospinal fluid, saliva, etc. is extremely low. Although a large amount of free hemoglobin may appear in these specimens under pathological conditions, its absolute value is still relatively low. The sensitivity of conventional measurement methods far cannot meet the performance requirements of these specimens. Although the colloidal gold method can be used as a confirmation method for occult blood test, it is currently limited to qualitative tests; although the test strip method and colorimetry have relatively high sensitivity, their specificity is poor, and they are both qualitative or semi - quantitative tests; although there are reports at home and abroad using dedicated instruments for nephelometry for occult blood quantitative detection, this instrument is only limited to the detection of fecal specimens, and it is not clear whether its methodological performance is applicable to the detection of other types of specimens. Summary of the Invention

[0004] The object of the present invention is to provide a latex enhanced immunoturbidimetric kit for detecting free hemoglobin, which is used to detect the content of free hemoglobin and improve the detection accuracy.

[0005] The object of the present invention can be achieved by the following technical solutions:

[0006] A latex enhanced immunoturbidimetric kit for detecting free hemoglobin, the kit includes R1 reagent and R2 reagent;

[0007] The R1 reagent includes 50 mM tris(hydroxymethyl)aminomethane, 0.5 - 2 g / L coagulant, 50 - 200 mg / L blocker, 0.5 - 1.5 g / L sodium chloride, 0.05 - 0.15 g / L bovine serum albumin, 0.25 - 0.75 g / L trehalose and 0.05 - 0.5 g / L preservative;

[0008] The R2 reagent includes 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 0.5 - 1.5 g / L sodium chloride, 0.05 - 0.15 g / L bovine serum albumin, 3 - 7 g / L glycerol, 1 - 3 g / L trehalose, 0.05 - 0.5 g / L preservative and 0.05 - 0.2 g / L latex particles sensitized with anti-human hemoglobin antibody.

[0009] Furthermore, the pH of the R1 reagent and the R2 reagent is 7.0 - 7.5.

[0010] Furthermore, the coagulant in the R1 reagent is one or a combination of several of PEG6000, PEG20000 and dextran.

[0011] Furthermore, the model of the blocker in the R1 reagent is one or a combination of several of GU Blocker-MM1, GU Blocker-MM2, GU Blocker-MM3, GU Blocker-MM4, GU Blocker-MM5, GU Blocker-MM6, GU Blocker-MM7 and Beijing Jingda HBR-5; the blockers GU Blocker-MM1-7 are from Anhui Global Gene Technology Co., Ltd.

[0012] Furthermore, the latex particles sensitized with anti-human hemoglobin antibody are prepared by the following steps:

[0013] (1) Pre-activation: Add EDC to ultrapure water and mix well to make an EDC solution; Mix the activation solution, 10% JSR carboxyl microspheres and the EDC solution, and place them in a shaker at 30 °C and 200 r / min for shaking and activation for 15 min to obtain solution A;

[0014] (2) Crosslinking: Add the coupling solution to Solution A, then add the anti-human hemoglobin antibody. After mixing evenly, shake and react for 20 min, and sonicate 10 times with an ultrasonic mixer; continue to react for 40 min and sonicate 10 times; continue to react for 60 min; after coupling for 120 min, add the blocking agent CE510 and sonicate 15 times, and continue to mix evenly for 60 min;

[0015] (3) Storage: After blocking, sonicate 15 times, then centrifuge twice at 16500 rpm for 30 min. After removing the supernatant, latex particles sensitized with anti-human hemoglobin antibody are obtained.

[0016] Furthermore, the carboxyl microspheres are one or a combination of several of the types P0219, P0113, and P0220.

[0017] Furthermore, the concentration of the EDC solution is 5 - 15 mg / mL.

[0018] Furthermore, the activation solution is a 10 mM 2-morpholinoethanesulfonic acid solution with a pH of 5.5 - 6.5.

[0019] Furthermore, the coupling solution is a 20 mM Tris solution with a pH of 7.5 - 8.5.

[0020] A method for detecting free hemoglobin, using the above latex enhanced immunoturbidimetry kit, includes the following steps:

[0021] S1. Centrifuge the sample to be tested at 4°C and 12000 rpm for 5 min, take the supernatant to obtain the test sample;

[0022] S2. Establish a calibration curve using the calibrator and quality control; set the project parameters, place the sample and reagents on each plate respectively, and start the detection;

[0023] Mix the test sample with Reagent R1, incubate for 5 - 10 min, then add Reagent R2, start to detect the OD value A1, detect the OD value A2 again after reacting for 5 - 10 min, calculate the reactivity ΔA = A2 - A1, and substitute the reactivity ΔA into the calibration curve to calculate the concentration.

[0024] Furthermore, the sample to be tested includes blood samples and urine samples.

[0025] Furthermore, the calibrator and quality control are prepared by the following steps:

[0026] (1) Dilute human hemoglobin antigen to 0 mg / L, 20 mg / L, 50 mg / L, 200 mg / L, 500 mg / L, and 1000 mg / L using the calibrator and quality control diluent;

[0027] (2) Use vials to dispense the prepared calibrators and quality control products (0.5 mL per vial), cover with a soft rubber stopper, do not completely seal it, leave a certain air-permeable gap, and place it in a -80 °C refrigerator for pre-freezing overnight; turn on the freeze dryer, turn on the refrigerator to pre-cool to -45 °C, place the pre-frozen calibration and quality control products in the freeze drying rack, and freeze dry for 12 - 16 h to obtain freeze-dried calibrators and quality control products;

[0028] (3) Place the freeze-dried calibrators and quality control products in a 37 °C incubator for hot compress for 1 day to obtain calibrators and quality control products.

[0029] Furthermore, the diluent of the calibrators and quality control products includes ultrapure water, bovine serum albumin, and sucrose, and the mass ratio of ultrapure water, bovine serum albumin, and sucrose is (85 - 95):(2 - 5):(5 - 15).

[0030] Advantages of the present invention:

[0031] (1) The present invention provides a latex-enhanced immunoturbidimetric kit for detecting free hemoglobin. Through the chemical coupling of hemoglobin monoclonal antibody and carboxyl microspheres, a monoclonal antibody-microsphere-monoclonal antibody conjugate is formed. This conjugate specifically binds to hemoglobin antigen in the sample through the hemoglobin monoclonal antibody to form an antigen-monoclonal antibody-microsphere-monoclonal antibody-antigen complex system; the synergistic effect of the high specific binding ability of the monoclonal antibody and the signal amplification effect of the microspheres is used to establish a calibration curve of concentration-OD value through calibration and quality control products. Substituting the OD value of the complex detected by the turbidimetry method into the calibration curve can quantitatively calculate the hemoglobin content in the sample.

[0032] (2) The latex particles sensitized with anti-human hemoglobin antibody used in the present invention have high sensitivity and specificity, and can specifically detect extremely low levels of free hemoglobin; it can be applied to a biochemical analyzer and can quickly and conveniently perform automated quantitative detection of different types of samples (such as blood, urine samples). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described below with reference to the accompanying drawings.

[0034] Figure 1 is the correlation curve of the blood sample value - the measured value of the invention reagent and the HPLC result;

[0035] Figure 2 is the correlation curve of the urine sample value - the measured value of the invention reagent and the HPLC result. DETAILED DESCRIPTION OF THE INVENTION

[0036] The following describes the specific embodiments of the present invention in detail, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0037] Example 1

[0038] This embodiment provides a latex enhanced immunoturbidimetric kit for detecting free hemoglobin, and the kit includes R1 reagent and R2 reagent;

[0039] The R1 reagent includes 50 mM tris(hydroxymethyl)aminomethane, 1 g / L PEG6000, 100 mg / L GU Blocker-MM1, 1 g / L sodium chloride, 0.1 g / L bovine serum albumin, 0.5 g / L trehalose, and 0.1 g / L preservative, with a pH of 7.4;

[0040] The R2 reagent includes 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 0.1 g / L sodium chloride, 0.1 g / L bovine serum albumin, 5 g / L glycerol, 2 g / L trehalose, 0.1 g / L preservative, and 0.1 g / L latex particles sensitized with anti-human hemoglobin antibody, with a pH of 7.4.

[0041] The latex particles sensitized with anti-human hemoglobin antibody are prepared by the following steps:

[0042] (1) Pre-activation: Add EDC to ultrapure water and mix well to prepare an EDC solution with a concentration of 10 mg / mL; add the activation solution, 10% JSR carboxyl microspheres P0219, and the EDC solution, shake and mix well, and place it in a shaker at 30 °C and 200 r / min for 15 min of shaking activation to obtain solution A;

[0043] (2) Cross-linking: Add the coupling solution to solution A, then add the anti-human hemoglobin antibody, mix well and react with shaking for 20 min, and sonicate with a sonicator for 10 times; continue to react for 40 min and sonicate for 10 times; continue to react for 60 min; after coupling for 120 min, add the blocking agent CE510 and sonicate for 15 times, and continue to mix well for 60 min;

[0044] (3) Storage: After the blocking is completed, sonicate 15 times, then centrifuge twice at 16500 rpm for 30 min, remove the supernatant to obtain the latex particles sensitized with anti-human hemoglobin antibody.

[0045] The activation solution is a 10 mM 2-(N-morpholino)ethanesulfonic acid solution with a pH of 5.5 - 6.5;

[0046] The coupling solution is a 20 mM Tris solution with a pH of 7.5 - 8.5.

[0047] Example 2

[0048] This embodiment provides a method for detecting free hemoglobin. Using the above latex enhanced immunoturbidimetric kit, it includes the following steps:

[0049] S1. Preparation of calibration standards and quality control samples: Dilute human hemoglobin antigen to 0 mg / L, 20 mg / L, 50 mg / L, 200 mg / L, 500 mg / L, and 1000 mg / L using calibration standard and quality control diluent (obtained by mixing 85 parts by weight of ultrapure water, 4 parts by weight of bovine serum albumin, and 10 parts by weight of sucrose); dispense the prepared calibration standards and quality control samples into vials (0.5 mL per vial) using ampoules, cover with soft rubber stoppers, not completely seal, leaving a certain air-permeability gap, and place in a -80°C refrigerator for pre-freezing overnight; turn on the freeze dryer, turn on the refrigerator to pre-cool to -45°C, place the pre-frozen calibration and quality control samples in the freeze-drying rack, and freeze-dry for 12 h to obtain freeze-dried calibration standards and quality control samples; place the freeze-dried calibration standards and quality control samples in a 37°C incubator for hot compress for 1 day to obtain calibration standards and quality control samples;

[0050] S2. Centrifuge the blood sample to be tested at 4°C and 12000 rpm for 5 min, take the supernatant to obtain the test blood sample;

[0051] S3. Establish a calibration curve using calibration standards and quality control samples; set the project parameters, place the samples and reagents on each plate respectively, and start the detection;

[0052] Mix the test blood sample with R1 reagent, after incubation for 5 min, add R2 reagent, start detecting the OD value A1, after reacting for 5 min, detect the OD value A2 again, calculate the reactivity ΔA = A2 - A1, and substitute the reactivity ΔA into the calibration curve to deduce the concentration.

[0053] The blood sample values obtained from the above tests - the measured values of the invention reagent and the HPLC results are shown in Table 1, and the correlation curve graph is as Figure 1 shown.

[0054] Example 3

[0055] The difference between this example and Example 2 is that the blood sample is changed to a urine sample, and the specific implementation steps are as follows:

[0056] S1. Preparation of calibration standards and quality control samples: Dilute human hemoglobin antigen to 0 mg / L, 20 mg / L, 50 mg / L, 200 mg / L, 500 mg / L, and 1000 mg / L using calibration standard and quality control diluent (obtained by mixing 85 parts by weight of ultrapure water, 4 parts by weight of bovine serum albumin, and 10 parts by weight of sucrose); dispense the prepared calibration standards and quality control samples into vials (0.5 mL per vial) using ampoules, cover with soft rubber stoppers, not completely seal, leaving a certain air-permeability gap, and place in a -80°C refrigerator for pre-freezing overnight; turn on the freeze dryer, turn on the refrigerator to pre-cool to -45°C, place the pre-frozen calibration and quality control samples in the freeze-drying rack, and freeze-dry for 12 h to obtain freeze-dried calibration standards and quality control samples; place the freeze-dried calibration standards and quality control samples in a 37°C incubator for hot compress for 1 day to obtain calibration standards and quality control samples;

[0057] S2, centrifuging the urine sample to be tested at 4°C and 12000 rpm for 5 min, taking the supernatant to obtain a test urine sample;

[0058] S3. Use calibrators and quality control products to establish a calibration curve; set project parameters, place samples and reagents on each plate, and start testing;

[0059] The test urine sample was mixed with R1 reagent, incubated for 5 minutes, then R2 reagent was added to start detecting OD value A1. After 5 minutes of reaction, OD value A2 was detected again, and the reaction degree ΔA=A2-A1 was calculated. The reaction degree ΔA was substituted into the calibration curve to calculate the concentration.

[0060] The remaining raw materials and preparation process remain the same as in Example 1.

[0061] The urine sample values ​​obtained from the above test and the HPLC results of the invention reagent test values ​​are shown in Table 2, and the correlation curve is shown in Figure 2 shown.

[0062] Example 4

[0063] Compared with Example 2, this embodiment differs in that the blood sample is changed to a quality control sample, and the specific implementation steps are as follows:

[0064] S1. Prepare calibration products and quality control products: dilute human hemoglobin antigen to 0 mg / L, 20 mg / L, 50 mg / L, 200 mg / L, 500 mg / L and 1000 mg / L using calibration product and quality control product diluent (85 parts by weight of ultrapure water, 4 parts by weight of bovine serum albumin and 10 parts by weight of sucrose mixed evenly); use vials to pack the prepared calibration products and quality control products (0.5 mL per bottle), cover them with soft rubber stoppers, not completely cover them, leave a certain air permeable gap, and pre-freeze them in a -80°C refrigerator overnight; turn on the freeze dryer, turn on the refrigerator to pre-cool to -45°C, put the pre-frozen calibration quality control products into the freeze drying rack, freeze-dry for 12 hours, and obtain freeze-dried products of calibration products and quality control products; put the freeze-dried products of calibration products and quality control products into a 37°C incubator for hot compress for 1 day to obtain calibration products and quality control products;

[0065] S2. Dissolve the quality control sample to be tested in ultrapure water and let it stand for 0.5 h to obtain a test quality control sample;

[0066] S3. Use the calibration products and quality control products to establish a calibration curve; set the project parameters, place the samples and reagents on each plate, start the test, and test the concentration of the quality control products to be (30±6) mg / L-(500±100) mg / L;

[0067] Mix the test quality control sample with the R1 reagent. After incubation for 5 minutes, add the R2 reagent and start detecting the OD value A1. After reacting for 5 minutes, detect the OD value A2 again. Calculate the reactivity ΔA = A2 - A1, and substitute the reactivity ΔA into the calibration curve to deduce the concentration.

[0068] The sample values obtained from the above tests - the HPLC results of the invention reagent measurements are shown in Table 3.

[0069] Table 1

[0070]

[0071]

[0072] Table 2

[0073]

[0074] Table 3

[0075]

[0076]

[0077] Combined with Table 1 and Figure 1 From the HPLC test results of the detection values of this reagent for blood samples, it can be seen that the detection values of the reagent of the present invention have good clinical correlation, R 2 = 0.9947;

[0078] Combined with Table 2 and Figure 2 From the HPLC test results of the detection values of this reagent for urine samples, it can be seen that the detection values of the reagent of the present invention have good clinical correlation, R 2 = 0.9929;

[0079] From the test results of the repeatability of this reagent for the quality control product in Table 3, it can be seen that the precision of the reagent of the present invention is good.

[0080] In summary, a latex enhanced immunoturbidimetric kit for detecting free hemoglobin provided by the present invention, and the detection method using this kit is an automated quantitative detection method with high sensitivity, high specificity and applicable to multiple sample types.

[0081] The above-disclosed are only several specific embodiments of the present invention. However, the embodiments of the present invention are not limited thereto, and any changes that can be conceived by those skilled in the art should fall within the protection scope of the present invention.

Claims

1. A latex enhanced immunoturbidimetric kit for detecting free hemoglobin, characterized in that, The kit includes R1 reagent and R2 reagent; The R1 reagent includes 50 mM tris(hydroxymethyl)aminomethane, 0.5 - 2 g / L coagulant, 50 - 200 mg / L blocker, 0.5 - 1.5 g / L sodium chloride, 0.05 - 0.15 g / L bovine serum albumin, 0.25 - 0.75 g / L trehalose, and 0.05 - 0.5 g / L preservative; The R2 reagent includes 50 mM 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid, 0.5 - 1.5 g / L sodium chloride, 0.05 - 0.15 g / L bovine serum albumin, 3 - 7 g / L glycerol, 1 - 3 g / L trehalose, 0.05 - 0.5 g / L preservative, and 0.05 - 0.2 g / L latex particles sensitized with anti-human hemoglobin antibody.

2. The latex enhanced immunoturbidimetric kit for detecting free hemoglobin according to claim 1, characterized in that, The pH of the R1 reagent and the R2 reagent is 7.0 - 7.

5.

3. A latex enhanced immunoturbidimetric kit for detecting free hemoglobin according to claim 1, characterized in that, The coagulant in the R1 reagent is one or a combination of several of PEG6000, PEG20000, and dextran.

4. A latex enhanced immunoturbidimetric kit for detecting free hemoglobin according to claim 1, characterized in that, The model number of the blocker in the R1 reagent is one or a combination of several of GU Blocker-MM1, GU Blocker-MM2, GU Blocker-MM3, GU Blocker-MM4, GU Blocker-MM5, GU Blocker-MM6, GU Blocker-MM7, and Beijing Jingda HBR-5.

5. A latex enhanced immunoturbidimetric kit for detecting free hemoglobin according to claim 1, characterized in that, The latex particles sensitized with anti-human hemoglobin antibody are prepared by the following steps: (1) Pre-activation: Add EDC to ultrapure water and mix well to prepare an EDC solution; Add the activation solution, 10% JSR carboxyl microspheres, and the EDC solution, shake and mix well, and place in a shaker at 30 °C and 200 r / min for 15 min of activation to obtain solution A; (2) Cross-linking: Add the coupling solution to solution A, then add anti-human hemoglobin antibody, mix well and shake and react for 20 min, sonicate with a sonicator for 10 times; Continue to react for 40 min and sonicate 10 times; Continue to react for 60 min; After coupling for 120 min, add the blocking agent CE510 and sonicate 15 times, and continue to mix well for 60 min; (3) Storage: After the blocking is completed, sonicate 15 times, then centrifuge at 16500 rpm for 30 min twice, remove the supernatant to obtain the latex particles sensitized with anti-human hemoglobin antibody.

6. The latex enhanced immunoturbidimetric kit for detecting free hemoglobin according to claim 5, wherein The model number of the carboxyl microspheres is one or a combination of several of P0219, P0113, and P0220.

7. A latex enhanced immunoturbidimetric kit for detecting free hemoglobin according to claim 5, characterized in that, The concentration of the EDC solution is 5 - 15 mg / mL; the activation solution is a 10 mM 2-morpholinoethanesulfonic acid solution with a pH of 5.5 - 6.5; the coupling solution is a 20 mM Tris solution with a pH of 7.5 - 8.

5.

8. A method for detecting free hemoglobin, which uses the above-mentioned latex-enhanced immunoturbidimetric kit for detecting free hemoglobin, is characterized in that, It includes the following steps: S1. Centrifuge the test sample at 4 °C and 12000 rpm for 5 min, take the supernatant to obtain the test sample; S2. Use the calibrator and quality control product to establish a calibration curve; Set the item parameters, place the sample and reagents on each plate respectively, and start the detection; Mix the test sample with Reagent R1, incubate for 5 - 10 min, then add Reagent R2 and start detecting the OD value A1. After reacting for 5 - 10 min, detect the OD value A2 again, calculate the reactivity ΔA = A2 - A1, and substitute the reactivity ΔA into the calibration curve to deduce the concentration; The sample to be tested includes blood samples and urine samples.

9. The detection method of free hemoglobin according to claim 8, wherein The calibrator and the quality control product are prepared by the following steps: (1) Dilute human hemoglobin antigen to 0 mg / L, 20 mg / L, 50 mg / L, 200 mg / L, 500 mg / L, and 1000 mg / L using the calibrator and quality control product diluent; (2) Aliquot the prepared calibrator and quality control product (0.5 mL per vial) using vials, cover with soft rubber stoppers, do not completely seal, leave a certain air - permeable gap, and place in an - 80°C refrigerator for pre - freezing overnight. Turn on the freeze - dryer, pre - cool the refrigerator to - 45°C, place the pre - frozen calibration and quality control products in the freeze - drying rack, and freeze - dry for 12 - 16 h to obtain freeze - dried calibrator and quality control products; (3) Place the freeze - dried calibrator and quality control products in a 37°C incubator for hot compress for 1 day to obtain the calibrator and quality control product.

10. The detection method of free hemoglobin according to claim 8, characterized in that, The diluent for the calibrator and the quality control product includes ultrapure water, bovine serum albumin, and sucrose, and the mass ratio of ultrapure water, bovine serum albumin, and sucrose is (85 - 95):(2 - 5):(5 - 15).

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