Complement C1q latex immunoturbidimetric assay kit

Abstract

The present invention belongs to the field of medical examination, and in particular to a latex immunoturbidimetric assay kit for complement C1q. The kit of the present invention includes reagent 1 and reagent 2, wherein the reagent 1 is composed of tris(hydroxymethyl)aminomethane, sodium chloride, Triton X‑100, bovine serum albumin, a liquid biological preservative and zinc chloride; the reagent 2 is composed of tris(hydroxymethyl)aminomethane, sodium chloride, C1q antibody latex particles, bovine serum albumin and alanine. The present invention also provides a preparation method and a detection method of the above-mentioned kit, which has the advantages of simple detection method, wider linear range, higher precision and accuracy. The test results meet the clinical diagnosis criteria, and the method can be effectively applied to the diagnosis of diseases such as acute inflammation, early infectious diseases, tissue damage, and acute myocardial infarction.

Description

Technical Field

[0001] The invention belongs to the field of medical testing, and in particular relates to a latex immunoturbidimetric assay kit for complement C1q. Background Art

[0002] Complement C1q is a protein synthesized by the liver with multiple physiological functions. It is mainly involved in immune regulation in the body, can promote the proliferation and differentiation of immune cells, and can also promote the phagocytosis of phagocytes. Elevated complement C1q is often seen in acute inflammation, early infectious diseases, tissue damage, acute myocardial infarction, etc., while decreased complement C1q is often seen in congenital complement deficiency, systemic lupus erythematosus, acute glomerulonephritis, etc.

[0003] At present, the main detection methods of complement C1q are one-way immunodiffusion, rocket immunoelectrophoresis (RIE), enzyme-linked immunosorbent assay (ELISA) and immunoturbidimetry (TIA), but there is no recognized standard in the industry. Among them, one-way immunodiffusion is a traditional and outdated method. Due to the many factors that affect it, it has defects such as complex operation and low accuracy, and its clinical application is limited; the enzyme-linked immunosorbent assay has cumbersome operation steps, and the detection time is more than 90 minutes. The results are greatly affected by human operation factors, especially temperature and time factors. The results have a great influence on the results, the reproducibility is poor, and it is difficult to automate. RIE has important guiding significance for the detection of C1q in serum, but due to its poor reproducibility, long time consumption, and manual operation, there is currently no specialized commercial equipment, and this method is rarely used in clinical laboratories. ELISA requires manual operation and is time-consuming, so it has not been used on a large scale in clinical practice.

[0004] Immunoturbidimetry is currently the method most commonly used in clinical practice. Immunoturbidimetry is an antigen-antibody specific binding reaction. It quantifies the turbidity changes produced by the antigen-antibody immune complex formed by the detection reaction. It utilizes the specific binding between antigen and antibody and has the advantages of good stability, simple and convenient operation, rapid detection, good repeatability, high accuracy, and no pollution. It is used in automatic biochemical analyzers. Currently, immunoturbidimetry has been widely used in the detection of apolipoprotein B, prealbumin, fibronectin, etc., which has brought good news to patients.

[0005] However, there are various factors in immunoturbidimetry that affect the absorbance and product color changes of many biochemical reactions, which significantly interfere with the biochemical results detected by turbidimetry and colorimetry, thereby affecting the sensitivity, linear range and precision of the detection. Therefore, there is an urgent need in the art to provide a kit for detecting C1q content with a wide linear range, higher precision and higher accuracy. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a kit for detecting C1q content with a wide linear range, higher precision and higher accuracy.

[0007] In one aspect, the present invention provides a kit for measuring complement C1q, comprising reagent 1 and reagent 2;

[0008] Specifically, the reagent 1 is composed of tris(hydroxymethyl)aminomethane, sodium chloride, Triton X-100, bovine serum albumin, a biological preservative and zinc chloride;

[0009] The reagent 2 is composed of tris(hydroxymethyl)aminomethane, sodium chloride, C1q antibody latex particles, bovine serum albumin and alanine.

[0010] More specifically, the concentration of tris(hydroxymethyl)aminomethane in the reagent 1 may be 100-120 mM, the concentration of sodium chloride may be 100-140 mM, the concentration of Triton X-100 may be 0.5-2 mL / L, the concentration of bovine serum albumin may be 4-7 g / L, the concentration of the biological preservative may be 1-2 mL / L, and the concentration of zinc chloride may be 0.2 g / L-0.8 g / L.

[0011] Preferably, in the reagent 1, the concentration of tris(hydroxymethyl)aminomethane is 100 mM, the concentration of sodium chloride is 140 mM, the concentration of Triton X-100 is 2 mL / L, the concentration of bovine serum albumin is 4 g / L, the concentration of the biological preservative is 2 mL / L, and the concentration of zinc chloride is 0.8 g / L.

[0012] More specifically, the concentration of tris(hydroxymethyl)aminomethane in the reagent 2 may be 100-120 mM, the concentration of sodium chloride may be 100-140 mM, the concentration of C1q antibody latex particles may be 3-5 g / L, the concentration of bovine serum albumin may be 4-7 g / L, and the concentration of alanine may be 1-5 g / L.

[0013] Preferably, in the reagent 2, the concentration of tris(hydroxymethyl)aminomethane is 120 mM, the concentration of sodium chloride is 100 mM, the concentration of C1q antibody latex particles is 3 g / L, the concentration of bovine serum albumin is 7 g / L, and the concentration of alanine is 5 g / L.

[0014] Specifically, the C1q antibody latex particles are microsphere-coupled antibodies.

[0015] More specifically, the size of the microspheres may be 50-200 nm.

[0016] Preferably, the size of the microspheres is 100 nm.

[0017] More specifically, the preparation method of the C1q antibody latex particles is:

[0018] (1) Mixing microspheres with buffer;

[0019] (2) Activated microspheres;

[0020] (3) Add conjugated antibody to obtain C1q antibody latex particles.

[0021] Preferably, the buffer described in step (1) includes, but is not limited to, one or more of MES, MOPS, BES, CAPS, BES or BICINE buffer.

[0022] Further preferably, the buffer solution in step (1) is MES.

[0023] Preferably, a coupling agent is added during the activation in step (2).

[0024] Further preferably, the coupling agent in step (2) is selected from at least one of EDC, NHS, EDAC, WLS or PAM.

[0025] More preferably, the coupling agent in step (2) may be EDC and NHS;

[0026] The added mass ratio of EDC and NHS is 1:1.

[0027] Preferably, the C1q antibody latex particles are goat anti-human C1q antibody latex particles, rabbit anti-human C1q antibody latex particles or mouse anti-human C1q antibody latex particles.

[0028] More preferably, the C1q antibody latex particles are goat anti-human C1q antibody latex particles.

[0029] Specifically, the kit also includes calibrators and quality control products.

[0030] More specifically, the calibrators and quality control products include complement C1q, bovine serum albumin and biological preservatives.

[0031] More specifically, the biological preservative can be a liquid preservative; preferably proclin300.

[0032] Preferably, the complement C1q concentration of the calibrator can be 10-500 mg / L;

[0033] The complement C1q concentration of the quality control product can be 50-300 mg / L;

[0034] The concentration of bovine serum albumin can be 4-7 g / L;

[0035] The concentration of proclin300 can be 1-2mL / L.

[0036] Further preferably, the C1q concentration is 100-300 mg / L;

[0037] The concentration of bovine serum albumin can be 5 g / L;

[0038] The concentration of proclin300 can be 2mL / L.

[0039] Specifically, the aforementioned kit determination steps include:

[0040] S1, add reagent 1 and sample or calibrator and incubate together;

[0041] S2, add reagent 2, and read the first reading after 25-35 seconds;

[0042] S3. Continue the reaction for 4-5 minutes and then take the second reading.

[0043] Specifically, the sample described in step S1 includes, but is not limited to: peripheral blood, whole blood or peripheral blood.

[0044] Specifically, the first reading time in step S2 is 30 seconds.

[0045] Specifically, the second reading time in step S3 is 4.5 minutes.

[0046] The technical effects achieved by the present invention are:

[0047] (1) Good linearity and wider linear range, R of the fitting equation 2 The linear range is 10-500 mg / L.

[0048] (2) It has higher precision, with the CV value of repeatability being 1.07 or less and the CV value of inter-batch precision being 2.69 or less.

[0049] (3) High accuracy, the results of human serum complement C1q measurement meet the clinical diagnostic standards.

[0050] (4) The detection method is simple and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is the standard curve for detecting complement C1q content. DETAILED DESCRIPTION

[0052] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0053] Example 1

[0054] A latex immunoturbidimetric assay kit for complement C1q comprises reagent 1, reagent 2, a calibrator and a quality control product.

[0055] Instrument: Hitachi 7600 fully automatic biochemical analyzer.

[0056] The reagent information used in the present invention is shown in Table 1:

[0057] Table 1

[0058]

[0059]

[0060] 1.1 Reagent 1

[0061] Prepare according to the formula in Table 2, stir thoroughly and store at 2-8°C. The remaining solvent is purified water.

[0062] Table 2

[0063] Element concentration Example 1 Use concentration Tris(hydroxymethyl)aminomethane 100-120mM 100mM Sodium chloride 100-140mM 140mM Triton X-100 0.5-2mL / L 2mL / L BSA 4-7g / L 4g / L Liquid biological preservative (proclin300) 1-2mL / L 2mL / L Zinc chloride 0.2g / L-0.8g / L 0.8g / L

[0064] 1.2 Reagent 2

[0065] Prepare according to the formula in Table 3, stir thoroughly and store at 2-8°C. The remaining solvent is purified water.

[0066] Table 3

[0067] Element concentration Example 1 Use concentration Tris(hydroxymethyl)aminomethane 100-120mM 120mM Sodium chloride 100-140mM 100mM Sheep anti-human C1q antibody latex particles 3-5g / L 3g / L BSA 4-7g / L 7g / L Alanine 1-5g / L 5g / L

[0068] Among them, the preparation process of sheep anti-human C1q antibody latex particles is as follows:

[0069] (1) Take 0.1 mL of microspheres and add 0.9 mL of MES (0.1 mol / L, pH 6.0) and mix;

[0070] (2) Add 5 mg of EDC and NHS to the above mixture for activation, stir gently at room temperature for 15 min, centrifuge at 10,000 r / min for 15 min, wash repeatedly with 0.1 mol / L PBS for 3 times, discard the supernatant, resuspend the precipitate in 0.1 mol / L PBS, shake, and ultrasonically treat to obtain an activated microsphere solution.

[0071] (3) Take 40 μL of goat anti-human C1q antibody (10 mg / mL) and add it to 1 mL of activated microsphere solution. Gently stir at room temperature for 4 h, centrifuge at 10,000 r / min for 15 min, wash the precipitate three times with PBS containing 0.05% Tween20, and then re-dissolve it with 0.1 mol / L PBS to obtain goat anti-human C1q antibody latex particles.

[0072] 1.3 Calibrators

[0073] The composition of the calibrator is shown in Table 4 below:

[0074] Table 4

[0075] Element concentration Complement C1q 10-500mg / L BSA 5g / L Liquid biological preservative (proclin300) 2mL / L

[0076] 1.4 Quality Control Products

[0077] The composition of the quality control product is shown in Table 5 below:

[0078] Table 5

[0079] Element concentration Complement C1q 50-300mg / L BSA 5g / L Liquid biological preservative (proclin300) 2mL / L

[0080] The above reagent 1, reagent 2, calibrator and quality control product are packaged separately to obtain a latex immunoturbidimetric assay kit for complement C1q.

[0081] 1.5 Determination method

[0082] 1.5.1 Specific steps for detecting complement C1q content using this kit

[0083] Reaction and detection conditions: temperature 37°C; main wavelength 570nm, secondary wavelength 800nm; sample volume 2μL, reagent 1 volume 200μL, reagent 2 volume 50μL; reaction direction: ascending reaction.

[0084] The sample is serum or plasma, and heparin is recommended as the anticoagulant.

[0085] The detection steps are:

[0086] (1) 2 μL of sample or calibrator and 200 μL of reagent 1 were incubated for 5 min;

[0087] (2) Add 50 μL of reagent 2 and read A1 for the first time after 30 seconds;

[0088] (3) Continue the reaction for 4.5 minutes and then read A2 for the second time.

[0089] The blank sample was a calibrator without the addition of complement C1q.

[0090] A 测定=A2-A1, A 校准 =A2-A1, A 空白 =A2-A1.

[0091] 1.5.2 Calculation

[0092] The absorbance change of the calibration material is △A(A 校准 -A 空白 ) is the ordinate, and its corresponding concentration C 校准 Draw a calibration curve as the horizontal axis. According to the △A (A 测定 -A 空白 ) Find the corresponding sample concentration on the calibration curve.

[0093] 1.5.3 Inspection result determination

[0094] (1) The C1q content of the sample is 153mg / L-233mg / L, which is within the normal range and is considered a negative result;

[0095] (2) The C1q content of the sample is greater than 233 mg / L, which is within the abnormal range and is judged as a positive result.

[0096] Example 2 Linear range determination

[0097] Using the kit and the determination method of Example 1, six different concentrations of C1q were set: 10 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L, with three replicates for each concentration.

[0098] The test results are shown in Table 6:

[0099] Table 6

[0100]

[0101] The standard curve was established with 6 different concentrations of C1q as the horizontal axis and the average concentration as the vertical axis. Figure 1 As shown, the fitting equation of the curve is y = 1.0148x-1.0611, R 2 =0.9999. The results show that the kit and the detection method of the present invention have very good linearity and can accurately quantify the concentration of C1q in the sample.

[0102] Example 3 Precision Determination

[0103] 3.1 Repeatability

[0104] The kit and the determination method of Example 1 were used to measure three low, medium and high concentrations of C1q (100 mg / L, 300 mg / L, 500 mg / L), and each concentration was measured 10 times. The test results are shown in Table 7:

[0105] Table 7

[0106]

[0107] 3.2 Inter-batch precision

[0108] The kit and determination method of Example 1 were used to measure three low, medium and high concentrations of C1q (100 mg / L, 300 mg / L, 500 mg / L). The kits were from three different batches. Each sample was measured 5 times using the same kit. The mean values, SD and CV% of different samples were calculated. The results are shown in Table 8.

[0109] Table 8

[0110]

[0111]

[0112] The above results show that the SD values ​​of different batches of the kits are below 10, and the CV% values ​​are below 3, indicating that the kit of the present invention has good precision and can be used for accurate detection of C1q.

[0113] Example 4 Method Verification

[0114] 4.1 Testing of healthy people

[0115] The C1q content of 30 healthy subjects (confirmed not to have lupus erythematosus, acute myocardial infarction, rheumatoid arthritis, renal fibrosis, osteomyelitis, etc.) was detected using the kit and the determination method of Example 1.

[0116] Test results: C1q content was between 153mg / L-233mg / L, and was judged to be negative.

[0117] 4.2 Testing of patients with rheumatoid arthritis

[0118] The C1q content of 20 patients diagnosed with rheumatoid arthritis was detected using the kit and the determination method of Example 1.

[0119] The diagnostic criteria for rheumatoid arthritis refer to the "Guidelines for Clinical Research of New Chinese Medicines":

[0120] (1) Clinical manifestations: Most patients have a history of streptococcal infection. In the acute phase, patients may experience multiple and migratory joint pain, which often occurs in large joints and may be accompanied by redness, swelling, pain, nodules, or erythema. In the chronic phase, patients may only feel joint pain.

[0121] (2) Laboratory tests: Anti-O is above 1:500; or the erythrocyte sedimentation rate is increased; or the anti-streptokinase is more than 80 units; or the hyaluronidase is above 128 units; or the C-reactive protein is positive.

[0122] Test results: C1q content was >233 mg / L, which was judged as positive.

[0123] 4.3 Testing of patients with osteomyelitis

[0124] The C1q content of 10 patients diagnosed with osteomyelitis was detected using the kit and the determination method of Example 1.

[0125] Diagnostic criteria for osteomyelitis:

[0126] 1. Diagnostic principles of osteomyelitis:

[0127] The diagnosis is mainly based on the patient's medical history, clinical manifestations, combined with routine blood tests, bacterial culture and drug sensitivity tests, blood cultures, as well as imaging examinations such as X-ray examinations, CT examinations, and radioactive bone scans. If necessary, a pathological examination of the patient's lesion location can be performed during surgery. This is the gold standard for diagnosing osteomyelitis.

[0128] 2. Related inspections:

[0129] (1) Laboratory examination:

[0130] 1) Routine blood examination: Erythrocyte sedimentation rate and C-reactive protein are elevated, white blood cell count is often normal, and in some patients the white blood cell count is elevated.

[0131] 2) Bacterial culture and drug sensitivity test: Purulent bacteria can be seen in the puncture fluid of acute suppurative osteomyelitis.

[0132] 3) Blood culture: Pathogenic bacteria can be detected. The positive rate of blood culture is higher when the patient has chills and high fever.

[0133] (2) Imaging examination:

[0134] 1) X-ray examination: 3-4 weeks after infection, irregular bone thickening and hardening may occur, with residual bone resorption areas or cavities, which may contain dead bones of varying sizes, and sometimes the bone marrow cavity cannot be seen.

[0135] 2) CT examination: can determine the formation of diseased bones.

[0136] 3) Radioactive bone scan: Radioactive bone scan can show the location of the lesion in the early stage of the disease.

[0137] Test results: C1q content was >233 mg / L, which was judged as positive.

[0138] 4.4 Detection of patients with renal fibrosis

[0139] The C1q content of 7 patients diagnosed with renal fibrosis was detected using the kit and the assay method of Example 1.

[0140] Diagnostic criteria for renal fibrosis:

[0141] (1) Clinical symptoms and signs: Patients experience symptoms such as low back pain, frequent urination, oliguria, hematuria, and hypertension. In terms of physical signs, percussion pain in the renal area, cystic masses in the renal area, and enlarged kidneys may occur.

[0142] (2) Imaging examination: B-ultrasound can show the size, shape, echo of the kidneys, and whether there are abnormal changes such as masses or cysts; CT and MRI can observe the structure and anatomy of the kidneys in more detail, including whether there are fibrotic changes in the renal tubules, nephrons, and renal interstitium.

[0143] (3) Laboratory examination: Commonly used indicators include routine urinalysis, urine microalbumin, serum creatinine, creatinine clearance, etc. Routine urinalysis can detect whether there are abnormalities such as protein, red blood cells, and white blood cells in the urine; urine microalbumin can help evaluate the glomerular filtration function; serum creatinine and creatinine clearance can reflect the status of the glomerular filtration function.

[0144] (4) Renal biopsy: By obtaining a kidney tissue specimen for pathological examination, the extent and type of renal interstitial fibrosis can be accurately determined.

[0145] Test results: C1q content was >233 mg / L, which was judged as positive.

[0146] 4.5 Testing of patients with acute myocardial infarction

[0147] The C1q content of 20 patients diagnosed with acute myocardial infarction was detected using the kit and the determination method of Example 1.

[0148] Diagnostic criteria for acute myocardial infarction:

[0149] (1) The patient experiences persistent and severe chest pain and tightness, accompanied by palpitations, shortness of breath, sweating, nausea and vomiting, and irritability;

[0150] (2) The electrocardiogram shows obvious ST segment elevation or depression with dynamic evolution;

[0151] (3) Myocardial enzymes such as creatine kinase, creatine kinase isoenzymes, and troponin I were significantly elevated and showed dynamic changes.

[0152] Patients who meet two of the above three criteria can be diagnosed with acute myocardial infarction.

[0153] Test results: C1q content was >233 mg / L, which was judged as positive.

[0154] In summary, the kit of the present invention can effectively detect the C1q content in serum, and the detection result meets the clinical diagnosis standard.

[0155] Comparative Example

[0156] The repeatability of the 300 mg / L C1q standard was determined using the kits of Comparative Examples 1-4 with reference to 3.1 in Example 3, and the determination was repeated 10 times, and the mean values, SD and CV% of different samples were calculated respectively. The differences between the kits of Comparative Examples 1-3 and Example 1 are shown in Table 9 below:

[0157] Table 9

[0158] Comparative Example Differences from Example 1 Comparative Example 1 No zinc chloride added Comparative Example 2 No added alanine Comparative Example 3 Replace the alanine in reagent 2 with an equal amount of glycine Comparative Example 4 No added zinc chloride and alanine

[0159] The test results are shown in Table 10:

[0160] Table 10

[0161] Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Mean 319.46 325.19 330.44 328.58 SD 17.58 20.36 25.67 23.11 CV% 5.50 6.26 7.77 7.03

[0162] The results show that the detection method of the embodiment of the present invention is significantly better than that of Comparative Examples 1-4, and it can be seen from Comparative Examples 1-2 and Comparative Example 4 that the zinc chloride in reagent 1 and the alanine in reagent 2 have a synergistic effect and jointly improve the detection performance of C1q.

Claims

1. A kit for measuring complement C1q, characterized in that: Includes reagent 1 and reagent 2; The reagent 1 is composed of tris(hydroxymethyl)aminomethane, sodium chloride, Triton X-100, bovine serum albumin, biological preservative and zinc chloride; The reagent 2 is composed of tris(hydroxymethyl)aminomethane, sodium chloride, C1q antibody latex particles, bovine serum albumin and alanine; In the reagent 1, the concentration of tris(hydroxymethyl)aminomethane is 100-120 mM, the concentration of sodium chloride is 100-140 mM, the concentration of Triton X-100 is 0.5-2 mL / L, the concentration of bovine serum albumin is 4-7 g / L, the concentration of the biological preservative is 1-2 mL / L, and the concentration of zinc chloride is 0.2 g / L-0.8 g / L; In the reagent 2, the concentration of tris(hydroxymethyl)aminomethane is 100-120 mM, the concentration of sodium chloride is 100-140 mM, the concentration of C1q antibody latex particles is 3-5 g / L, the concentration of bovine serum albumin is 4-7 g / L, and the concentration of alanine is 1-5 g / L.

2. The kit according to claim 1, characterized in that In the reagent 1, the concentration of tris(hydroxymethyl)aminomethane is 100 mM, the concentration of sodium chloride is 140 mM, the concentration of Triton X-100 is 2 mL / L, the concentration of bovine serum albumin is 4 g / L, the concentration of the biological preservative is 2 mL / L, and the concentration of zinc chloride is 0.8 g / L.

3. The kit according to claim 2, characterized in that In the reagent 2, the concentration of tris(hydroxymethyl)aminomethane is 120 mM, the concentration of sodium chloride is 100 mM, the concentration of C1q antibody latex particles is 3 g / L, the concentration of bovine serum albumin is 7 g / L, and the concentration of alanine is 5 g / L.

4. The kit according to claim 3, characterized in that The C1q antibody latex particles are goat anti-human C1q antibody latex particles, rabbit anti-human C1q antibody latex particles or mouse anti-human C1q antibody latex particles.

5. The kit according to claim 1, characterized in that The kit also includes calibration products and quality control products.

6. The kit according to claim 5, characterized in that The calibration products and quality control products include complement C1q, bovine serum albumin and biological preservatives.

7. The kit according to any one of claims 1 to 6, characterized in that The measurement steps include: S1, add reagent 1 and sample or calibrator and incubate together; S2, add reagent 2, and read the first reading after 25-35 seconds; S3. Continue the reaction for 4-5 minutes and then take the second reading.

8. The kit according to claim 7, characterized in that The sample in step S1 includes peripheral blood, whole blood or peripheral blood.

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