A method, device and kit for detecting H factor concentration

Abstract

The present invention belongs to the field of bioengineering, and provides a method, device and kit for detecting the concentration of factor H, which is used to detect the concentration level of factor H in human plasma / serum / urine. It includes preparing a kit for detecting the concentration level of factor H; adding the reagent I to the sample to be tested and the standard respectively, and detecting the absorbance values ​​of the sample to be tested and the standard respectively; continuing to add the reagent II to the sample to be tested and the standard, and detecting the absorbance values ​​of the sample to be tested and the standard; using the absorbance values ​​of the sample to be tested and the standard, the concentration level of factor H in the sample to be tested is obtained. The detection range of the method for detecting the concentration level of factor H provided by the present invention is wide, and the concentration of factor H within the concentration range of 4.66-800 mg / l can be detected, the detection efficiency is high, the detection operation steps are simple, the detection cost is low, the universality is high, and it can meet the needs of large-scale clinical detection.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and in particular relates to a method, equipment and kit for detecting the level of factor H. Background Art

[0002] H factor (Complement factor H) was first discovered and reported by Nilson et al. in 1965. According to the electrophoretic position, it was named β1f-globulin (Nilsson, UR and HJ Mueller-Eberhard, Isolation of Beta If-Globulin from Human Serum and Its Characterization as the Fifth Component of Complement. J Exp Med, 1965. 122: p. 277-98.), which is the most characteristic member of the H factor protein family. The H factor family protein gene is located in the 1q32 human complement activation regulatory gene cluster region. It is a group of glycoproteins in plasma, all of which are composed of independently foldable short consensus repeat (SCR) domains ( Figure 1 and Figure 2 ). Factor H is a glycoprotein composed of 1213 amino acid residues, with a molecular weight of about 150kDa. It consists of 20 SCRs domains. Each SCR consists of about 60 amino acids, forming a globular structure. It contains four conserved cysteine ​​residues (Ⅰ, Ⅱ, Ⅲ, Ⅳ), and forms disulfide bonds between Ⅰ~Ⅲ and Ⅱ~Ⅳ, which is conducive to maintaining its spatial conformation. The liver is its main synthetic organ. In addition, the mesangial cells, podocytes and tubular epithelial cells of the kidney, retinal pigment epithelial cells, platelets, peripheral mononuclear cells, glial cells, fibroblasts, endothelial cells, etc. of the kidney also express it. The content of factor H in normal human blood circulation is abundant, with a concentration of about 300-800mg / l. Its level is affected by genetic and environmental factors.

[0003] Different SCR regions of factor H can bind to heparin, mucopolysaccharides on the cell surface, protein components on the surface of microorganisms, C-reactive protein (CRP), and complement molecules such as C3b and C3d, respectively, to exert different physiological functions ( Figure 3). The N-terminus and C-terminus of factor H have different biological functions: the SCR1-4 region at its N-terminus mainly participates in the degradation of the alternative pathway C3 convertase (C3bBb) in the liquid phase through the auxiliary factor I, and can also competitively bind to C3b to inhibit the formation of C3 convertase (C3bBb); its C-terminus mainly binds to the mucopolysaccharide on the surface of the host cell, which helps the SCR1-4 at the N-terminus to inhibit the excessive activation of the complement alternative pathway on the surface of the host cell and protect the host cell from damage. The latest research has found that factor H can also bind to apoptotic cell fragments, DNA, histones, nucleosomes and annexin-II to regulate the elimination of apoptotic cells.

[0004] In addition, factor H can also bind to complement binding proteins on the surface of a variety of bacteria, viruses, fungi and parasites to mediate bacterial "immune escape"; factor H can also bind to cell surface receptors such as CR3, integrins and L-selectins expressed by neutrophils, B lymphocytes and monocytes, mediating cell adhesion and cytokine production to exert an immunomodulatory effect.

[0005] Recent studies have found that the occurrence and development of many cardiovascular diseases, tumor diseases, infectious diseases, blood diseases, kidney diseases, rheumatic diseases, ophthalmic diseases and obstetric diseases are related to abnormal H factor regulation, that is, the patients may have genetic abnormalities in the "complement activation regulatory protein region" of chromosome 1q32. If the level of complement H factor in the patient's blood can be accurately detected, it can help with disease diagnosis, indicate the severity of the disease, guide clinical treatment to a certain extent, and improve patient prognosis.

[0006] In the prior art, there are two main methods for detecting the level of complement factor H in human circulation, one is enzyme-linked immunosorbent assay (ELISA), and the other is radioimmunodiffusion. The ELISA method has a long detection time and complicated operation steps; although the radioimmunodiffusion method has a short detection time, it requires radioactive protection. Moreover, the sample amount that can be detected at a single time in both methods is small, so both detection methods cannot achieve rapid and accurate detection of the level of complement factor H in batches, and cannot meet clinical needs. Summary of the invention

[0007] In order to solve the technical problems of long detection time, complicated operation steps, small sample size for single detection in the prior art, the present invention adopts the principle of enhanced latex immunoturbidimetry to provide a method that can quickly detect the level of factor H in plasma / serum / urine, can detect a large number of samples at the same time, and is simple to operate. The method has a wide detection range, relatively broad requirements for sample storage conditions, and simple requirements for sample anticoagulation methods, and has good clinical application value.

[0008] To achieve the purpose of the present invention, the present invention provides a method for detecting the concentration level of factor H, comprising:

[0009] preparing a kit comprising reagent I and reagent II for detecting the concentration level of factor H in a sample to be tested;

[0010] Adding the reagent I to the sample to be tested, obtaining a first test sample after incubation, and obtaining a first absorbance value A1 of the sample to be tested after testing; adding the reagent II to the first test sample, obtaining a second test sample after incubation, and obtaining a second absorbance value A2 of the sample to be tested;

[0011] Add the reagent I to the standard, incubate to obtain a first detection standard, and detect to obtain a first absorbance value B1 of the standard; add the reagent II to the first detection standard, incubate to obtain a second detection standard, and detect to obtain a second absorbance value B2 of the standard;

[0012] The concentration level of factor H in the sample to be tested is obtained by using the first absorbance value A1 and the second absorbance value A2 of the sample to be tested and the first absorbance value B1 and the second absorbance value B2 of the standard.

[0013] Wherein, obtaining the concentration level of factor H in the sample to be tested by using the first absorbance value A1 and the second absorbance value A2 of the sample to be tested and the first absorbance value B1 and the second absorbance value B2 of the standard substance comprises:

[0014] The difference between the first absorbance value A2 of the sample to be tested and the second absorbance value A1 of the sample to be tested is divided by the difference between the second absorbance value B2 of the standard and the first absorbance value B1 to obtain a ratio K;

[0015] Multiply the known concentration of the standard factor H by the obtained ratio K to obtain the concentration value C of the factor H in the sample to be tested.

[0016] Wherein, the reagent I comprises Tris buffer and PEG200 with a mass percentage concentration of 50-90 g / L; the reagent II comprises PEG200 with a mass percentage concentration of 50-90 g / L and a latex particle suspension of factor H antibody.

[0017] Preferably, the reagent I comprises Tris buffer and PEG200 with a mass percentage concentration of 70 g / L; the reagent II comprises PEG200 with a mass percentage concentration of 20 g / L and a suspension of factor H antibody latex particles.

[0018] The detection conditions set during the detection are a temperature of 37°C, a main wavelength of 546nm or 600nm, and a sub-wavelength of 700nm.

[0019] In particular, in one measurement, the amount of the sample to be tested is 3 μl, the amount of the standard is 3 μl, the amount of reagent I is 240 μl, and the amount of reagent II is 60 μl.

[0020] Wherein, the sample to be tested is human plasma, human serum or human urine.

[0021] Wherein, the storage temperature of the sample to be tested is -80 to 38°C.

[0022] Wherein, the storage time of the sample to be tested is 0-28 days.

[0023] Among them, the concentration of H factor in the samples to be tested is 4.68-2000mg / L.

[0024] Among them, the amount of samples to be tested is 500-1000.

[0025] The incubation temperature of the incubation treatment is 37° C. and the incubation time is 5 min.

[0026] The serum anti-H factor antibody is derived from one of sheep anti-human H factor, rabbit anti-human H factor, mouse anti-human H factor, horse anti-human H factor or donkey anti-human H factor, and has a titer of 1:64.

[0027] Furthermore, the sheep anti-human factor H, rabbit anti-human factor H, mouse anti-human factor H, horse anti-human factor H or donkey anti-human or horse anti-human factor H is a monoclonal antibody or a polyclonal antibody.

[0028] To achieve the purpose of the present invention, the present invention provides, on the other hand, a device for detecting the concentration level of factor H, comprising:

[0029] preparing a kit comprising reagent I and reagent II for detecting the concentration level of factor H in a sample to be tested;

[0030] Absorbance value detection device, used for:

[0031] After adding the reagent I to the sample to be tested, a first test sample is obtained, and absorbance value detection is performed to obtain a first absorbance value A1 of the sample to be tested;

[0032] Performing absorbance detection on a second test sample obtained by adding the reagent II to the first test sample to obtain a second absorbance value A2 of the test sample;

[0033] After adding the reagent I to the standard product, the first detection standard product is obtained, and the absorbance value is detected to obtain the first absorbance value B1 of the standard product;

[0034] Performing absorbance detection on a second detection standard obtained by adding the reagent II to the first detection standard to obtain a second absorbance value B2 of the standard;

[0035] The H factor concentration level calculation device is used to calculate the H factor concentration level in the sample to be tested by using the first absorbance value A1 and the second absorbance value A2 of the sample to be tested and the first absorbance value B1 and the second absorbance value B2 of the standard.

[0036] Wherein, the reagent I comprises Tris buffer and PEG200 with a mass percentage concentration of 50-90 g / L; the reagent II comprises PEG200 with a mass percentage concentration of 10-30 g / L and a suspension of factor H antibody latex particles.

[0037] Preferably, the reagent I comprises Tris buffer and PEG200 with a mass percentage concentration of 70 g / L; the reagent II comprises Tris buffer, PEG200 with a mass percentage concentration of 2 g / L and a suspension of factor H antibody latex particles.

[0038] In particular, the serum anti-factor H antibody is derived from one of sheep anti-human factor H, rabbit anti-human factor H, mouse anti-human factor H, horse anti-human factor H or donkey anti-human factor H, and has a titer of 1:64.

[0039] Furthermore, the sheep anti-human factor H, rabbit anti-human factor H, mouse anti-human factor H, horse anti-human factor H or donkey anti-human factor H is a monoclonal antibody or a polyclonal antibody.

[0040] Wherein, the absorbance value detection device is a biochemical analysis instrument or any other device that can realize absorbance value detection.

[0041] The H factor concentration level calculation device is a computer or any other device that can perform calculation functions.

[0042] The detection parameters of the absorbance detection device are set as temperature 37°C, main wavelength 546nm or 600nm, and sub-wavelength 700nm.

[0043] To achieve the purpose of the present invention, the present invention provides a kit for detecting factor H in another aspect, wherein the kit can be used in a method for detecting the concentration level of factor H or in an apparatus for detecting the concentration level of factor H, comprising:

[0044] Reagent I comprising Tris buffer and PEG200 solution;

[0045] Reagent II comprising PEG200 solution and factor H antibody latex particle suspension.

[0046] Wherein, the mass percentage concentration of the PEG200 solution in the reagent I is 50-90 g / L, preferably 70 g / L.

[0047] Wherein, the mass percentage concentration of the PEG200 solution in the reagent II is 10-30 g / L, preferably 20 g / L.

[0048] Particularly, the volume concentration of the factor H antibody latex particle suspension is 400-800 ml / L, preferably 600 ml / L.

[0049] In particular, the serum anti-factor H antibody is derived from one of sheep anti-human factor H, rabbit anti-human factor H, mouse anti-human factor H, horse anti-human factor H or donkey anti-human factor H, and has a titer of 1:64.

[0050] Furthermore, the sheep anti-human factor H, rabbit anti-human factor H, mouse anti-human factor H, horse anti-human factor H or donkey anti-human factor H is a monoclonal antibody or a polyclonal antibody.

[0051] The kit and method of the present invention adopt the determination principle of latex enhanced immunoturbidimetry, utilizing the combination of H factor antigen in serum and H factor antibody cross-linked on latex particles to form an insoluble immune complex, making the reaction solution turbid. The turbidity, that is, the decrease in transmittance and the increase in absorbance, reflects the concentration of H factor in human serum samples. The concentration of H factor can be calculated from a dose-response curve made by a calibrator.

[0052] Advantages of the present invention:

[0053] 1. The method provided by the present invention has a wide detection range and can detect factor H levels within the concentration range of 4.86-2000 mg / l.

[0054] 2. The method of the present invention has high detection efficiency and can detect the concentration level of factor H in a sample within 5-10 minutes.

[0055] 3. The method of the present invention has low requirements for the storage conditions of blood. Blood samples stored at a temperature of -80 to 37°C, blood samples stored for up to four weeks, and blood samples that have been repeatedly frozen and thawed less than four times can all be tested, and the test results will not be affected.

[0056] 4. The method of the present invention has no anticoagulation requirement for blood, and samples anticoagulated with EDTA or heparin can be tested, and the test results are accurate and unaffected.

[0057] 5. The method provided by the present invention is simple to operate. The level of factor H in human plasma / serum / urine can be determined by only measuring and calculating the absorbance before and after the sample to be tested and the standard. Therefore, the detection cost is low and the universality is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 It is a schematic diagram of the gene structure of the H factor family described in the background technology of the present invention;

[0059] Figure 2 It is a schematic diagram of the SCR structure of the factor H family described in the background technology of the present invention;

[0060] Figure 3 It is a schematic diagram of the binding of factor H to various molecules described in the background technology of the present invention;

[0061] Figure 4 It is a dose response curve of factor H concentration and absorbance made by the standard substance described in the examples of the present invention. Specific implementation methods

[0062] 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. It should be noted that 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.

[0063] Example 1 Kit

[0064] The reagents in the kit are prepared into reagent I and reagent II according to the following ratios:

[0065] Reagent I: Tris buffer

[0066] PEG200 70g / L.

[0067] Reagent II: Tris buffer

[0068] PEG200 20g / L

[0069] Factor H antibody latex particle suspension 600ml / L.

[0070] The factor H antiserum provided by the present invention is one of goat anti-human factor H, rabbit anti-human factor H, mouse anti-human factor H, horse anti-human factor H or donkey anti-human factor H, and the goat anti-human factor H, rabbit anti-human factor H, mouse anti-human factor H, horse anti-human factor H or donkey anti-human factor H is a monoclonal antibody or a polyclonal antibody.

[0071] Example 2 Kit

[0072] The reagents in the kit are prepared into reagent I and reagent II according to the following ratios:

[0073] Reagent I: Tris buffer

[0074] PEG200 50g / L.

[0075] Reagent II: Tris buffer

[0076] PEG200 10g / L

[0077] Factor H antibody latex particle suspension 400ml / L.

[0078] The factor H antiserum provided by the present invention is one of goat anti-human factor H, rabbit anti-human factor H, mouse anti-human factor H, horse anti-human factor H or donkey anti-human factor H, and the goat anti-human factor H, rabbit anti-human factor H, mouse anti-human factor H, horse anti-human factor H or donkey anti-human factor H is a monoclonal antibody or a polyclonal antibody.

[0079] Example 3 Kit

[0080] The reagents in the kit are prepared into reagent I and reagent II according to the following ratios:

[0081] Reagent I: Tris buffer

[0082] PEG200 90g / L.

[0083] Reagent II: Tris buffer

[0084] PEG200 30g / L

[0085] Factor H antibody latex particle suspension 800ml / L.

[0086] The factor H antiserum provided by the present invention is one of goat anti-human factor H, rabbit anti-human factor H, mouse anti-human factor H, horse anti-human factor H or donkey anti-human factor H, and the goat anti-human factor H, rabbit anti-human factor H, mouse anti-human factor H, horse anti-human factor H or donkey anti-human factor H is a monoclonal antibody or a polyclonal antibody.

[0087] Alternatively, the ratio of the above components may be:

[0088] Alternatively, the ratio of the above components may be:

[0089] Reagent I: Tris buffer PEG200 50g / L

[0090] Reagent II: Tris buffer PEG200 10g / L

[0091] H factor antibody latex particle suspension 400ml / L;

[0092] Reagent I: Tris buffer PEG200 50g / L

[0093] Reagent II: Tris buffer PEG200 10g / L

[0094] H factor antibody latex particle suspension 600ml / L;

[0095] Reagent I: Tris buffer PEG200 50g / L

[0096] Reagent II: Tris buffer PEG200 10g / L

[0097] H factor antibody latex particle suspension 800ml / L;

[0098] Reagent I: Tris buffer PEG200 50g / L

[0099] Reagent II: Tris buffer PEG200 20g / L

[0100] H factor antibody latex particle suspension 400ml / L;

[0101] Reagent I: Tris buffer PEG200 50g / L

[0102] Reagent II: Tris buffer PEG200 20g / L

[0103] H factor antibody latex particle suspension 600ml / L;

[0104] Reagent I: Tris buffer PEG200 50g / L

[0105] Reagent II: Tris buffer PEG200 20g / L

[0106] H factor antibody latex particle suspension 800ml / L;

[0107] Reagent I: Tris buffer PEG200 50g / L

[0108] Reagent II: Tris buffer PEG200 30g / L

[0109] H factor antibody latex particle suspension 400ml / L;

[0110] Reagent I: Tris buffer PEG200 50g / L

[0111] Reagent II: Tris buffer PEG200 30g / L

[0112] H factor antibody latex particle suspension 600ml / L;

[0113] Reagent I: Tris buffer PEG200 50g / L

[0114] Reagent II: Tris buffer PEG200 30g / L

[0115] H factor antibody latex particle suspension 800ml / L;

[0116] Reagent I: Tris buffer PEG200 70g / L

[0117] Reagent II: Tris buffer PEG200 10g / L

[0118] H factor antibody latex particle suspension 400ml / L;

[0119] Reagent I: Tris buffer PEG200 70g / L

[0120] Reagent II: Tris buffer PEG200 10g / L

[0121] H factor antibody latex particle suspension 600ml / L;

[0122] Reagent I: Tris buffer PEG200 70g / L

[0123] Reagent II: Tris buffer PEG200 10g / L

[0124] H factor antibody latex particle suspension 800ml / L;

[0125] Reagent I: Tris buffer PEG200 70g / L

[0126] Reagent II: Tris buffer PEG200 20g / L

[0127] H factor antibody latex particle suspension 400ml / L;

[0128] Reagent I: Tris buffer PEG200 70g / L

[0129] Reagent II: Tris buffer PEG200 20g / L

[0130] H factor antibody latex particle suspension 800ml / L;

[0131] Reagent I: Tris buffer PEG200 70g / L

[0132] Reagent II: Tris buffer PEG200 30g / L

[0133] H factor antibody latex particle suspension 400ml / L;

[0134] Reagent I: Tris buffer PEG200 70g / L

[0135] Reagent II: Tris buffer PEG200 30g / L

[0136] H factor antibody latex particle suspension 400ml / L;

[0137] Reagent I: Tris buffer PEG200 70g / L

[0138] Reagent II: Tris buffer PEG200 30g / L

[0139] H factor antibody latex particle suspension 600ml / L;

[0140] Reagent I: Tris buffer PEG200 70g / L

[0141] Reagent II: Tris buffer PEG200 30g / L

[0142] H factor antibody latex particle suspension 800ml / L;

[0143] Reagent I: Tris buffer PEG200 90g / L

[0144] Reagent II: Tris buffer PEG200 10g / L

[0145] H factor antibody latex particle suspension 400ml / L;

[0146] Reagent I: Tris buffer PEG200 90g / L

[0147] Reagent II: Tris buffer PEG200 10g / L

[0148] H factor antibody latex particle suspension 600ml / L;

[0149] Reagent I: Tris buffer PEG200 90g / L

[0150] Reagent II: Tris buffer PEG200 10g / L

[0151] H factor antibody latex particle suspension 800ml / L;

[0152] Reagent I: Tris buffer PEG200 90g / L

[0153] Reagent II: Tris buffer PEG200 20g / L

[0154] H factor antibody latex particle suspension 400ml / L;

[0155] Reagent I: Tris buffer PEG200 90g / L

[0156] Reagent II: Tris buffer PEG200 20g / L

[0157] Factor H antibody latex particle suspension 600ml / L

[0158] Reagent I: Tris buffer PEG200 90g / L

[0159] Reagent II: Tris buffer PEG200 20g / L

[0160] H factor antibody latex particle suspension 800ml / L;

[0161] Reagent I: Tris buffer PEG200 90g / L

[0162] Reagent II: Tris buffer PEG200 30g / L

[0163] H factor antibody latex particle suspension 400ml / L;

[0164] Reagent I: Tris buffer PEG200 90g / L

[0165] Reagent II: Tris buffer PEG200 30g / L

[0166] H factor antibody latex particle suspension 600ml / L;

[0167] Any one of the above, the performance of the prepared kit is close to the performance of the kit prepared in the preferred embodiment.

[0168] Furthermore, the factor H antiserum used in the above-mentioned kit is one of sheep anti-human factor H, rabbit anti-human factor H, mouse anti-human factor H, horse anti-human factor H or donkey anti-human factor H, and the sheep anti-human factor H, rabbit anti-human factor H, mouse anti-human factor H, horse anti-human factor H or donkey anti-human factor H is a monoclonal antibody or a polyclonal antibody.

[0169] It should be noted that, since the present invention uses liquid PEG200, the reagents I and II provided by the present invention are in liquid form, so the reagents have high solubility, strong ability to bind to the sample to be tested, fast polymerization promotion, and sufficient and rapid reaction. They can react with the sample to be tested within 2 minutes to obtain the test results with an accuracy rate of more than 99%.

[0170] The reagents prepared by the present invention are suitable for biochemical analyzers of different technical specifications, for example:

[0171] A kit consisting of one 80ml reagent I and one 20ml reagent II can be used in Beckman Coulter automatic biochemical analyzer to detect factor H concentration;

[0172] A test kit consisting of two 60ml reagent I and two 15ml reagent II can be used for Hitachi fully automatic biochemical analyzer to detect H factor concentration;

[0173] A test kit consisting of three 67ml reagent I and two 20ml reagent II can be used for Olympus automatic biochemical analyzer to detect H factor concentration;

[0174] A test kit consisting of four 3.7 ml reagent I and one 3.7 ml reagent II can be used for the German Siemens fully automatic biochemical analyzer to detect the H factor concentration.

[0175] Example 4 Determination of kit performance

[0176] The performance of reagents I and II in the kits prepared in Examples 1-3 was evaluated to determine whether the kits met the relevant standards of the Clinical Laboratory Standards Institute of the United States, ie, the CLSI standards.

[0177] 1. Reagent appearance

[0178] Reagent I and reagent II in the kits of Examples 1-3 were visually inspected under light conditions. Reagent I was a colorless, clear, transparent liquid, and reagent II was a light yellow, clear liquid. No precipitation or impurities were found, indicating that the reagents in the kits were normal.

[0179] 2. Reagent blank absorbance

[0180] Take R of Examples 2-4 respectively Ⅰ 240 μl, add 5 μl of distilled water, incubate at 37℃ for 5 min, and then add the corresponding R Ⅱ 60 μl, incubated at 37°C for 5 min, and measured on a 7060 automatic biochemical analyzer at a wavelength of 340 nm, and the absorbance values ​​of Examples 2-4 were all 0.01, less than 0.04, indicating that the kit was a normal blank control.

[0181] 3. Precision determination

[0182] 3.1 Intra-batch precision test method

[0183] Under normal working conditions of the instrument, use the same batch of reagents to continuously test (about 200 mg / L) samples 20 times and calculate the average value of the measured values The coefficient of variation (CV%) was calculated by the following formula, and the coefficient of variation was 1.2%, which is much smaller than 10%. Therefore, the kit of the present application is stable.

[0184] SD={∑(X i -X) 2 / (N-1)} 1 / 2

[0185] I=1

[0186]

[0187] Where: SD-standard deviation; CV-coefficient of variation; The mean of the measured values; X i is the i-th

[0188] Measured value; N – number of measurements.

[0189] 3.2 Batch precision test method

[0190] Under normal working conditions of the instrument, take three batches of reagents, one set from each batch, and measure the samples (about 200 mg / L) three times each. Then calculate the mean of the measurement results of each batch. The total mean of the results of the three batches of reagents The relative range (%) of the measured mean values ​​of the three batches of reagents was calculated according to the following formula, and the relative range was 2.1%, which is less than 10%. Therefore, the test kit of the present application has a good effect.

[0191] Relative range

[0192] Where: for The maximum value, for The minimum value in .

[0193] 4. Accuracy determination

[0194] Under normal working conditions of the instrument, the reagent was calibrated with the standard provided by the Department of Nephrology of Peking University First Hospital, and the samples of known concentration were measured. The measurement was repeated 10 times and the relative deviation of the mean of the measurement results was 1.0%. Therefore, the error was small and the measurement accuracy was high.

[0195] 5. Analytical sensitivity determination

[0196] R Ⅰ240 μl of reagent, add 5 μl of 200 mg / L sample, incubate at 37°C for 5 min, then add R Ⅱ 60μl, incubated at 37℃ for 5min, and measured the absorbance value at 340nm wavelength (sub-wavelength 700nm) on 7060 automatic biochemical analyzer, which was 720, less than 0.10. Therefore, this kit can sensitively reflect the absorbance of the sample.

[0197] 6. Stability determination

[0198] The blank absorbance, precision, accuracy and analytical sensitivity of the reagents stored at 2-8°C under the specified storage conditions within two months before and after the expiration date were tested, and the blank absorbance was less than 0.10; the coefficient of variation of the intra-batch precision was less than 0.1%, the relative range of the inter-batch precision was less than 15%; the accuracy was within the range of ±10%; the analytical sensitivity was greater than 0.04, which met the manufacturer's product technical requirements and could be stored at 2-8°C for at least 12 months. In addition, the linear correlation of this product is >0.9900, and the linear correlation is good.

[0199] The above measurements are all regular operations, the containers used are calibrated, the wavelength, sensitivity and stability of the biochemical analyzer meet the requirements, the temperature is accurate, the distilled water is qualified, and the target values ​​of the standard substance content or fixed-value serum are verified.

[0200] It has been determined that the test kits provided in this application all meet the relevant standards of the American Clinical Laboratory Standards Organization, namely the CLSI standards.

[0201] Example 5 Detection of H factor concentration of samples under different environmental conditions

[0202] In this example, the kit provided in Example 1 was used to detect the concentration of factor H.

[0203] 1. Sample preparation

[0204] 160 fresh blood samples were collected and arranged into treatment groups as shown in Table 1 according to the blood storage temperature T, storage time t and freeze-thaw times K, with the factor H concentration of the standard as the measurement basis.

[0205] Table 1 Environment sample settings

[0206]

[0207]

[0208] 2. Sample processing and testing

[0209] Use a biochemical analyzer to process and measure samples at a temperature of 15-35°C and a humidity of 45-85%.

[0210] That is, 240 μl of the reagent I solution prepared in Example 2 was added to 5 μl of the sample, and after incubation at 37° C. for 5 min in a biochemical analyzer, its absorbance value A1 was measured under the conditions of a main wavelength of 340 nm and a sub-wavelength of 700 nm. After the measurement, the reagent II solution containing the factor H antiserum prepared in Example 2 was added to the sample, and incubated at 37° C. for 5 min in a biochemical analyzer. After an immune reaction occurred, its absorbance value A2 was measured under the conditions of a main wavelength of 340 nm and a sub-wavelength of 700 nm. The difference A was obtained based on the absorbance values ​​before and after the reaction of the sample, that is, A=A2-A1.

[0211] 3. Calculation of H factor concentration

[0212] 3.1 Treatment and determination of standard samples

[0213] The operating conditions and the operating process are the same as those in step 2, that is, 240 μl of the reagent I solution prepared in Example 2 is added to 5 μl of the standard, and after incubation at 37° C. for 5 min in a biochemical analyzer, its absorbance value B1 is measured under the conditions of a main wavelength of 340 nm and a sub-wavelength of 700 nm. After the measurement, the reagent II solution containing the factor H antiserum prepared in Example 2 is added to the standard, and incubated at 37° C. for 5 min in a biochemical analyzer. After an immune reaction occurs, its absorbance value B2 is measured under the conditions of a main wavelength of 340 nm and a sub-wavelength of 700 nm. The difference B is obtained based on the absorbance values ​​of the standard before and after the reaction, that is, B=B2-B1.

[0214] 3.2 Calculation of H factor concentration

[0215] The concentration of factor H C (mg / L) was calculated based on the difference A before and after the sample reaction, the difference B before and after the standard reaction, and the concentration c (mg / L) of the standard, that is: C = A / B·c, to obtain the concentration of factor H in the sample.

[0216] The analysis results shown in Tables 2 and 3 were obtained based on the result values ​​of the H factor concentration.

[0217] Table 2 Analysis results of H factor concentration values ​​Ⅰ

[0218]

[0219] Table 3 Analysis results of H factor concentration values ​​Ⅱ

[0220]

[0221]

[0222] As shown in the concentration value analysis result I in Table 2, it can be seen that the detection results of the kit of the present invention for samples with different freeze-thaw times, samples at different temperatures, and samples with different storage times are not significantly different, that is, the kit and detection method for detecting the H factor concentration provided by the present invention obtain consistent detection results under the influence of different freeze-thaw times, temperatures and storage times, indicating that the kit and detection method provided by the present invention have extremely low requirements for samples, and the detection results are accurate and can meet clinical testing standards.

[0223] In order to further verify the accuracy and sensitivity of the kit, the present application conducted a multi-factor analysis of variance. The analysis results are shown in Table 2. Under the influence of the cross factors of freeze-thaw times and temperature, freeze-thaw times and storage time, temperature and storage time, and freeze-thaw times and temperature and storage time, the H factor concentration values ​​still did not differ. That is, even if the samples are affected by multiple factors, the results obtained by the kit and method provided by the present application are still consistent, further indicating that the H factor concentration detection kit and detection method provided by the present invention are suitable for sample detection under different conditions, and the detection results are accurate and can fully meet the clinical test standards.

[0224] Example 6 Detection of H factor concentrations in samples of different concentrations

[0225] Under normal working conditions of the instrument, samples with known concentrations as shown in Table 4 were measured, and each sample was measured 10 times. The relative deviation between the mean of the measured results and the known concentration was only 1.0%. It can be seen that the kit provided by the present application can detect samples with a concentration range of 4.66-800 mg / L, and the test results are accurate and meet the clinical test standards.

[0226] Table 4 Concentration sample settings

[0227] serial number concentration serial number concentration serial number concentration serial number concentration 1 4.66 7 160 13 350 19 700 2 10 8 190 14 400 20 720 3 30 9 220 15 500 21 740 4 60 10 250 16 600 22 760

[0228] 5 100 11 280 17 650 23 780 6 130 12 300 18 680 24 800

[0229] The present invention is characterized in that the method provided by the present invention has a wide detection range, and the H factor level within the concentration range of 4.86-2000 mg / l can be detected; the detection efficiency is high, the detection time only takes 1 minute, and the entire detection process only takes 5-10 minutes; the requirements for the storage conditions of blood are low, and blood samples stored at a temperature of -80 to 37°C, blood samples stored for up to four weeks, and blood samples that have been repeatedly frozen and thawed within 4 times can all be tested, and the test results are not affected, there is no requirement for anticoagulation of blood, and samples anticoagulated with EDTA or heparin can be tested.

Claims

1. A method for detecting the concentration of factor H, characterized in that: include: A kit for detecting the concentration level of factor H in a sample to be tested is prepared, comprising a reagent I and a reagent II containing a factor H antibody; wherein the reagent I is composed of a Tris buffer and a PEG200 solution, and the reagent II is composed of a PEG200 solution and a latex particle suspension of the factor H antibody; Adding the reagent I composed of Tris buffer and PEG200 solution to the sample to be tested, obtaining a first test sample after incubation, and obtaining a first absorbance value A1 of the sample to be tested after testing; adding the reagent II containing factor H antibody to the first test sample, obtaining a second test sample after incubation, and obtaining a second absorbance value A2 of the sample to be tested; Adding the reagent I composed of Tris buffer and PEG200 solution to the standard, obtaining a first detection standard after incubation, and obtaining a first absorbance value B1 of the standard after detection; adding the reagent II composed of PEG200 solution and latex particle suspension of factor H antibody to the first detection standard, obtaining a second detection standard after incubation, and obtaining a second absorbance value B2 of the standard after detection; Using the obtained first absorbance value A1 and second absorbance value A2 of the sample to be tested and the first absorbance value B1 and second absorbance value B2 of the standard substance, the concentration level of factor H in the sample to be tested is obtained; Wherein, the mass percentage concentration of PEG200 in the reagent I is 50-90 g / L; the mass percentage concentration of PEG200 in the reagent II is 10-30 g / L; Wherein, the volume concentration of the latex particle suspension of the factor H antibody is 400-800 ml / L.

2. The method according to claim 1, characterized in that The method of obtaining the concentration level of factor H in the sample to be tested by using the first absorbance value A1 and the second absorbance value A2 of the sample to be tested and the first absorbance value B1 and the second absorbance value B2 of the standard substance comprises: The difference between the first absorbance value A2 of the sample to be tested and the second absorbance value A1 of the sample to be tested is divided by the difference between the second absorbance value B2 of the standard and the first absorbance value B1 to obtain a ratio K; Multiply the known concentration of the standard factor H by the obtained ratio K to obtain the concentration value C of the factor H in the sample to be tested.

3. The method according to claim 1, characterized in that The detection conditions set during the detection are a temperature of 37° C., a main wavelength of 546 nm or 600 nm, and a sub-wavelength of 700 nm.

4. The method according to claim 1, characterized in that The factor H antiserum is derived from one of goat anti-human factor H, rabbit anti-human factor H or mouse anti-human factor H, and its titer is 1:

64.

5. A device for detecting the concentration of H factor, characterized in that: include: A kit for detecting the concentration level of factor H in a sample to be tested is prepared, comprising a reagent I composed of a Tris buffer and a PEG200 solution and a reagent II composed of a PEG200 solution and a latex particle suspension of a factor H antibody; and an absorbance value detection device, used for: After adding the Tris buffer and the PEG200 solution reagent I to the sample to be tested, a first test sample is obtained for absorbance detection to obtain the sample to be tested. Measure the first absorbance value A1 of the sample; Performing absorbance detection on a second test sample obtained by adding the reagent II composed of the PEG200 solution and the latex particle suspension of the factor H antibody to the first test sample to obtain a second absorbance value A2 of the test sample; After adding the reagent I composed of Tris buffer and PEG200 solution to the standard product, the first detection standard product is obtained, and the absorbance value is detected to obtain the first absorbance value B1 of the standard product; Adding the reagent II composed of the PEG200 solution and the latex particle suspension of the factor H antibody to the first detection standard to obtain a second detection standard, and performing absorbance detection on the second detection standard to obtain a second absorbance value B2 of the standard; A device for calculating the concentration level of factor H in the sample to be tested, for calculating the concentration level of factor H in the sample to be tested using the first absorbance value A1 and the second absorbance value A2 of the sample to be tested and the first absorbance value B1 and the second absorbance value B2 of the standard; Wherein, the mass percentage concentration of PEG200 in the reagent I is 50-90 g / L PEG200; the mass percentage concentration of PEG200 in the reagent II is 10-30 g / L PEG200; Wherein, the volume concentration of the latex particle suspension of the factor H antibody is 400-800 ml / L.

6. The device according to claim 5, characterized in that The serum anti-factor H antibody is derived from one of sheep anti-human factor H, rabbit anti-human factor H, mouse anti-human factor H, horse anti-human factor H or donkey anti-human factor H, and has a titer of 1:

64.

7. A kit for detecting the concentration of factor H, characterized in that: The kit can be used in the method of claim 1 or the device of claim 6, comprising: Reagent I comprising Tris buffer and PEG200 solution; Latex particle suspension reagent II containing PEG200 solution and factor H antibody; Wherein, the mass percentage concentration of PEG200 in the reagent I is 50-90 g / L PEG200; the mass percentage concentration of PEG200 in the reagent II is 10-30 g / L PEG200; Wherein, the volume concentration of the latex particle suspension of the factor H antibody is 400-800 ml / L.

8. The kit according to claim 7, characterized in that The serum anti-H factor antibody is derived from one of sheep anti-human H factor, rabbit anti-human H factor or mouse anti-human H factor, and its titer is 1:64.

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