CN112485447A

CN112485447A - Kit for determining complement C1q

Info

Publication number: CN112485447A
Application number: CN202011295934.4A
Authority: CN
Inventors: 姚浩; 李赛; 胥安庆
Original assignee: Chongqing Zhongyuan Huiji Biotechnology Co Ltd
Current assignee: Chongqing Zhongyuan Huiji Biotechnology Co Ltd
Priority date: 2020-11-18
Filing date: 2020-11-18
Publication date: 2021-03-12
Anticipated expiration: 2040-11-18
Also published as: CN112485447B

Abstract

The invention discloses a kit for determining complement C1q, which comprises: reagent R1 and reagent R2, reagent R1 contains buffer solution, surfactant, antiseptic, inorganic salt ion and accelerator, reagent R2 contains buffer solution, recombinant streptococcal protein G and complement C1q antiserum. According to the invention, a small amount of recombinant streptococcal protein G is added into the reagent R2, so that the precipitation of hybrid protein in C1q antiserum can be accelerated obviously under the condition of not influencing antigen-antibody reaction atopy, the processing time of the production stage of the kit is shortened obviously, and the production efficiency is improved obviously on the basis of ensuring the detection accuracy and precision of the kit.

Description

Kit for determining complement C1q

Technical Field

The invention relates to the field of medical inspection, in particular to a kit for determining complement C1 q.

Background

Complement C1q is a glycoprotein composed of a group of enzymatically active heterotetramers present on the surface of blood, body fluids and cell membranes, is a subunit of the first complement component C1 of the classical complement pathway, acts as a promoter of the classical complement activation pathway, recognizes and binds to immune complexes to initiate the activation pathway, and has the ability to modulate a variety of immune cellular responses. Can be used as the index for diagnosing Systemic Lupus Erythematosus (SLE) and predicting the activity of Lupus Nephritis (LN), and plays an important role in diagnosing diseases such as rheumatoid arthritis, kidney diseases, renal transplant rejection, atherosclerosis, mixed connective tissue diseases and the like.

At present, the detection means of complement C1q mainly include Rocket Immunoelectrophoresis (RIE), enzyme-linked immunosorbent assay (ELISA) and immunoturbidimetry (TIA), but at present, no accepted standard exists in the industry. RIE has important guiding significance for detecting C1q in serum, but due to poor repeatability, long time consumption, manual operation and no special commercial equipment, the method is rarely used in clinical laboratories. ELISA requires manual operations, is time-consuming and has not been used clinically on a large scale.

At present, the content of C1q in serum is generally detected by using a common turbidimetry method. The conventional turbidimetry method generally directly uses antiserum to prepare the R2 reagent, but a large amount of nonspecific proteins exist in the antiserum, and the R2 reagent generates obvious white precipitates in the presence of a coagulant, which is particularly serious on goat anti-C1 q serum. The conventional method for solving this problem is to leave the R2 reagent for a long time and then filter the precipitate to obtain a supernatant, or to prepare the R2 reagent using an antibody purified by a protein G gel column. The former has long treatment time, and particularly has poor effect on goat anti-C1 q serum, and the filtrate still has precipitate; the latter effect is significant but adds significantly to the cost of the reagents.

Disclosure of Invention

According to the kit for determining complement C1q, a small amount of recombinant streptococcal protein G is added into a reagent R2, so that the precipitation of foreign proteins in serum of C1q is obviously accelerated, the preparation time of the kit is shortened, and the precision and the stability of reagent detection are improved.

In order to achieve the purpose, the invention adopts the following technical means: a kit for determining complement C1q, which comprises a reagent R1 and a reagent R2, and is characterized in that the reagent R2 comprises recombinant streptococcal protein G.

Preferably, the reagent R2 further comprises a buffer and complement C1q antiserum.

Preferably, the recombinant streptococcal protein G content is 0.02-0.1G/L.

Preferably, the reagent R2 further comprises inorganic salt ions, an accelerator and a preservative.

Preferably, the reagent R1 comprises a buffer, a surfactant, a preservative, inorganic salt ions, and an accelerator.

Preferably, the reagent R1 comprises 30-50mmol/L buffer solution, 10-20g/L surfactant, 0.5-1.5g/L preservative, 10-250mmol/L inorganic salt ion and 40-80g/L accelerator.

Preferably, the reagent R2 comprises 30-50mmol/L buffer solution, 200-400mmol/L inorganic salt ions, 10-30g/L accelerator agent, 0.5-1.5g/L preservative and 10-30g/L complement C1q antiserum.

Preferably, the buffer in the reagent R1 is at least one selected from Tris or phosphate buffer; the surfactant is at least one selected from Triton X-100, Brij 58 or Tween-80; the preservative is at least one selected from Proclin-300, sodium azide and gentamicin; the inorganic salt ions are selected from at least one of sodium chloride or potassium chloride; the accelerator is at least one selected from dextran, PEG6000 and PEG 8000.

Preferably, the buffer in the reagent R2 is selected from one of Tris or phosphate buffer; the inorganic salt ions are selected from at least one of sodium chloride or potassium chloride; the accelerator is selected from at least one of dextran, PEG6000 or PEG 8000; the preservative is at least one selected from Proclin-300, sodium azide and gentamicin.

Use of recombinant streptococcal protein G as a procoagulant agent for the preparation of a kit for the determination of complement C1 q. The invention has the beneficial effects that:

according to the invention, a small amount of recombinant streptococcal protein G is added into the reagent R2 of the serum complement C1q detection kit, so that the precipitation of the foreign protein in the serum of the C1q antibody can be obviously accelerated under the condition of not influencing the idiosyncratic reaction of the antigen and the antibody, the processing time of the production stage of the kit is obviously shortened, the production efficiency is obviously improved on the basis of ensuring the detection accuracy and precision of the kit, and the production cost is reduced.

Drawings

FIG. 1 is a standard curve graph of the concentration of C1q standard substance versus the measured value provided in example 3 of the present invention;

FIG. 2 is a graph showing the linear relationship between the amount of recombinant streptococcal protein G added to reagent R2 and the blank set, according to example 4 of the present invention;

FIG. 3 is a graph of the incubation time of reagent R2 as provided in example 5 of the present invention plotted linearly against the blank set.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following examples are included to more clearly and clearly illustrate the technical solutions of the present invention by way of illustration. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The specific embodiments of the present invention are merely illustrative of the invention and are not intended to limit the invention in any way.

Example 1 preparation of C1q test kit

The kit for measuring the serum complement C1q comprises a reagent R1 and a reagent R2 which are independent of each other.

1. Preparation of reagent R1

The preparation is carried out according to the following formula, and the mixture is fully stirred, evenly mixed and stored at the temperature of 2-8 ℃.

Reagent R1:

the rest of the solvent is purified water

2. Preparation of reagent R2

Reagent R2

The rest of the solvent is purified water

Example 2 method of Using the kit

(1) Mixing the serum sample to be detected with the reagent R1 and the reagent R2, and stirring and uniformly mixing the mixture to make the mixture fully react;

(2) measuring absorbance difference (main wavelength of 340nm, sub wavelength of 700nm, instrument reading point of 16-34) after reaction with a full-automatic biochemical analyzer (Hitachi 7180);

(3) the concentration of complement C1q in the sample was calculated from the absorbance change values.

The detection principle of the invention is as follows: the complement C1q in serum sample can combine with anti-C1 q antibody in reagent to form antigen-antibody complex, generate certain turbidity, the turbidity is proportional to the antigen content when certain antibody exists, the turbidity is measured under certain wavelength and the complement C1q can be quantitatively measured by multi-point calibration curve.

Complement C1q (mg/L) ═ C in the samples_S×ΔA_T/ΔA_S(mg/L)

(in the formula,. DELTA.A_TSample tube absorbance value, Δ A, against blank tube absorbance_SCalibration tube absorbance values, C, against blank tube absorbance_SConcentration of complement C1q in calibration solutions

Example 3 kit Performance test

The serum complement C1q test kit prepared in example 1 was performance tested according to the method of use described in example 2.

(1) Detection result of C1q standard substance

Using the kit prepared in example 1, various concentrations of C1q standard (seven different concentrations, 30mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L, 300mg/L, 400mg/L, 3 replicates for each concentration) were assayed, and signals were read by a fully automatic biochemical analyzer (Hitachi 7180). The results are shown in the following table:

TABLE 1C1q test results for Standard

The concentration of the C1q standard substance is used as an abscissa, the measured average concentration is used as an ordinate, a logarithmic equation is established and a standard curve is fitted, the result is shown in figure 1, the result shows that the detection result is good in linearity when the kit is used for detection, and the concentration of C1q in a sample can be quantitatively analyzed through the standard curve.

(2) Precision degree

Selecting a low-value sample, a medium-value sample and a high-value sample of clinical C1q, determining by using the kit prepared in example 1 and the method used in example 2, repeating the determination for 10 times for each concentration, calculating a determination mean value and a standard deviation, calculating a variation coefficient, and performing repeated investigation, wherein the detection results are shown in the following table:

TABLE 2C 1q precision of test kit

The coefficient of variation is 1.15%, 1.14% and 1.34%, and meanwhile, the accuracy examination is performed by calculating the relative deviation by (mean/standard value-1) × 100%, and the results show that the relative deviation is 0.50%, 0.50% and 0.73%, and the experimental results show that the kit obtained by adopting the preparation scheme described in the embodiment has better precision and repeatability.

(3) Stability of

Taking the C1q detection kit to carry out a conventional storage stability test, and standing at 2-8 ℃ for 2, 4, 6, 8, 9, 10, 11, 12, 13 and 14 months respectively to carry out detection; the uncapping stability test is carried out according to the test of placing for 0 day, 7 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, 30 days and 32 days at the temperature of 2-8 ℃. The result shows that the C1q detection kit is stored in a light-shielding environment at the temperature of 2-8 ℃ and has the validity period of 12 months. The product is stored in a dark environment at the temperature of 2-8 ℃ after being uncovered, and the validity period is 30 days.

(4) Interference immunity

Preparing a C1q standard substance with a concentration of 200mg/L, adding different amounts of interferents into the standard substance, respectively, then using the detection kit prepared in example 1, detecting the samples by using the method in example 1, repeating the detection for 3 times for each group of samples, calculating the average value, and simultaneously calculating the relative deviation by (average value/standard value-1) × 100% for accuracy examination. The results are shown in the following table:

TABLE 3C 1q detection kit anti-interference

The results show that when the interfering substances are added to the C1q standard: when bilirubin, haemolytic hemoglobin and chyle are used, the relative deviation of the measured concentration and the theoretical concentration is respectively 3.18%, 1.78% and 1.31%, and the relative deviation is less than 5%, and the result shows that the interferents basically have no influence on the reaction system, and the kit prepared by the invention has better anti-interference performance.

Example 4 Effect of recombinant Streptococcus G addition on kit Performance

In this example, six experiments were performed, wherein the kit used in each experiment was prepared in the same manner as in example 1 except that the recombinant streptococcal bacterium G content in the reagent R2 was different from that in example 1. The specific cases are as follows:

TABLE 4

0mg, 0.1mg, 0.5mg, 2.5mg and 12.5mg of recombinant streptococcal protein G are respectively added into 5ml of reagent R2, and then the five groups of experiments are incubated for 24 hours at 4 ℃, and the precipitation amount of impurity proteins and the precision of detection results are detected.

(1) Effect of addition of recombinant Streptococcus G to reagent R2 on the amount of impurity protein precipitated

The experimental result shows that the white precipitation amount in the reagent R2 is obviously improved along with the increase of the content of the recombinant streptococcal protein G. The measured dry weight of the precipitate is shown in the following table:

TABLE 5 Effect of recombinant Streptococcus protein G addition on impurity protein precipitation

(2) Influence of addition of recombinant Streptococcus G to reagent R2 on test results

In this example, to verify whether the addition amount of recombinant streptococcal protein G affects the specific reaction of antigen and antibody (i.e., the accuracy of the detection result), 22 clinical constant value samples were selected, the above 5 kits were used for detection, and 22 fresh samples with hospital measured values from small to large were selected to examine the correlation with the blank group.

After blank calibration using a fully automatic biochemical analyzer (hitachi 7180), the reactivity of the samples was measured using the above 5 kits, and the results are shown in the following table:

TABLE 6 sample detection reactivity

Note: the reactivity of 22 fresh samples was tested using Hitachi 7180: blank calibration (K value modified to 10000) with a calibration type of Linear.

Comparing the above 4 kit detection results with blank kit detection results and performing correlation mapping (shown in figure 2), wherein the correlation R between the A group and the blank group²0.9866, group B R²0.9751, group C R²0.4428, group D R²Is 0.3097. The experimental result shows that the addition of a small amount of recombinant streptococcal protein G (such as group A) in the reagent R2 does not obviously influence the specific reaction of antigen and antibody, and can accelerate the precipitation of impurity protein; however, addition of an excess of recombinant streptococcal protein G (e.g.groups C and D) will bind to the targetA target protein, resulting in a significantly reduced correlation with the blank test results; when 0.1G/L of recombinant streptococcal protein G (group B) was added to reagent R2, the specific reaction of antigen and antibody was not affected, and the amount of protein precipitated was approximately 7 times that of the blank group (conventional method).

Example 5 Effect of incubation time on kit Performance after addition of recombinant Streptococcus G to reagent R2

In this example, five experiments were performed, wherein the amount of recombinant streptococcal protein G added to the reagent R2 used in the blank group was different from that in example 1, the incubation time of the reagent R2 used in the reagent groups A to D was different from that in example 1, and the remaining preparation methods of the reagent groups were the same as those in example 1. The specific cases are as follows:

TABLE 7

Blank group: adding 0G/L of recombinant streptococcal protein G into 5ml of reagent R2, and incubating for 24h at 4 ℃; groups A to D: adding 0.1G/L of recombinant streptococcal protein G into 5ml of reagent R2 respectively, and then incubating at 4 ℃, wherein group A is incubated for 2 hours, group B is incubated for 5 hours, group C is incubated for 10 hours, and group D is incubated for 20 hours, and detecting the precipitation amount of impurity proteins and the precision of detection results in the five experiments.

(1) Effect of different incubation times on the amount of protein precipitated as impurities

The experimental results show that when 0.1G/L recombinant streptococcal protein G is added to reagent R2, the precipitation amount increases with the increase of the incubation time, and the increase speed of the precipitation amount is slowed down after 5h of incubation. The measured dry weight of the precipitate is shown in the following table:

TABLE 8 Effect of incubation time on the amount of impurity protein precipitated

(2) Effect of different incubation times on the assay results

In this example, in order to verify whether different incubation times affect the specific reaction of the antigen and antibody (i.e., the accuracy of the detection result) after adding a small amount of recombinant streptococcal protein G, 22 fresh samples with hospital measured values from small to large were selected to examine the correlation with the blank group.

TABLE 9 sample detection reactivity

Comparing the above 4 kit detection results with blank kit detection results and performing correlation mapping (shown in figure 3), wherein the correlation R between the A group and the blank group²0.9879, group B R²0.9857, group C R²0.9898, group D R²Is 0.9724. The experimental result shows that the addition of a small amount of recombinant streptococcal protein G into the reagent R2 can not affect the specific reaction of the antigen and the antibody, the white precipitation amount is 9.7mg which is about 7 times of the precipitation amount of a blank group (a traditional method) when the incubation time is 5h (such as a group A), the incubation time is greatly shortened, and the experimental result shows that the precipitation speed of the impurity protein in the reagent R2 can be accelerated after the small amount of recombinant streptococcal protein G is added into the reagent R2, the processing time in the production stage is remarkably shortened, and the production efficiency is remarkably improved on the basis of keeping the detection accuracy and precision of the kit.

Example 4

In this example, 4 sets of experiments were set up, wherein each set of experiments used a kit different from example 1 only in the kind of surfactant in the reagent R1, and the remaining kits were prepared in the same manner as in example 1. The four kits are adopted for detection at the same time, and the detection results are shown in the following table:

watch 10

Note: the surfactant in the reagent R1 in group A in the embodiment is 15g/L of Brij 58; group B is 15g/L Tetronic 1307; group C is Tween-80 of 15 g/L; the group D is 15g/L of Tween-20, and the rest components and the preparation process are the same as those of the group D in the example 1.

The experimental result shows that the detection precision of the kit is higher by adopting the 4 surfactants in the reagent R1.

Example 5

In this example, 5 sets of experiments were set up, wherein each set of experiments was carried out using a kit differing from example 1 only in the concentration of the surfactant Triton X-100 in the reagent R1, and the remaining kits were prepared in the same manner as in example 1. The five kits are simultaneously adopted for detection, and the detection results are shown in the following table:

TABLE 11

Note: the surfactant in the reagent R2 in group A in the embodiment is Triton X-100 with the concentration of 2 g/L; group B is TritonX-100 of 5 g/L; group C is TritonX-100 of 10 g/L; group D is 20g/L Triton X-100; group E is TritonX-100 of 30g/L, and the rest of the components and the preparation process are the same as those in example 1.

The experimental result shows that when the concentration range of the surfactant Triton X-100 in the reagent R1 is 10-20g/L, the detection precision of the kit is improved.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Claims

1. A kit for determining complement C1q, which comprises a reagent R1 and a reagent R2, and is characterized in that the reagent R2 comprises recombinant streptococcal protein G.

2. The kit for determining complement C1q of claim 1, wherein the reagent R2 further comprises a buffer and complement C1q antiserum.

3. The kit for determining complement C1q according to claim 2, wherein the recombinant streptococcal protein G is present in an amount of 0.02-0.1G/L.

4. The kit for detecting complement C1q according to claim 3, wherein the reagent R2 further comprises inorganic salt ions, an accelerator, and a preservative.

5. The kit for detecting complement C1q according to any one of claims 1 to 4, wherein the reagent R1 comprises a buffer, a surfactant, a preservative, an inorganic salt ion, and an accelerator.

6. The kit for detecting complement C1q, wherein the reagent R1 comprises 30-50mmol/L buffer solution, 10-20g/L surfactant, 0.5-1.5g/L preservative, 10-250mmol/L inorganic salt ion and 40-80g/L accelerator.

7. The kit for detecting complement C1q, wherein the reagent R2 comprises 30-50mmol/L buffer solution, 200-400mmol/L inorganic salt ions, 10-30g/L accelerator, 0.5-1.5g/L preservative and 10-30g/L complement C1q antiserum.

8. The kit for detecting complement C1q, according to claim 6, wherein the buffer in reagent R1 is at least one selected from Tris or phosphate buffer; the surfactant is at least one selected from Triton X-100, Brij 58 or Tween-80; the preservative is at least one selected from Proclin-300, sodium azide and gentamicin; the inorganic salt ions are selected from at least one of sodium chloride or potassium chloride; the accelerator is at least one selected from dextran, PEG6000 and PEG 8000.

9. The kit for detecting complement C1q, according to claims 7-8, wherein the buffer in reagent R2 is selected from one of Tris or phosphate buffer; the inorganic salt ions are selected from at least one of sodium chloride or potassium chloride; the accelerator is selected from at least one of dextran, PEG6000 or PEG 8000; the preservative is at least one selected from Proclin-300, sodium azide and gentamicin.

10. Use of recombinant streptococcal protein G as a procoagulant agent for the preparation of a kit for the determination of complement C1 q.