CN116063482A - C-reactive protein (CRP) urine detection kit - Google Patents

Abstract

The invention relates to a C-reactive protein (CRP) urine detection kit. The ELISA double-antibody sandwich detection kit is prepared by combining the enzyme-labeled antibody and the coated antibody which are well paired by screening aiming at CRP specificity screening and preparing the CRP monoclonal antibody, and the kit can be used for CRP detection and has good specificity and detection effect.

Description

C-reactive protein (CRP) urine detection kit

Technical Field

The invention relates to the field of biology, in particular to the field of detection of C-reactive protein, and in particular relates to a C-reactive protein (CRP) urine detection kit.

Background

The C-reactive protein is also called CRP, is an acute phase-reactive protein, and can be synthesized by liver cells. When the pathogen is slightly stimulated or tissue is damaged, the synthesis of liver cells is rapidly started, the concentration of C-reactive protein reaches pathological level (more than 8 mg/L) within 6h, and the peak value is reached within 48-72 h, so that the early-stage tissue inflammatory necrosis is directly reflected. CRP, an extremely sensitive index of acute phase response, has various biological activities, and its concentration and secretion level are not changed by feeding and anti-inflammatory drugs, etc. Thus, the changes in the level of C-reactive protein have an important correlation with the inflammatory response and severity of the disease, and C-reactive protein is the most effective and most versatile inflammatory response marker. The detection of C-reactive protein has certain significance in various diseases such as bacterial and viral infection, tissue trauma, cerebral infarction, myocardial infarction, cardiovascular diseases and the like.

Specific applications of CRP in certain diseases include the following. Lower respiratory tract infection: CRP is significantly higher than viral infection at bacterial infection. CRP is greatly increased during bacterial pneumonia, and the average value can reach 200mg/L. CRP values depend on the duration of the infection. Viral pneumonia is complicated with bacterial infection, if CRP value is low, observation and detection are also needed before antibiotic treatment is administered, if the value is high, antibiotic treatment should be administered immediately. Continuous monitoring of CRP level decrease is an indicator of satisfactory therapeutic effect. CRP levels decreased within 3-4 days and returned to normal near levels within 2-4 weeks, indicating good therapeutic efficacy. The clinical symptoms of bacterial and viral infections of the respiratory tract are sometimes difficult to distinguish, and CRP can help make decisions as to whether to treat early antibiotics. CRP values may be normal if the onset is <12 hours, even with a clear manifestation of bacterial infection. However, after 12 hours, the CRP value was abnormal and confirmed as a bacterial infection. Urinary system infection: CRP level of patients with cystitis is <30-50mg/L, pyelonephritis is >10-20mg/L, median value is 75mg/L, and maximum value is 230mg/L, and CRP detection has good prompt effect particularly for patients incapable of expressing symptoms (infants, unconscious, dementia patients and the like). In addition, it has an important role in evaluating the severity of urinary system infection. Infection of gynaecology and pregnant women: CRP in pregnant women rises from 6mg/L at pregnancy to 20mg/L at delivery, to 60 mg/L24 hours later, to 25 mg/L24 hours later, and to 150mg/L on average 48 hours later after caesarean section. Higher levels of CRP indicate infection. Gynecological inflammatory diseases such as adnexitis, CRP values for acute and chronic pelvic inflammatory disease are significantly higher than those for non-inflammatory disease. Acute and chronic inflammatory CRP values are significantly different and are related to WBC fractions. After pelvic inflammatory disease treatment, the change amplitude value of CRP before and after antibiotic treatment is obviously larger than the WBC value, which indicates that CRP sensitivity is higher. The dynamic observation of CRP can better guide clinical medication. Single observations of WBC values may mask observations of disease development and treatment outcome. Infant and childhood infections: the clinical features of severe infections in infancy are often obscured. The CRP increase has a higher specificity and sensitivity to infant infection. Continuous CRP assays are more valuable for diagnosis. A febrile infant may be suspected of having a viral infection if the symptoms persist for >12 hours at a CRP level <20mg/L, a clinically undiagnosed bacterial infection, or a CRP value between 8-12 hours may exclude or confirm the possibility of a bacterial infection. Sepsis, endocarditis, meningitis and osteomyelitis: the CRP has higher value level and average value of 150-200mg/L. Rapid decline of CRP to normal is a sign of disease improvement and effective antibiotic treatment. In older children, adults with bacterial meningitis, symptoms are more than 12 hours, CPR is rarely less than 50mg/L, and symptoms are less than 12 hours, CRP may be normal. High CRP values occur in a few hours. Acute appendicitis: CRP may be normal with less than 12 hours of onset, and a second test several hours later may yield diagnostically valuable results. And WBC count analysis is the best indicator for diagnosis. Zheng Jianfang reports 400 columns of CRP and WBC suggest that the study of the diagnostic value of acute appendicitis: acute appendicitis can be excluded for both CRP and WBC normal, with a predicted efficiency of 100%. The cases adopt conservative treatment according to clinical symptoms as much as possible, CRP and WBC are measured for multiple times, and when a certain index is raised, whether operation is performed or not is comprehensively considered. Tissue damage: CRP started to increase 6-8 hours after surgery, reaching the highest level for 2-4 days. Choledochoscope, open cholecystectomy, the former with CRP mean 20mg/L and the latter with CRP mean 100mg/L. The acute liver transplant rejection reaction does not cause CRP elevation, and the CRP can be greatly elevated by postoperative bacterial infection. Patients with reduced liver function have protein synthesis failure and CRP will be lower than normal. The liver function effect should be considered in assessing CRP. Post-operative infection: CRP >75 mg/L6 days after surgery or 6 days later clearly suggests that complications are likely. Suspected acute ileus, CRP continues to rise high or continuously, suggesting that complications may be possible. Dynamic observations of daily CRP determinations are valuable for finding infections. Cardiovascular disease: for the prediction of the first cardiovascular event in healthy people, the hs-CRP is incorporated into the conventional cholesterol screening (1), the level of the prediction of cardiovascular risk is improved, and the prediction of LDL-C is not independently relied on (2), and the rise of the concentration of the hs-CRP can screen the prediction of the recurrent cardiovascular event of coronary heart disease patients with high risk asymptomatic patients with normal cholesterol level, but future cardiovascular events: when the concentration of hs-CRP in the serum of the coronary heart disease patient is increased by one standard deviation, the relative risk of occurrence of non-fatal myocardial infarction is increased by 45 percent, and the hs-CRP can be used as an independent predictor for the disease deterioration of the coronary heart disease patient and the risk of occurrence of myocardial infarction, which suggests that the hs-CRP can be utilized to distinguish high-risk patients from low-risk patients. Content of hs-CRP in AMI and UAP blood: the decisive role of UAP (7.28+/-2.03) mg/lAMI (22.19+/-7.64) mg/l in the normal group (2.35+/-0.58) in the formation of ACS is well established, hs-CRP being a powerful, independent predictor of the occurrence of acute coronary events, the rise of hs-CRP being related to the activity of inflammation and to the steady state of arteriosclerotic plaques, a highly sensitive inflammatory marker. Evaluation of hs-CRP for cardiovascular inflammation: for unstable angina, myocardial infarction patients with non-Q wave type, even if cTnI is not increased, the rise of hs-CRP occurs, and the corresponding death rate within 14 days is increased. The detection of hs-CRP in the treatment process of diabetics can well prevent acute myocardial infarction. When the condition is improved or cured, detection of a low level of hs-CRP indicates a better prognosis. Mortality of all factors is predicted: studies report that patients with hs-CRP concentrations >5mg/L at admission have mortality rates raised by 50% to 330% for any reason, e.g. patients with hs-CRP concentrations >8mg/L at admission have a double increase in mortality risk. As a unique marker for risk of death, hs-CRP should be routinely tested at the time of patient admission and high risk patients requiring special care should be sorted based on the test results.

It is based on the importance of CRP that it becomes particularly important to detect this indicator. The methods mainly applied in the market at present are divided into three main categories: 1. immunological turbidimetry (scattering, transmission) is a process of measuring protein concentration by using an antigen and an antibody to react in a reaction solution and then irradiating the reaction solution with a light beam to scatter or transmit the light beam. The method is characterized in that whether the antigen and the antibody fully react or not, in addition, different factors are larger on the light source of the instrument of each manufacturer, compared with an immune transmission nephelometry method and an immune scattering nephelometry method, the immune projection nephelometry method has excellent precision, similar analysis sensitivity and detection range, good correlation and small systematic error, and is more practical and convenient. 2. A method in which an antigen or antibody is bound to an enzyme as a probe by a labeling immunoassay (RIA radioimmunoassay, enzyme-labeled immunoadhesion assay ELISA), and the presence or absence of the antibody or antigen is examined by using the activity of the enzyme, and the radiolabel in the Radioimmunoassay (RIA) is replaced with the enzyme. Since no radioactive substance is used, the sensitivity is relatively safe and only slightly inferior, but the sensitivity approximately equal to that of RIA has been gradually reached due to improvements in the kind of enzyme used and the binding method and the like. POCT (immunodiafiltration and immunochromatography, enzyme labelling, biosensors, biochips) is a rapid detection method that has evolved relatively rapidly in recent years. The POCT detection method has the advantages of high detection speed, simple operation and capability of giving out results only by a few minutes, and can be used for bedside detection. However, the types of monoclonal antibodies currently selectable against CRP are not sufficiently abundant.

Disclosure of Invention

The present invention overcomes the deficiencies of the prior art and provides an improved CRP monoclonal antibody.

The antibodies are paired antibodies, 1B6 and 4C5, respectively.

Further, the light chain variable region sequence of the 1B6 antibody is shown in SEQ ID NO:1, the heavy chain variable region sequence is shown as SEQ ID NO. 2; the light chain variable region sequence of the 4C5 antibody is shown in SEQ ID NO:1, and the heavy chain variable region sequence is shown as SEQ ID NO. 2.

Further, in some specific embodiments, the heavy chain variable region and the light chain variable region are sequences having at least 80% identity or up to 15 amino acid differences.

In some specific embodiments, the at least 80% identity is preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; the up to 3 mutations are preferably up to 3, 2, 1 or 0 mutations; the up to 15 mutations are preferably up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations; preferably, the mutation is an insertion, a deletion or a substitution, preferably a conservative amino acid substitution, preferably a back mutation or a hot spot mutation.

The term "monoclonal antibody" herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variants (e.g., containing naturally occurring mutations or produced during production of the formulation, such variants typically being present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. The modifier "monoclonal" herein is not to be construed as requiring production of the antibody or antigen binding molecule by any particular method. For example, monoclonal antibodies can be made by a variety of techniques including, but not limited to, hybridoma techniques, recombinant DNA methods, phage display techniques, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and other methods known in the art.

Furthermore, the monoclonal antibodies of the invention may also be labeled with a recognizable label.

Furthermore, the invention also provides a kit for CRP detection, which is characterized in that the kit contains monoclonal antibodies specific to CRP.

Furthermore, the invention also provides the application of the monoclonal antibody specific to CRP in preparing a kit for CRP detection.

Further, the kit of the present invention is an ELISA detection kit.

The ELISA detection kit further comprises: coating antibody, enzyme-labeled antibody, sample diluent, 10-fold washing solution, negative control, positive control, TMB substrate solution and stop solution.

Advantageous effects

The invention aims at screening CRP specificity and preparing CRP monoclonal antibody, and obtains better paired enzyme-labeled antibody and coated antibody through screening, and the ELISA double-antibody sandwich detection kit is prepared by combining the two antibodies, and can be used for detecting CRP, and the kit has better specificity and detection effect.

Drawings

FIG. 1A diagram of the results of antibody specificity identification

The kit of FIG. 2 is directed to sample detection results

Detailed Description

The present disclosure is further described below in conjunction with specific embodiments, and advantages and features of the present disclosure will become apparent as the description proceeds. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. The disclosed embodiments are merely exemplary and do not constitute any limitation on the scope of the present disclosure. It will be understood by those skilled in the art that various changes and substitutions may be made in the details and form of the technical solutions of the present disclosure without departing from the spirit and scope of the present disclosure, but these changes and substitutions fall within the scope of the present disclosure.

EXAMPLE 1 preparation of CRP monoclonal antibodies

Animal immunization and hybridoma cell strain selection: the purchased CRP protein (QITAI organism, cat No. A006) was fully emulsified with 50% Freund's complete adjuvant, 4 female BALB/c mice were immunized, intraperitoneally injected, the first dose 100 μg/mouse, re-immunized 10d apart, 100 μg/mouse, 3 needles total immunized, 4d booster injection 1 needle 150 μg/mouse prior to fusion. The mouse tail blood was collected after 3 immunization needles and the antibody titer was detected by indirect ELISA. Wherein the antibody titer of the blood of the No. 2 mouse reaches more than 1:12800, the female mouse is selected, the female mouse is taken out in a sterile way, cell fusion is carried out according to the conventional hybridoma fusion technology, the HAT selection medium is screened, the cell supernatant is detected by an indirect ELISA method, and strong positive Kong Xibao is selected for cloning. 9 96-well plates are fused together, the fusion rate reaches 100%, the Europan positive wells are cloned, and cell strains 1B6, 2C7, 6D2, 7B12 and 4C5 of strong positive stable secretion specific mAb are obtained through 3-4 cloning by a limiting dilution method.

The 5 cells were cultured in an expanded manner and cell pellets were collected, and liquid paraffin-sensitized female mice were injected intraperitoneally, and after 14 days, ascites were collected and ELISA titer was measured on the ascites, and the results are shown in Table 1. 4mL of each mAb ascites is taken, and crude extraction is carried out by preliminary salting-out by a 50% saturated ammonium sulfate precipitation method. Dissolving ammonium sulfate precipitate after salting out for the 2 nd time, purifying by S-200 chromatography column, eluting with 0.05mol/L Tris-HCl (pH8.0), collecting elution peak in sections, and detecting by reducing SDS-PAGE electrophoresis to find that only 2 protein bands of light and heavy chains are present, which shows that the purity is better to be more than 95%. The protein concentration was measured by the Lowry method and is shown in table 1.

TABLE 1 abdominal water titer and antibody protein concentration

Antibody name	Abdominal water titer	Antibody protein concentration (g/L)
			1B6	256000	5.14
2C7	512000	3.97
			6D2	256000	4.86
7B12	256000	5.62
			4C5	128000	6.34

Example 2 selection of paired antibodies

HRP was labeled by the simple sodium periodate method. The basic test procedure for detecting CRP antigen by ELISA double antibody sandwich method is adopted by chessboard titration test. The purified 5 antibodies are respectively diluted to 10mg/L, 5mg/L, 2.5 mg/L and 1.25mg/L, enzyme-labeled strips are respectively coated on each dilution of each antibody, the plates are washed overnight at 4 ℃, and then reacted with Pfs25 purified antigens with the concentrations of 50mg/L and 10mg/L, meanwhile, CRP standard antigens are used as positive control, and saline negative control is provided. And respectively adding 4 other enzyme-labeled antibodies different from the coated antibody, wherein the dilutions are 1:2000, 1:4000, 1:8000 and 1:16000, the A450 value of the TMB is measured, and the optimal collocation of the coated antibody and the enzyme-labeled antibody is determined to be 1B6, 4C5 and 1.5mg/L according to the saturated concentration of the coated antibody and the enzyme-labeled antibody corresponding to the maximum A value.

Example 3 subclass and specific identification of 1B6 and 4C5 antibodies

Subclass and specificity determination of the two antibodies were performed using the immunoglobulin standard subclass identification kit from Sigma, HRP-labeled rabbit anti-mouse IgA, igM, igG, igG1, igG2a, igG2b, igG3, λ and κ, the specific procedure for subclass identification being followed by kit instructions. The results show that the 1B6 antibody is of the IgG1 class, the light chain type is kappa, the 4C5 antibody is of the IgG1 class, and the light chain type is kappa.

The above 2 monoclonal antibodies are respectively reacted with CRP, BSA, HBsAg, PD-1, mouse serum and other antigen coating strips, and the A450 value of the reaction of mAb and each antigen is measured by indirect ELISA. Saline (NS) negative control was set. The results are shown in FIG. 1.

As can be seen from the results of FIG. 1, the 2 mAbs all react with CRP antigen strongly, have higher A450 value, but do not react with BSA, HBsAg, PD-1, mouse serum and other antigen-coated antigens, and show the same A450 value as negative control Normal Saline (NS), which indicates that the two antibodies of the invention have better specificity.

Example 4 affinity and sequence identification of 1B6 and 4C5 antibodies

Anti-human CRP antibodies were captured using a ProteinA chip (GEHelthcare; 29-127-558). The sample and run buffer was HBS-EP+ (10mM HEPES,150mM NaCl,3mM EDTA,0.05%surfactantP20) (GEHealthcare; BR-1006-69). The flow-through cell was set at 25 ℃. The sample block was set at 16 ℃. Both were pretreated with running buffer. In each cycle, the antibody to be tested was first captured with a ProteinA chip, then a single concentration of CRP antigen protein was injected, the binding and dissociation processes of the antibody and antigen protein were recorded, and finally chip regeneration was completed with Glycine H1.5 (GEHelthcare; BR-1003-54). Binding was measured by injecting different concentrations of human CRP in solution for 250 seconds with a flow rate of 30 μl/min starting from 200nM (see detailed results for the actual concentration tested), at 1:1 dilution, total 5 concentrations. Dissociation phases were monitored for up to 600 seconds and triggered by switching from sample solution to running buffer. The surface was regenerated by washing with 10mM glycine solution (pH 1.5) at a flow rate of 30. Mu.l/min for 40 seconds. Bulk refractive index (bulk refractive index) differences were corrected by subtracting the response obtained from the goat anti-human Fc surface. Blank injections (=double reference) were also subtracted. To calculate apparent KD, langmuir1 was used: model 1. The dissociation constants of the antibodies and human CRP proteins are shown in table 2.

TABLE 2 dissociation constant of monoclonal antibody

Antibody name	Dissociation constant (nM)
		1B6 antibodies	12.35±0.13
4C5 antibodies	8.63±0.06

From the results in table 2, it can be seen that all 2 monoclonal antibodies prepared by the invention have better binding capacity.

Amplifying and sequencing identification of the light and heavy chain variable region sequence by using an antibody light and heavy chain identification kit to obtain the light chain variable region sequence of the 1B6 antibody as shown in SEQ ID NO:1, the heavy chain variable region sequence is shown as SEQ ID NO. 2; the light chain variable region sequence of the 4C5 antibody is shown in SEQ ID NO:1, and the heavy chain variable region sequence is shown as SEQ ID NO. 2.

Examples 5 1B6 and 4C5 double antibody sandwich assays

The coated antibody is 1B6, the enzyme-labeled antibody is 4C5, and a double-antibody sandwich ELISA quantitative detection kit aiming at CRP is established. And drawing a standard curve at the same time, and calculating the content of CRP in the sample according to the standard curve. The result shows that the sensitivity of the kit can reach 1ng/mL, and the linear range is 0-40ng/mL.

CRP levels in urine of 20 patients with urinary pyelonephritis infection and 10 normal persons were measured using the established CRP ELISA test kit. Urine was collected in a clean tube at 2500r/min, centrifuged for 5min, and the supernatant was collected for detection. 100. Mu.L of 1B6 was added to each well of the ELISA plate at 1.5mg/L overnight at 4℃using 1B6 as a coating antibody. Phosphate buffer (PBST, containing 2mL/L Tween 20) was washed 3 times, 100. Mu.L of standard substance or 1:500 diluted plasma sample to be tested was added, respectively, and incubated at 37℃for 1h. PBST was washed 3 times, 100. Mu.L of HRP-labeled 4C5 antibody diluted 1:1000 was added, and incubated at 37℃for 1h. After 3 times of PBST washing, 100 mu L of ABTS chromogenic solution is added to each well, and the mixture is incubated at room temperature for 15min for chromogenic reaction. The absorbance (A) was measured on a microplate reader using a 405nm filter with a blank control zeroed. And calculating the concentration of CRP in the plasma to be detected by using a standard curve. Statistical analysis data were plotted using Graphpad prism5 software, and were processed using SPSS17.0 statistical analysis software using

Representing the measurement data, and comparing and analyzing 2 groups by adopting t test, wherein P < 0.05 is statistically significant. The results are shown in FIG. 2.

CRP sandwich ELISA was used to detect CRP content in urine of 20 patients with urinary tract pyelonephritis infection and 10 normal people. The results show that CRP levels in 20 patients with the infection of the urinary tract pyelonephritis are (5.01+/-0.21) mg/L, normal artificial (6.24+/-0.17) mug/L, CRP levels in urine of the patients with the infection of the urinary tract pyelonephritis are obviously increased, and the difference is statistically significant (P is less than 0.01, and is shown in figure 2).

The above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (6)

1. A group of monoclonal antibodies for double-antibody sandwich detection, which is characterized by consisting of 1B6 and 4C5, wherein the coating antibody is 1B6, and the enzyme-labeled antibody is 4C5;1B6 antibody has the light chain variable region sequence shown in SEQ ID NO:1, the heavy chain variable region sequence is shown as SEQ ID NO:2 is shown in the figure; the light chain variable region sequence of the 4C5 antibody is shown in SEQ ID NO:3, the heavy chain variable region sequence is shown as SEQ ID NO: 4.

2. The set of monoclonal antibodies for use in a double antibody sandwich assay according to claim 1, wherein the enzyme-labeled antibodies in the monoclonal antibodies are labeled with chromogenic enzymes.

3. A kit for CRP urine detection comprising a set of monoclonal antibodies for use in a double antibody sandwich assay according to claim 1.

4. The kit of claim 3, further comprising a sample diluent, a wash solution, a negative control, a positive control, a TMB substrate solution, and a stop solution.

5. Use of a set of monoclonal antibodies for the sandwich detection of diabodies according to claim 1 for the preparation of a kit for the detection of CRP.

6. The use according to claim 5, wherein the kit further comprises a sample diluent, a wash solution, a negative control, a positive control, a TMB substrate solution and a stop solution.

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