CN1271409C - Raid quantitative determination of cardiac muscle troponin I by three-anti method - Google Patents

Abstract

The patent of the present invention relates to a new method for rapid and quantitative determination of cardiac troponin I content in human peripheral blood or tissue based on the principle of ELISA. Because the content of cardiac troponin I in the blood is very small, the sensitivity of the current common enzyme-linked method is low. 1 hour or more. In order to improve the measurement speed and measurement sensitivity, the present invention has successfully developed a three-antibody method cardiac troponin I rapid quantitative assay kit, which uses two monoclonal antibodies to replace one monoclonal antibody in the traditional method as a capture antibody, which improves the antibody concentration. The capture ability, combined with the streptavidin-biotin amplification system, effectively shortens the detection time and improves the measurement sensitivity, so that the rapid quantitative detection of cardiac troponin I on ordinary enzyme-linked instruments and other instruments can meet the inspection and clinical requirements .

Description

Rapid Quantitative Determination of Cardiac Troponin I by Three Antibody Method

Technical Field The patent of this invention relates to a new method for rapid quantitative determination of human peripheral blood or tissue cardiac troponin I based on the principle of ELISA.

Background Art Myocardial ischemic injury, especially acute myocardial infarction (AMI) is one of the major diseases that threaten human life. Clinical chemists and clinical scientists have been working hard to find and explore serum indicators with good specificity and high sensitivity. Cardiac troponin I (cTnI) has a high degree of myocardial specificity. When cardiomyocytes are damaged, cTnI appears early in the blood and lasts for a long time, which is closely related to the degree of myocardial injury and prognosis. Therefore, it can be used as a specific indicator of myocardial injury, and has very important significance in the diagnosis and prognosis of heart disease.

The determination of cardiac troponin I can be divided into two methods: qualitative and quantitative. The qualitative determination mainly uses the principle of chromatography and is completed by making test strips. It has been clinically applied at present. Its advantages are fast and easy to operate, but its disadvantage is that it only Can display positive and negative results, without specific values, cannot observe its dynamic changes and judge infarction time, area and prognosis based on values, and has low sensitivity and is easily interfered by various factors. Even the improved semi-quantitative small instrument measurement method also has the above disadvantages. Quantitative determination is mainly designed based on the principle of ELISA and chemiluminescence, and most of them use two monoclonal antibodies (a few use only one monoclonal antibody or one of them uses polyclonal antibody), and one monoclonal antibody is used to capture Antibody, another monoclonal antibody is a labeled antibody, which is determined by an enzyme-linked sandwich method. The relying and auxiliary instruments in the determination include automated and non-automated chemiluminescence instruments, fluorescence analyzers, and enzyme-linked instruments, among which the price of enzyme-linked instruments is low, ranging from several thousand yuan to tens of thousands of yuan. However, chemiluminescence instruments and fluorescence measuring instruments are expensive, ranging from hundreds of thousands to hundreds of thousands of yuan. Several large companies abroad have produced large-scale automated chemiluminescence or fluorescence instruments to complete quantitative measurements, but they are expensive and require supporting equipment. Reagents are still difficult to popularize and apply in most hospitals in China. Therefore, it is very necessary to establish a quantitative enzyme-linked method for cardiac troponin I based on an enzyme-linked instrument, but the sensitivity of the assay for cardiac troponin I established by an ordinary enzyme-linked method is low, and the minimum detection sensitivity is about 0.2ng/ml, which is very low. It is difficult to reach below 0.1ng/ml, and the determination speed is also slow, requiring more than 1 hour.

Summary of the invention Because the content of cardiac troponin I in the blood is very small, in order to improve the measurement speed and sensitivity, the present invention has successfully developed a three-antibody rapid quantitative assay kit for cardiac troponin I, using two monoclonal antibodies to replace the traditional A monoclonal antibody in the method is a capture antibody, which improves the antibody capture ability. At the same time, combined with the streptavidin-biotin amplification system, it effectively shortens the detection time and improves the detection sensitivity. The rapid quantitative detection of cardiac troponin I meets the inspection and clinical requirements.

The technical scheme of the present invention: two monoclonal antibodies acting on cardiac troponin I are capture antibodies, referred to as Ab1+Ab2 for short, after linking with biotin or long-chain biotin, referred to as Ab1-Bio+Ab2-Bio for short. Another monoclonal antibody that acts on cardiac troponin I is a labeled antibody, referred to as Ab3, which is referred to as Ab3-POD after coupling with horseradish peroxidase, and Ab3 after coupling with biotin or long-chain biotin -Bio. Ab1, Ab2, and Ab3 are required to act on different sites of the cTnI antigen. The pairing of Ab1+Ab2 and Ab3 has a better binding force and higher specificity to the cTnI antigen. Monoclonal antibodies and other raw materials can come from qualified commercial products (such as Sigma, Roch, etc.). Using the principle of enzyme-linked immunosorbent immunoassay and its reaction pathway

Specifically, the following options are included:

Option One:

(1). 96-well enzyme-linked plates, enzyme-linked strips or other solid supports are linked to streptavidin or avidin (abbreviated as S), and albumin or other blocking solutions are blocked.

(2).Ab1-Bio+Ab2-Bio combined with S, cardiac troponin I antigen (including known cardiac troponin I standard, quality control, and cardiac troponin I in the sample to be tested, referred to as cTnI) is combined with Ab1-Bio+Ab2-Bio, and cTnI is combined with Ab3-POD. A reaction chain of S～Ab1-Bio+Ab2-Bio～cTnI～Ab3-POD is formed successively.

(3). POD makes chromogenic substrates (such as tetramethylbenzidine, TMB, etc.) chromogenic or chemiluminescent substrates emit light, and then is measured by corresponding instruments, and the content of cardiac troponin I in the sample to be tested is calculated according to the standard curve.

Option II:

(1). 96-well enzyme-linked plates, enzyme-linked strips or other solid supports are linked to Ab1+Ab2, and blocked with albumin or other blocking solutions.

(2). Ab1+Ab2 binds to cTnI, and cTnI binds to Ab3-POD, forming a reaction chain of Ab1+Ab2～cTnI～Ab3-POD successively.

(3). Same as (3) in Scheme 1.

third solution:

(1). Same as 1 in Scheme 2.

(2). Ab1+Ab2 binds to cTnI, cTnI binds to Ab3-Bio, Ab3-Bio binds to streptavidin-horseradish peroxidase (S-POD for short), and successively forms Ab1+Ab2～cTnI～Ab3- Bio～S-POD reaction chain.

(3). Same as (3) in Scheme 1.

After the above-mentioned innovations, the determination time of this method can be shortened to about 35 minutes, and its minimum detection sensitivity is 0.05ng/ml, which is equal to or exceeds the level of similar foreign kits, and it can be determined only by an ordinary enzyme-linked instrument, and the detection result can be issued quickly , which can be flexibly used in batches for the detection of different numbers of cases in the emergency department and routine, which is more convenient for clinical application and is suitable for wide application in large, medium and small hospitals. .

Specific implementation methods According to the above three schemes, the specific implementation methods are:

Option One:

(1). 96-well enzyme-linked plates or enzyme-linked strips are linked with streptavidin, and sealed with 0.5-1% albumin or other blocking solutions for later use.

(2). Add a certain amount of Ab1+Ab2-Bio, incubate at 37°C for a certain period of time (preferably 60 minutes) or overnight at 4°C, seal it and pack it in a kit, and open it for sample measurement.

(3). The known cardiac troponin I standard substance or the sample to be tested in the hole during determination (the known cardiac troponin I standard substance can also be pre-frozen in the set standard well, and the standard diluent is added during detection. Just dissolve), then add a certain amount of Ab3-POD reaction solution, shake lightly and incubate at 37°C for a certain period of time (preferably 20 minutes).

(4). Pour off the liquid in the wells, wash with washing solution (PBS, containing 0.05% Tween-20) several times, and the optimum amount of washing solution added to each well is 250-300 μl each time.

(5). Add tetramethylbenzidine (TMB), a chromogenic solution, and incubate at 37° C. for a certain period of time (preferably 15 minutes).

(6). Add 50 μl of stop solution (0.5-2M H ₂ SO ₄ ) to each well or instill one drop, shake gently for 10 minutes and read the OD value at 450 nm on an enzyme-linked instrument.

(7). Calculate the cardiac troponin I content of the sample to be tested according to the standard curve.

Option II:

(1). Add a certain amount of Ab1 and Ab2 to the 96-well enzyme-linked plate or enzyme-linked strip to pre-coat, incubate at 37°C for a certain period of time (preferably 60 minutes) or overnight at 4°C, with 0.5-1% albumin or other blocking solutions Closed and ready for use.

(2). The following steps are the same as (3) to (7) in Scheme 1.

third solution:

(1). Same as (1) in scheme two.

(2). The known cardiac troponin I standard substance or the sample to be tested in the hole when measuring (the known cardiac troponin I standard substance can also be pre-frozen in the set standard well, and the standard diluent is added during detection. Just dissolve), then add a certain amount of the mixed reaction solution of Ab3 and S-POD, shake lightly and incubate at 37°C for a certain period of time (preferably 20 minutes).

(3). The following steps are the same as (3) to (7) in Scheme 1.

In the method described above, the pH of the reaction solution is between 6.5 and 7.5, preferably 6.8 to 7.2. The pH of the lotion used is 6.5-7.5, preferably 6.8-7.2; the temperature is 2-37°C, preferably lower than 15°C; the lotion is washed 3-6 times, preferably 5 times.

The composition and operating instructions of the three-antibody method cardiac troponin I rapid quantitative assay kit processed according to the first plan:

Kit composition

1. One enzyme-linked rack.

2. There are 12 enzyme-linked strips, including 6 standard strips and 6 sample strips (two standard strips and two sample strips for trial products). The first well of the standard strip is the blank control, the 2nd to 6th wells are the standard points (the concentrations of cardiac troponin I are 3.2, 1.6, 0.8, 0.4, 0.2 ng/ml, which have been freeze-dried in the wells), and the 7th well is Sample well, can be used for emergency or routine case detection. The 2nd to 7th holes of the sample strip are the detection sample holes. The quantity ratio of standard strips and sample strips can be specially ordered according to the needs of users, and it is still based on the number of wells measured, and there is no additional charge.

3. Standard diluent 10ml (white bottle with blue cap).

4, 4. Lotion 200ml (large bottle).

5. Reaction solution 10ml (white bottle with red cap).

6. 6. Chromogenic solution A (white bottle with black cap).

7. Chromogenic solution B 20ml (white bottle with purple cap).

8. Stop solution 10ml (dropping bottle).

9. One instruction manual.

Operating Instructions

1. Principle

Using three high-specificity and high-sensitivity anti-human cardiac troponin I monoclonal antibodies, two of which are capture antibodies and one is a labeled antibody, combined with the streptavidin-biotin amplification system, the principle of ELISA is applied Detect the content of cardiac troponin I (cTnI) in human blood or other samples, and it is used for the diagnosis of myocardial injury caused by various diseases.

2. Sample collection and processing

Take 2ml of blood in dry tube or heparin anticoagulant tube. The drying tube needs to be placed at room temperature for 30 minutes and then centrifuged (2000rpm, 5 minutes), and the serum is taken for detection. The heparin anticoagulant tube can be centrifuged immediately (same as above), and the plasma is taken for testing. If the sample needs to be stored for a long time, the plasma or serum should be separated and stored below -20°C.

3. Instrument

Enzyme-linked instrument Micro-sampler 37℃ constant temperature water bath

4. Operation steps

(1) The kits used recently can be thawed at 2-8°C one day in advance or quickly thawed at room temperature. Before the detection, take out the enzyme-linked strips used, open and remove the sealing film of the seventh hole of the standard strip, and then press it onto the enzyme-linked frame for detection. Take 6ml of the washing solution and add it to the reaction solution bottle (red cap) and mix well (do not freeze and thaw again, store at 2-8°C after use).

Note: ① After the kit is thawed, store it at 2-8°C and use it up within 2-3 weeks. Avoid repeated freezing and thawing;

② Do not unpack the enzyme-linked strips that are not used for this test;

③ Keep the reagents at 2-8°C after application.

(2) Add 100 μl of standard diluent to wells 1 to 6 of the standard strip.

Add 100 μl of plasma or serum to the 7th well of the standard strip and the 2nd to 7th detection wells of the sample strip.

(3) Add 50 μl of reaction solution to each well, shake for 10-30 seconds, and place in a 37°C water bath for 20 minutes (the bottom of the enzyme-linked strip touches the water surface or is partially immersed in water). Note: The time interval between adding samples in steps (2) and (3) is less than 3 minutes.

(4) Pour off the liquid in the well, and wash with the lotion 5 times. The optimum amount of lotion added to each well is 250-300μl. Water quality cannot be guaranteed).

(5) Take the required chromogenic solution A and chromogenic solution B at a volume ratio of 1:20 and mix thoroughly, add 150 μl to each well, and bathe in water at 37°C for 15 minutes.

(6) Add 50 μl of stop solution to each well or instill one drop, take the first well of the standard strip as the blank control, shake gently for 10 minutes and read the OD value at 450 nm on an enzyme-linked instrument.

5. Calculation results

Standard calculation: According to the OD values of standard wells 2 to 6 and the corresponding cTnI concentrations (3.2, 1.6, 0.8, 0.4, 0.2ng/ml) as the X-axis and Y-axis respectively, calculate the linear regression equation Y=aX±b, Y is the cTnI concentration, X is the OD value), and the formula is substituted into the sample OD value to calculate the cTnI concentration (ng/ml) of the sample, which can be automatically processed by a computer or an enzyme-linked analyzer if conditions permit.

Simple calculation: select the sample to be tested and the standard point OD similar to the direct comparison calculation

Sample cTnI concentration (ng/ml)=(standard point concentration×OD value of sample to be tested)/standard point OD value.

6. Result judgment

Statistical analysis of 200 cases of healthy people shows that the normal reference value is <0.15ng/ml, ≥0.15ng/ml indicates elevated cTnI, 0.15～0.5ng/ml indicates mild myocardial injury, and ≥0.5ng/ml indicates acute myocardial infarction or Severe heart muscle damage. <0.15ng/ml is normal. The detection range is 0.05-4.0ng/ml, and the minimum detection value is 0.05ng/ml. If the result is >4.0ng/ml, it needs to be diluted with negative serum to an appropriate multiple for detection.

Claims (3)

1. A method based on three strains of anti-human cardiac troponin I monoclonal antibodies to establish rapid quantitative determination of cardiac troponin I content is characterized in that two strains of monoclonal antibodies acting on cardiac troponin I are The capture antibody uses another monoclonal antibody acting on cardiac troponin I as the labeled antibody, combined with the streptavidin-biotin amplification system, and uses the principle of enzyme-linked immunosorbent immunoassay to establish a rapid quantitative detection method for cardiac troponin I . 2. According to the method according to claim 1, the two capture antibodies are referred to as Ab1 and Ab2 respectively, and after being linked with biotin or long-chain biotin, they are referred to as Ab1-Bio and Ab2-Bio respectively; the other labeled antibody is referred to as Ab2-Bio for short. Ab3, linked with horseradish peroxidase, referred to as Ab3-POD, linked with biotin or long-chain biotin, referred to as Ab3-Bio; streptavidin or avidin referred to as S; streptavidin spicy The root peroxidase is referred to as S-POD; the cardiac troponin I antigen includes the known cardiac troponin I standard, quality control and the cardiac troponin I in the sample to be tested, referred to as cTnI; the third antibody method The reaction pathway of the rapid quantitative determination of cardiac troponin I method specifically includes the following schemes: Option One: (1) 96-well enzyme-linked plates, enzyme-linked strips or other solid supports are linked to S, and albumin or other blocking solutions are blocked; (2) Ab1-Bio and Ab2-Bio bind to S, cTnI binds to Ab1-Bio and Ab2-Bio, cTnI binds to Ab3-POD to form

reaction chain; (3) POD causes the chromogenic substrate to develop color or the chemiluminescent substrate to emit light, and then is measured by a corresponding instrument, and the cardiac troponin I content of the sample to be tested is calculated according to the standard curve; Option II: (1) 96-well enzyme-linked plates, enzyme-linked strips or other solid supports are connected to Ab1 and Ab2, and albumin or other blocking solutions are blocked; (2) Ab1 and Ab2 bind to cTnI, and cTnI binds to Ab3-POD to form

reaction chain; (3) POD causes the chromogenic substrate to develop color or the chemiluminescent substrate to emit light, and then is measured by a corresponding instrument, and the cardiac troponin I content of the sample to be tested is calculated according to the standard curve; third solution: (1) 96-well enzyme-linked plates, enzyme-linked strips or other solid supports are connected to Ab1 and Ab2, and albumin or other blocking solutions are blocked; (2) Ab1+Ab2 binds to cTnI, cTnI binds to Ab3-Bio, Ab3-Bio binds to S-POD to form

reaction chain; (3) POD causes the chromogenic substrate to develop color or the chemiluminescent substrate to emit light, and then is measured by a corresponding instrument, and the content of cardiac troponin I in the sample to be tested is calculated according to the standard curve. 3. The method according to claim 2, characterized in that the chromogenic substrate is tetramethylhydrazine TMB.