Protein Chip, Kit and Preparation Method thereof for Detecting Abnormal Decarboxy Prothrombin in Serum

Abstract

A protein chip, kit and preparation method thereof for detecting abnormal decarboxy prothrombin in serum, a substrate carrier of the protein chip is provided with a plurality of detection subareas; wherein each detection subarea is used for detecting a serum sample, and is internally provided with a detection-spot area and a control-spot area, and the detection-spot area has a detection spot formed by spraying a trace amount of a DCP-specific antibody, the control-spot area has a control spot formed by spraying a bovine serum albumin; all the detection spots within one of the detection-spot area have the same material concentration, to form each of the detection spots, a total volume of 3-5 nl of the DCP-specific antibody with a concentration of 3-5 mg/mL is used; each of the detection spots is formed by a non-contact spotter, performing 6-10 spot sprays and spraying 300-500 pL in each spray.

Claims

1. A high-throughput protein chip for detecting abnormal decarboxy prothrombin in serum, characterized in that: a plurality of detection subregions are provided on a substrate carrier of the protein chip, and each of the detection subregions is configured to detect a serum sample; each of the detected subregions is provided with a detection spot area and a control spot area, the detection spot area has a detection spot formed by spraying a trace of DCP specific antibody, and the control spot area has a control spot formed by spraying bovine serum albumin; substances on all the detection spots have the same concentration in the same detection spot area; and the total amount of DCP specific antibody for forming each of the detection spots is 3-5 nl, and the concentration thereof is 3-5 mg/ml, each of the detection spots is formed by a non-contact point sampler in 6-10 times and spraying 300-500 pl each time, and the diameter of the detection spot is 0.5-1 mm.

2. The protein chip of claim 1, characterized in that each of the detection spot areas includes 4-8 mutually separated detection spots arranged in a row, and the control spot area includes 4-8 mutually and independently separated control spots arranged in a row; the detection spots and the control spots are arranged in two parallel columns.

3. The protein chip of claim 2, characterized in that the length, width and thickness of the chip are 76.4 mm, 25.2 mm and 1 mm; and 10 detection subregions are provided on the substrate carrier.

4. The protein chip of claim 2, characterized in that a protrusion is provided between the detection subregions as a physical partition.

5. The protein chip of claim 1, characterized in that the specific antibody of the DCP is a murine anti-human DCP.

6. A method of preparing the protein chip for detecting abnormal decarboxy prothrombin of any of claims 1 to 5, characterized in that: the total amount of DCP-specific antibody for forming each of the detection spots is 3 nl, and the concentration thereof is 4 mg/ml; a detection spot is formed by spraying the DCP-specific antibody in 6-10 times and 300-500 pl each time.

7. The preparation method of claim 6, characterized in that the temperature of the DCP-specific antibody is 4-8 C. during the spraying process.

8. The preparation method of claim 6 or 7, characterized in that spraying the antibody is performed by the non-contact point sampler.

9. (canceled)

10. A method for detecting abnormal decarboxy prothrombin in serum, characterized in that the use of protein chip of claims 1-6 comprises the steps of: diluting the serum samples to be tested and dropping onto the detection subregion of the protein chip; after incubation, washing the detection subregion with PBST to remove nonspecific binding substances. adding HRP-labeled prothrombin antibody diluted with PBS; after incubation, washing with PBST to remove the nonspecific binding substances; and adding HRP substrate luminescent solution, scanning the protein chip by a chemiluminescence scanner to obtain DCP luminescence pixel values in serum samples to be measured after dilution respectively; wherein the incubation refers to incubating at 37 C. for 30 minutes.

11. The preparation method of claim 7, characterized in that spraying the antibody is performed by the non-contact point sampler.

12. A method for detecting abnormal decarboxy prothrombin in serum, characterized in that the use of protein chip of claim 2 comprises the steps of: diluting the serum samples to be tested and dropping onto the detection subregion of the protein chip; after incubation, washing the detection subregion with PBST to remove nonspecific binding substances. adding HRP-labeled prothrombin antibody diluted with PBS; after incubation, washing with PBST to remove the nonspecific binding substances; and adding HRP substrate luminescent solution, scanning the protein chip by a chemiluminescence scanner to obtain DCP luminescence pixel values in serum samples to be measured after dilution respectively; wherein the incubation refers to incubating at 37 C. for 30 minutes.

13. A method for detecting abnormal decarboxy prothrombin in serum, characterized in that the use of protein chip of claim 3 comprises the steps of: diluting the serum samples to be tested and dropping onto the detection subregion of the protein chip; after incubation, washing the detection subregion with PBST to remove nonspecific binding substances. adding HRP-labeled prothrombin antibody diluted with PBS; after incubation, washing with PBST to remove the nonspecific binding substances; and adding HRP substrate luminescent solution, scanning the protein chip by a chemiluminescence scanner to obtain DCP luminescence pixel values in serum samples to be measured after dilution respectively; wherein the incubation refers to incubating at 37 C. for 30 minutes.

14. A method for detecting abnormal decarboxy prothrombin in serum, characterized in that the use of protein chip of claim 4 comprises the steps of: diluting the serum samples to be tested and dropping onto the detection subregion of the protein chip; after incubation, washing the detection subregion with PBST to remove nonspecific binding substances. adding HRP-labeled prothrombin antibody diluted with PBS; after incubation, washing with PBST to remove the nonspecific binding substances; and adding HRP substrate luminescent solution, scanning the protein chip by a chemiluminescence scanner to obtain DCP luminescence pixel values in serum samples to be measured after dilution respectively; wherein the incubation refers to incubating at 37 C. for 30 minutes.

15. A method for detecting abnormal decarboxy prothrombin in serum, characterized in that the use of protein chip of claim 5 comprises the steps of: diluting the serum samples to be tested and dropping onto the detection subregion of the protein chip; after incubation, washing the detection subregion with PBST to remove nonspecific binding substances. adding HRP-labeled prothrombin antibody diluted with PBS; after incubation, washing with PBST to remove the nonspecific binding substances; and adding HRP substrate luminescent solution, scanning the protein chip by a chemiluminescence scanner to obtain DCP luminescence pixel values in serum samples to be measured after dilution respectively; wherein the incubation refers to incubating at 37 C. for 30 minutes.

16. A method for detecting abnormal decarboxy prothrombin in serum, characterized in that the use of protein chip of claim 6 comprises the steps of: diluting the serum samples to be tested and dropping onto the detection subregion of the protein chip; after incubation, washing the detection subregion with PBST to remove nonspecific binding substances. adding HRP-labeled prothrombin antibody diluted with PBS; after incubation, washing with PBST to remove the nonspecific binding substances; and adding HRP substrate luminescent solution, scanning the protein chip by a chemiluminescence scanner to obtain DCP luminescence pixel values in serum samples to be measured after dilution respectively; wherein the incubation refers to incubating at 37 C. for 30 minutes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 is a structure diagram of DCP protein chip, including 1substrate carrier, 2detection subregion, 3detection spot, 4control spot, 5physical partition.

[0036] FIG. 2 is a flow chart of protein chip by DCP antibody sandwich method.

[0037] FIG. 3 is a scanning chart of liver cancer and normal serum samples detected by the DCP antibody point sample chip, including 1-8: liver cancer serum; 9 healthy control serum; 10 blank control.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] The invention is described in details below in combination with the embodiments, but the scope of the invention is not limited. Unless otherwise specified, the operations in the following embodiments are conventional, and the reagents used are commercially available.

Embodiment 1. Preparation and Validation of Protein Chips 1-4

Main Equipment

[0039] Chemiluminescence scanner, by GE USA.

Main Reagents and their Sources

[0040] Murine monoclonal antibody DCP antibody (FUJIREBIO INC, Japan), aldehyde chip (Shanghai BaiO Technology Co., Ltd.), HRP-labeled prothrombin rabbit antibody (Fitzgerald Inc, USA), HRP chemiluminescence substrate liquid A and liquid B mixed in 1:1 and freshly prepared. (Millipore Corporation, USA)

[0041] Abnormal prothrombin standard product: FUJIREBIO INC, Japan.

[0042] Reagents and instruments used in the experiment: DCP antibody (FUJIREBIO INC, Japan); HRP-labeled rabbit antibody (Fitzgerald Inc., USA); Chemiluminescence scanner (GE, USA)

[0043] PBS formula: 8 g sodium chloride (NaCl), 0.2 g potassium chloride (KCl), 1.44 g disodium hydrogen phosphate (Na2HPO4), 0.24 g potassium dihydrogen phosphate (KH2PO4), pH 7.4, constant volume 1 L

[0044] PBST formula: PBS, 1 L+tween-20, 1 ml

[0045] Substrate carrier 1 is an aldehyde chip (Shanghai BaiO Technology Co., Ltd.), and each chip contains 10 detection squares (detection subregions); one serum sample is detected in each square, and 10 serum samples are detected at a time. The lengthwidththickness of each square is 76.425.21 mm.

Step 1. Preparing Chips 1-4

[0046] In each of the detection subregions 2, DCP antibodies of mice were successively added on the chip, and the DCP antibody point sample concentration was 4 mg/ml.

[0047] Each of the detection subregions 2 was provided with a detection spot area and a control spot area. The detection spot area had a detection spot 3 formed by spraying a trace of DCP specific antibody, and the control spot area had a control spot 4 formed by spraying bovine serum albumin at the concentration of 4 mg/ml.

[0048] The scheme of pointing the capture antibody was as follows:

Chip 1: in each detection grid, the concentration of murine DCP monoclonal antibody was 4 mg/ml, and 10 ul antibody was applied for each detection spot; 10% bovine serum albumin (BSA) was used as a negative control, 10 ul for each control spot.
Chip 2: the concentration of murine DCP monoclonal antibody was 4 mg/ml, and 5 ul antibody was applied for each detection spot; 10% bovine serum albumin (BSA) was used as a negative control, 5 ul for each control spot.
Chip 3: the concentration of murine DCP monoclonal antibody was 4 mg/ml, and 1 ul antibody was applied for each detection spot; 10% bovine serum albumin (BSA) was used as a negative control, 1 ul for each control spot.
Chip 4: the point sample concentration of murine DCP monoclonal antibody was 4 mg/ml, and 100 nl antibody was applied for each detection spot; 10% bovine serum albumin (BSA) was used as a negative control, 100 nl for each control spot.

[0049] Conventional contact pointing method was adopted.

Step 2. Use of Standard Curve and Standard Product Verification Chip

[0050] Preparation of Standard Products:

[0051] Standard abnormal prothrombins and serum for testing with a series of gradient concentration were respectively added to normal serum:

[0052] Standard serum: 0 mAU/ml, 5 mAU/ml, 10 mAU/ml, 20 mAU/ml, 40 mAU/ml, 80 mAU/ml, 160 mAU/ml, 320 mAU/ml, 640 mAU/ml):

[0053] Serum for testing: 0 mAU/ml, 2 mAU/ml, 4 mAU/ml, 8 mAU/ml, 16 mAU/ml, 35 mAU/ml, 50 mAU/ml, 70 mAU/ml, 90 mAU/ml, 120 mAU/ml, 150 mAU/ml, 300 mAU/ml):

Operation Procedure of Protein Chip:

[0054] 10 ul sample under test (which can be obtained by diluting 2.5 ul serum 4 times with PBS) was added to each of the chip detection subregion for incubation at 37 C. for 30 minutes, so that the DCP in the serum and DCP antibodies formed antigen-antibody complexes by means of antigen and antibody binding features.

[0055] PBST was applied for washing four times to remove nonspecific binding, and the PBS diluted HRP-labeled rabbit primary antibody was added for incubation at 37 C. for 30 minutes. Rabbit antibody was binded with antigen to form DCP antibody-DCP-rabbit HRP-labeled prothrombin antibody complexes.

[0056] PBST was applied for washing four times to remove nonspecific binding, HRP luminous substrate was added for incubation at 37 C. for 30 minutes, and detection spot pixel values were obtained by scanning of a chemiluminescence scanner.

[0057] The chemiluminescence pixels on the solid phase carrier are positively correlated with the amount of antigen detected in the specimen. The chip capture point-sample antibody (murine primary antibody) and the antibody for detection (rabbit primary antibody) were respectively taken from animals of different species to target different epitopes of prothrombin. As shown in FIG. 2, a flow chart of protein chip by antibody sandwich method was provided.

[0058] With the concentration of the standard serum as the horizontal coordinate and pixel values as the vertical coordinate, the standard curve was drawn and the regression equation of the standard curve was generated.

[0059] The square value R.sup.2 of the correlation coefficient of the regression equation of the standard curve obtained by chips 1-4 was between 0.55 and 0.6, and the linear correlation between the concentration of the standard product and the pixel value was poor.

[0060] Another batch of standard serum for testing with different concentrations was used to verify the standard curve, and it was found that the results obtained were significantly different from the concentration of standard serum for testing itself, and the test values of the standard serum for testing with a concentration lower than 80 mAU/ml were repeatedly lower than 30 mAU/ml, which was lower than the diagnostic threshold value of 40 mAU/ml, showing a false negative.

Step 3. These chips was validated with clinical serum having a given DCP content, with the serum samples as follows:

[0061] 35 samples of liver cancer serum are collected from the specimen bank of You'an Hospital, Capital Medical University. It is known that the abnormal decarboxy prothrombin's concentration of these samples is higher than the diagnostic threshold value 40 mAU/ml (1 mAU/ml=1 ng/ml).

[0062] 28 normal healthy human serum samples; the concentration of abnormal decarboxy prothrombin is lower than the diagnostic threshold value.

[0063] 7 blank controls (blank control is 1PBS), and the test results are statistically shown in table 1 below:

TABLE-US-00001 TABLE 1 Detection results of chips 1-4 Chip 1 Chip 2 Chip 3 Chip 4 DCP 40 DCP < 40 DCP 40 DCP < 40 DCP 40 DCP < 40 DCP 40 DCP < 40 mAU/ml mAU/ml mAU/ml mAU/ml mAU/ml mAU/ml mAU/ml mAU/ml Liver 20 15 21 14 24 11 24 11 cancer serum (35) Healthy 0 28 0 28 0 28 0 28 serum (28) Blank 0 7 0 7 0 7 0 7 control (7)
As can be seen from the results in table 1, DCP is not detected in 7 blank control samples. No DCP is detected in 28 healthy serum samples. Only 20-24 of the 35 samples of HCC serum are detected with DCP higher than the critical value, that is, these chips have detection sensitivity of (20-24)/35=57.1%-68.6%, and the proportion of missed detection is very high.

Embodiment 2. Preparation and Validation of Chips 5-7

Step 1: Preparing the Chips

[0064] Chip 5: the point sample concentration of murine DCP monoclonal antibody was 4 mg/ml, and 10 nl antibody was applied for each detection spot; 10% bovine serum albumin (BSA) was used as a negative control, 10 nl for each control spot;

[0065] Control chip 6: the concentration of murine DCP monoclonal antibody was 4 mg/ml, and 5 nl antibody was applied for each detection spot; 10% bovine serum albumin (BSA) was used as a negative control, 5 nl for each control spot.

[0066] Control chip 7: the concentration of murine DCP monoclonal antibody was 4 mg/ml, and 2 nl antibody was applied for each detection spot; 10% bovine serum albumin (BSA) was used as a negative control, 2n1 for each control spot;

[0067] The control chips 5-7 adopted a non-contact point sampler with picoliter precision, i.e. Nano-Plotter NP2.1 of GESIM Company, Germany, with a piezoelectric point sampler needle, and the temperature of the cabin inside the point sampler controlled to be within 4-8 degrees Celsius.

Step 2: Procedure and Experimental Reagent Same as Embodiment 1.

[0068] The square value R.sup.2 of the correlation coefficient of the regression equation of the standard curve obtained by chips 5-7 was gradually increased to 6.5-7.5, indicating that the linear relationship between the concentration of the standard product and the pixel value was significantly improved.

[0069] Another batch of standard serum for testing with different concentrations was used to verify the standard curve, and it was found that the difference between the obtained results and the test standard serum concentration was reduced, and the minimum detection limit was up to 4 mAU/ml.

Step 3. Chip Validation by Given Clinical Serum

[0070] Serum is the same as step 3 of Embodiment 1, and the test results are statistically shown in table 2.

TABLE-US-00002 TABLE 2 Detection results of chip 5-7 Chip 8 Chip 9 DCP 40 DCP < 40 DCP 40 DCP < 40 mAU/ml mAU/ml mAU/ml mAU/ml Liver cancer 27 8 30 5 serum (35) Healthy serum 0 28 0 28 (28) Blank control 0 7 0 7 (7)
As can be seen from the results in table 2, DCP is not detected in 7 blank control samples. No DCP is detected in 28 healthy serum samples. DCP is detected in 27-30 of the 35 samples of liver cancer serum higher than the critical value, that is, these chips have detection sensitivity of (27-30)/35=77.1%-85.7%, and the detection rate is significantly improved, but there is still a considerable proportion of missed detection. Combined with the verification of the test standard serum in step 2, it can be preliminarily determined that the detection is missed due to insufficient detection limit.

[0071] In parallel embodiments, the point volume is further reduced, but no improvement in the linear relationship between the concentration of the standard substance and the pixel value is further observed in the standard curve obtained from preparation. The detection of test standard serum and clinical serum is not significantly improved compared with chips 5-7.

Embodiment 3. Fabrication and Verification of Chips 8-10

[0072] With multi-dimensional attempts such as antibody purification and reagent formulation to detection temperature and time, the inventor has not observed further improvement in the linear relationship between standard substance concentration and pixel value, and the detection rate is difficult to break through 90%. In the accidental experiment, the inventor tried to divide the antibody into several parts and repeat pointing by several times on one spot, the resulted chip unexpectedly jumped to 0.9-0.95 in the correlation coefficient R.sup.2 of the standard curve and 92-95% in the detection rate relative to the other conditions unchanged in Embodiment 2, as follows:

Step 1. Fabrication of Chips 8 and 9

[0073] Chip 8: the spot concentration of murine DCP monoclonal antibody was 4 mg/ml, and 3 nl antibody was applied for each detection spot; 10% bovine serum albumin (BSA) was used as a negative control, 5 nl for each control spot. The non-contact point sampler with picoliter precision, i.e. Nano-Plotter NP2.1 of GESIM Company, Germany was adopted, with a piezoelectric point sampler needle, and the temperature of the point sampler inner chamber controlled to 4-8 degrees Celsius. Each point was sprayed with 300 pl-500 pl at a time for 3-5 times, with a total of 3 nl antibodies sprayed, and the diameter of the detection spot was 0.5-1 mm. The chip's structure was as shown in FIG. 1.

[0074] Chip 9: the point sample concentration of murine DCP monoclonal antibody was 4 mg/ml, and 5 nl antibody was detected for each spot; 10% bovine serum albumin (BSA) was used as a negative control, 5 nl for each control spot. The non-contact point sampler with picoliter precision, i.e. Nano-Plotter NP2.1 of GESIM Company, Germany was adopted, with a piezoelectric point sampler needle, and the temperature of the point sampler inner chamber controlled to 4-8 degrees Celsius. Each point was sprayed with 300 pl-500 pl at a time for 6-10 times, with a total of 3 nl point-sample antibodies sprayed, and the diameter of the detection spot was 0.5-1 mm.

Step 2. Procedure and Experimental Reagent Same as Embodiment 1

[0075] The square value R.sup.2 of the correlation coefficient of the regression equation of the standard curve obtained by chip 8 or 9 jumped to 9.2-9.5, and the linear relationship between the concentration of the standard product and the pixel value was significantly improved.

[0076] Another batch of test standard serum with different concentrations was used to verify the standard curve, and it was found that the result obtained was only slightly different from the concentration of the test standard serum itself, which was within the allowable range of error, wherein the test result of the test standard serum with a concentration of 60 mAU/ml was lower than the defined value, showing a false negative.

Step 3. Chip Validation by Given Clinical Serum

[0077] Serum is the same as step 3 of Embodiment 1, and the test results are statistically shown in table 3.

TABLE-US-00003 TABLE 3 Detection results of chips 8 and 9 Chip 8 Chip 9 DCP 40 DCP < 40 DCP 40 DCP < 40 mAU/ml mAU/ml mAU/ml mAU/ml Liver cancer 33 2 34 1 serum (35) Healthy serum 0 28 0 28 (28) Blank control 0 7 0 7 (7)

[0078] As can be seen from the results in table 1, DCP is not detected in 7 blank control samples. No DCP is detected in 28 healthy serum samples. DCP is detected in 33 of the 35 samples of HCC serum higher than the critical value, that is, the detection sensitivity of these chips (33-34)/35=94.3%-97.1%, and the detection rate is almost 100%.

The optimal chip scheme is as follows:

[0079] In each detection grid (detection subregion), DCP antibody of rats was successively added on the chip. The DCP antibody sample concentration was 4 mg/ml, and four detection spots were added in a row. 10% bovine serum albumin (BSA) was used as a negative control, and also added four times to form control spots;

[0080] The non-contact point sampler with picoliter precision, i.e. Nano-Plotter NP2.1 of GESIM Company, Germany was adopted, with a piezoelectric point-sample needle, and the temperature of the inner chamber of the point-sample instrument controlled to 4-8 degrees Celsius. Each point was sprayed with 300 pl-500 pl at a time for 6-10 times, with a total of 3-5 nl point-sample antibody sprayed. Each detection area was provided with 4 detection points, requiring 12 nl capture antibody, and the diameter of detection spot was 0.5-1 mm.

Experiment Application Effect of Optimal Chip Scheme

[0081] The correlation coefficient R.sup.2 of the standard curve regression equation is 0.98.

Serum Samples:

[0082] 8 samples of liver cancer serum are collected from the specimen bank of Beijing You'an Hospital Affiliated to Capital Medical University (the known abnormal decarboxy prothrombin concentration is higher than the diagnostic threshold value 40 mAU/ml (1 mAU/ml=1 ng/ml).

[0083] 1 normal healthy human serum;

[0084] 1 blank control (blank control is 1PBS).

Detection Results of the Chip:

[0085] No abnormal prothrombin is detected in the blank control samples and healthy control samples: indicating that the chip used in this experiment is effective. There is no abnormal prothrombin in normal people and no prothrombin is detected in healthy serum. This means that the false positive detected by the chip and method provided by the invention is 0, and the detection result can accurately distinguish the serum of liver cancer from the normal serum.

[0086] The results of spot scanning and calculation of 8 samples of HCC serum show positive, indicating that abnormal decarboxy prothrombin is found in 8 samples of HCC serum, especially in patients with advanced HCC; the detection spots in the 8 positive detection subregions show different degrees of brightness, indicating that the 8 liver cancer serum contain different concentrations of abnormal decarboxy prothrombin, as shown in FIG. 3, wherein 3 and 5 show weak positive, and the detection spot brightness is weak, while 1, 2, 4, 6, 7, 8 show strong positive. The detection results indicate that the protein chip of the invention can accurately detect the serum of liver cancer and semi-quantitatively detect the concentration of abnormal decarboxy prothrombin in the serum. The above data shows that the chip and method of the invention are accurate and reliable.

[0087] In addition, the sensitivity and specificity of serum tests in dozens of outpatients with liver cancer are more than 90%.

The Kit of the Invention:

[0088] Including Chips shown in Embodiment 3, wherein a protrusion is provided between the preferable detection subregions 2 in some chips as a physical partition 5; and

HRP-labeled prothrombin polyclonal antibody, and HRP chemiluminescent substrate liquid; the
HRP-labeled prothrombin polyclonal antibody is a rabbit antibody, and the DCP-specific antibody fixed on the detection spot is originated from a different species.