Abstract:
A detection method for cancer is provided. Magnetic carbon beads are used. The carbon beads are highly specified to a cancer. Surface area of grafted antigen are broadened by grafting functional molecules. Number of antigen is increased on the surface. Thus, the present invention improves sensitivity and accuracy of disease detection and greatly saves cost. The present invention can be applied for sample purification or massive disease detection.

Description:
TECHNICAL FIELD OF THE INVENTION 
       [0001]    The present invention relates to a disease detection method; more particularly, relates to using magnetic beads having high specificity to a disease and grafting functional molecule on the magnetic beads to enhance surface for grafting antigen, where efficiency of disease detection using the antigen grafted on the nano beads is obviously improved; and, thus, the present invention can be applied in treatments using disease purification and in clinical regular mass in-vitro quantitative cancer diagnoses. 
       DESCRIPTION OF THE RELATED ART 
       [0002]    A prior art, enzyme-linked immunosorbent assay (ELISA), is shown in  FIG. 9 . A layer of an antigen  9  is only coated at bottom of a container  8 . Only a limited part of surface of the antigen  9  can be used. Sensitivity of detection cannot be effectively enhanced. Hence, the prior art does not fulfill all users&#39; requests on actual use. 
       SUMMARY OF THE INVENTION 
       [0003]    The main purpose of the present invention is to use magnetic beads having high specificity to a disease and use grafting functional molecule on the magnetic beads to enhance surface for grafting antigen, where efficiency of disease detection using the antigen grafted on the nano beads is obviously improved; and, thus, the present invention can be applied in treatments using disease purification and in clinical regular mass in-vitro quantitative cancer diagnoses. 
         [0004]    To achieve the above purpose, the present invention is a method of detection using a nano carbon carrier modified by ionizing radiation, comprising steps of: (a) obtaining a solution having a plurality of magnetic beads and adding an antigen into the solution to combine the antigen with the magnetic beads, where the magnetic bead is a radioactive nano carrier, comprising a nano bead; a plurality of grafting functional molecules distributed on the nano carbon bead; and a magnetic material distributed on the nano bead; (b) gathering the magnetic beads by using a magnetic field and removing un-reacted part of the antigen and extra part of the solution by an absorber to obtain the solution having the magnetic beads all combined with the antigen; (c) adding an under-testing sample to the solution having the magnetic beads combined with the antigen and processing a specific reaction between a primary antibody of the under-testing sample and the antigen of the magnetic beads to combine the primary antibody with the antigen and gathering the magnetic beads by using a magnetic field to separate and remove uncombined part of the under-testing sample; and (d) adding a secondary antibody to the solution to combine the secondary antibody with the primary antibody and processing a content detection through combining the secondary antibody with a radioactive isotope, an enzyme or a nucleic acid molecule (DNA). Accordingly, a novel method of detection using a nano carbon carrier modified by ionizing radiation is obtained. 
     
    
     
       BRIEF DESCRIPTIONS OF THE DRAWINGS 
         [0005]    The present invention will be better understood from the following detailed description of the preferred embodiment according to the present invention, taken in conjunction with the accompanying drawings, in which 
           [0006]      FIG. 1  is the flow view showing the preferred embodiment according to the present invention; 
           [0007]      FIG. 2  is the view showing the step (a); 
           [0008]      FIG. 3  is the view showing the step (b); 
           [0009]      FIG. 4  is the view showing the step (c); 
           [0010]      FIG. 5  is the view showing the step (d); 
           [0011]      FIG. 6  is the view showing the step (e); 
           [0012]      FIG. 7  is the view showing the step (f); 
           [0013]      FIG. 8  is the view showing the step (g); and 
           [0014]      FIG. 9  is the view of the prior art. 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0015]    The following description of the preferred embodiment is provided to understand the features and the structures of the present invention. 
         [0016]    Please refer to  FIG. 1  to  FIG. 8 , which are a flow view showing a preferred embodiment according to the present invention and views showing step (a) to step (g). As shown in the figures, the present invention is a method of detection using a nano carbon carrier modified by ionizing radiation, comprising the following steps: 
         [0017]    (a) Grafting antigen on magnetic beads  11 : In  FIG. 2 , a plurality of magnetic beads  3  is provided in a solution  20  contained in a container  2  and an antigen  40  is added into the solution  20  to be grafted with the magnetic beads  3 , where the magnetic bead is a radioactive nano carrier, comprising a nano bead  31 ; a plurality of grafting functional molecules  32  distributed on the nano bead  31 ; and a magnetic material  33  distributed on the nano bead  31 . 
         [0018]    (b) Gathering beads and washing  12 : In  FIG. 3 , a magnet  21  is put under the container  2  for gathering the magnetic beads  3  by using a magnetic field of the magnet  21 . Un-reacted part of the antigen  40  and the solution  20  is removed by an absorber (i.e. needle)  5  to form the magnetic beads  3  all grafted with the antigen  40 . 
         [0019]    (c) Grafting antibody sample  13 : In  FIG. 4 , an under-testing sample is added into the solution  20  containing the magnetic beads  3  grafted with the antigen  40 . A specific reaction is processed between a primary antibody  41  in the under-testing sample  4  and the antigen  40  on the magnetic beads  3  for grafting the primary antibody on the antigen. Then, like what has been done in step (b), the magnetic beads  3  are gathered by using a magnetic field to separate and remove un-grafted part of the under-testing sample  4 . 
         [0020]    (d) Grafting secondary antibody  14 : In  FIG. 5 , a secondary antibody  42  is added to be grafted with the solution  20  having the magnetic beads  3  for processing a content detection. The content detection is done by grafting the secondary antibody  42  with a signal molecule  6 , like a radioactive isotope, an enzyme or a nucleic acid molecule (DNA), as described in step (e), step (f) and step (e). 
         [0021]    (e) Processing RIA  15 : In  FIG. 6 , radioimmunoassay (RIA) is processed by coupling a radioactive isotope  6   a  of iodine(I)-125 on the secondary antibody  42  for detecting a strength of gamma ray (γ-ray) radiated from I-125 to obtain a content of the primary antibody  41  in the under-testing sample  4 . 
         [0022]    (f) Processing luminescence analysis  16 : In  FIG. 7 , chemiluminescence immunoassay (CLIA) or enzyme-linked immunosorbent assay (ELISA) is processed by coupling a luminescent colorimetric enzyme  6   b  on the secondary antibody  42  to be reacted with a luminescence substrate for detecting a photon strength of the luminescence substrate to obtain a content of the primary antibody  41  in the under-testing sample  4 . 
         [0023]    (g) Processing immuno PCR  17 : In  FIG. 8 , immuno polymerase chain reaction (PCR) is processed by combining a biotin  61   c  on the secondary antibody  42 ; combining another biotin  61   c  on a nucleic acid molecule  6   c;  connecting the nucleic acid molecule  6   c  with the secondary antibody  42  through streptavidin  62   c ; processing PCR with a tag enzyme for forming magnified reaction signals; and separating the nucleic acid molecule  6   c  to obtain a content of the primary antibody  41  in the under-testing sample  4 . 
         [0024]    The nano bead  31  is a nano carbon bead; the grafting functional molecule  32  has a functional group formed through an acid-alkali treatment and ionizing radiation and the functional group is —COOH, —NH 2 , —SH, —OH, —COH or —COO—; and, the magnetic material  33  is a magnet powder made of iron (Fe), cobalt (Co), nickel (Ni) or iron (II, III) oxide, (Fe 3 O 4 ). 
         [0025]    Thus, a novel method of detection using a nano carbon carrier modified by ionizing radiation is obtained. 
         [0026]    On using the present invention, the magnetic beads  3  are used to combine the antigen  40  on surface of the nano beads  31  by the grafting functional molecule  32 . Then, by using the magnet  21  put under the container  2 , the magnetic material  33  of the magnetic beads  3  is reacted with the magnetic field of the magnet  21  to be attracted and moved toward the magnet  21  and, then, an absorber  5  is used to absorb extra part of the solution. Thus, un-reacted part of the antigen  40  can be easily removed by washing to form the magnetic beads  3  all combined with the antigen  40 . Then, the under-testing sample  4 , i.e. serum of a nasopharyngeal cancer patient, is added. By using the magnetic beads  3  having adhesion specificity to the cancer, only the primary antibody in the under-testing sample  4 , i.e. anti-EBV IgA, is grafted on the magnetic beads  3 . In the same way, a magnetic field is used to gather the magnetic beads  3  and the other part of the serum is separated and removed. Then, the magnetic beads  3  grafted with anti-EBV IgA is grafted with the secondary antibody  42 , i.e. anti-Human IgA. Again, a magnetic field is used to gather the magnetic beads  3  for separating and removing the other un-adhered part of the secondary antibody  42 . At last, the secondary antibody  42  can be grafted with one of three different signal molecule  6  for processing RIA, a luminescence analysis or immuno PCR. 
         [0027]    On processing RIA, the secondary antibody  42  is coupled with a radioactive isotope  6   a  of I-125. γ-ray will be radiated from I-125. By detecting a γ-ray strength through a γ-ray detector, a content of the primary antibody (anti-EBV IgA) in the under-testing sample  4  is obtained. This detection method has high accuracy and low cost. 
         [0028]    On processing luminescence analysis, the secondary antibody  42  is coupled with a luminescent colorimetric enzyme  6   b,  like horse radish peroxidase (HRP) or alkaline phosphatase (AP). The luminescent colorimetric enzyme  6   b  is reacted with a luminescence substrate. A photomultiplier tube (PMT) detector  7   b  is used to detect a photon strength to obtain a content of the primary antibody  41  in the under-testing sample  4 . This detection method obtains magnified signals for good accuracy; and, furthermore, can achieve convenience by using an automatic serum immuno analyzer. 
         [0029]    On processing immuno PCR, the secondary antibody  42  is grafted with the biotin  61   c.  At the same time, the nucleic acid molecule  6   c  is grafted with another biotin  61   c . Streptavidin  62   c  has strong adhesion to biotin and can be grafted with four biotins. Therefore, streptavidin  62   c  is used to connect the nucleic acid molecule  6   c  with the secondary antibody  42 . Then, PCR is processed with a tag enzyme for obtaining magnified reaction signals. Then, gel electrophoresis  7   c  is used to separate the nucleic acid molecule  6   c  for obtaining a content of the primary antibody  41  in the under-testing sample  4 . This detection method can magnify reaction signals for 1 billion times after 30 cycles and, thus, is the most sensitive detection method. 
         [0030]    Hence, the present invention uses magnetic beads for obtaining high specificity to a disease, where grafting functional molecule is used to enhance surface for grafting antigen. Efficiency of ELISA for the antigen grafted on the nano beads is obviously improved. Consequently, the present invention can be applied in treatments using disease purification and in clinical regular mass in-vitro quantitative cancer diagnoses. 
         [0031]    To sum up, the present invention is a method of detection using a nano carbon carrier modified by ionizing radiation, where magnetic beads having high specificity to a disease is used and grafting functional molecule is used to enhance surface for grafting antigen; efficiency of detection method using the antigen grafted on the nano beads is obviously improved; and the present invention can be applied in treatments using disease purification and in clinical regular mass in-vitro quantitative cancer diagnoses. 
         [0032]    The preferred embodiment herein disclosed is not intended to unnecessarily limit the scope of the invention. Therefore, simple modifications or variations belonging to the equivalent of the scope of the claims and the instructions disclosed herein for a patent are all within the scope of the present invention.