臨床免疫學(xué)檢驗(yàn)實(shí)驗(yàn)：Tumor marker-AFP

1、Tumor marker AFP 1、conception It is produced by tumor cells or tissue cells which against tumor cells. It usually exists in serum as the form of antigen, enzyme or hormone metabolites. Tumor markers can exist on the cell membrane、cytoplasm 、inside or outside of cells. According to the biochemical an

2、d immunological characteristics, tumor marker can help us to identify and diagnose the tumors. There are two categories: Specific tumor marker: it is a symbol of a specific tumor. It only appears and rises significantly when a patient have a particular tumor. Relevant tumor marker: Healthy people ca

3、n also produce relevant tumor markers. in some malignant tumor, it will increase dramatically. But if patient has benign tumor, its concentration also increase lightly. So its specificity is worse than specific tumor marker. 2、Application tumor screening： Human chorionic gonadotropin(HCG)has been su

4、ccessfullyused for screeningthe choriocarcinoma (絨毛膜癌) . Alpha fetalprotein(AFP),has special significance in thediagnosis of primary hepatic carcinoma (原發(fā)性肝癌) and germinocarcinoma(生殖細(xì)胞癌）. Diagnosis anddifferential diagnosis of tumor ： When a patientissuspected ofsuffering from the tumor,thetumor mar

5、kers will play a important role in the diagnosisof benign and malignanttumors. Biologicalcharacteristics anddisease stage ofjudgment ： when patient need a clear diagnosis,determination ofthe basic level of tumor markers is necessary.In the different stages of disease,the concentration of tumor marke

6、rsis different and itcan providehelpon prognosis. Observation of curative effect andprognosis ： Beforetreatment, tumor marker usually increases. If they decreases after treatment that means thetreatmentis effective and successful; If tumor marker rises again aftertreatment that means the treatment i

7、s invalid or failed; Patient got treatment and recovered. After a period of time, tumor marker increases significantly again that means tumor has recrudesced or metastasized. Classification Embryonaltumor markers: AFP、CEA Carbohydrate markers: CA125、CA199 Enzymes markers: PAP、NSE、PSAHormonalmarkers：

8、HCG、PTH、PCTProteinmarkers: C-peptide、copper-protein Material preparation : 1、 immunosorbent: 96 /48 holes plate The plate is made from polystyrene. It has a strong ability to absorb protein so it is ideal material to be used as solid phase carrier.Polystyrene also has low blank value and high transp

9、arency on the bottom of the hole. The properties among the different plates or different holes in the same plates should be similar. Detection of AFP Enzyme： HRP is commonly used to label antigen or antibody. Substance：（TMB ColorDevelopmentSolution） Ingredient: liquid A: contain 0.5 H2O2 liquid B：co

10、ntain 0.26 TMB DH2 H2O2 D 2H2OHRP- Ab/Aghydrogen donatorhydrogen acceptor TMB: No colorProduct: orange red colorCleaning solution ： Cleaning sulution is the phosphate buffer with 0.05% Twain-20.Diluent: already prepared in reagent set.Stop buffer: the concentration of H2SO4 is 2mol/l.Controlset：prep

11、ared in the set. Double antibody sandwich method detected AFP (two-steps method)enzyme labelled Ab Stop buffersubstanceantibodyUnknow antigenIncubatewashIncubatewashIncubateDetection Steps: Preparation:Coating: antibody is diluted to the workingconcentration andadded 50L/ hole, incubated in 37, 4h o

12、r 4, 24h. Washing: abandon the liquid of the holes and wash with PBSfor 5 times,every time 1minutes. (in our experiment, the plates are already prepared for detecting )Detection Steps: 1、Adding sample: establishan appropriate concentration gradient: 1:2, 1:4, 1:8, 1:16(semi-quantitative)The diluteds

13、amplesadded to two or three holes so we can compare with result and reduce the error. 50L /hole. 2、incubating: put the plate at 37, 20min . 3、washing: abandon the liquid of the holes and wash with PBSfor 5 times,every time 1minutes4、Addingenzyme labelled antibody : enzyme labelled antibody is dilute

14、d to the working concentration andadded 50L/ hole5、Incubating: put the plate at 37, 20min. 6、Washing: discard the liquid of the holes and wash with PBSfor 5 times,every time 1minutes7、Adding substance: liquid A (50L/ hole) and liquid B (50L/ hole) mixed together, 8、 Incubating: put the plate at 37,

15、in dark, 10min 9、terminal reaction: Each holeadds the 50L H2SO4 to terminate reaction. The blue color will change to yellow. Testing the result in 20min. 10、result judging: Observing by eyes: the deeper the color , the higher the concentration Testing by equipment: The detection wavelength is 450nm.

16、 Before testing the result, we have to adjust the blank hole to “0” then put the plate into the equipment and read the result. Result and clinic significance Reference range: AFP 20 ng/mL In normal circumstances, AFP was mainly derived from embryoniccells and only appears in placental period. Its ab

17、out two weeksafter birth AFP will disappearfrom the blood,so thecontent of serumAFP is less than20 ng / mL in normal healthy people. Hepatitis: patient with hepatitis , the content of AFP is usually between 20-400 ng / ml. If patients with hepatitisviral infection, serum AFP increased slightly. Abou

18、t 20% chronic hepatitis patientshave elevated AFP.In explosive hepatitis patients, AFP usually increased significantly.AFP in pregnant woman andfetal state During pregnancy, AFP in mothers serum is also usually between 20-400 ng / ml. With the extension of the pregnant time, AFP will gradually incre

19、ase. When fetussuffering fromhypoxia（缺氧）,death, congenitalgenetic defects（先天性遺傳缺陷）,neural tube defects（神經(jīng)管缺陷）,spinal bifida（脊柱裂）,AFP in mothers serum will rise up significantly. AFP in pregnant woman andfetal state Reportedly,if pregnant womens serum AFPis higher than 800 ng / ml that means fetalis

20、endangeredor already dead. If AFP ismore than 1075 ng / ml, the rate of diagnosis for fetal deathis up to 80 %. Tumor screening : The elevation of AFPis one of the most important indexes for primary hepatic carcinoma and germinocarcinoma. Itis very specific and sensitive. patient suffering from somebenign tumor of liver,serum AF