Q:1. how to collect samples and preparation of ELISA? Performed by ELISA test is generally common clinical samples including blood (finger blood, blood), urine, feces, cerebrospinal fluid, pleural effusion, prostatic fluid, semen, vaginal secretions, which Some time of sample collection, preservation methods and has certain requirements. Collection (a) clinical specimens A, blood samples:Some physiological factors, such as smoking, eating, exercise, mood swings, pregnancy, postural changes in blood can affect certain ingredients, even some of diurnal variation. Therefore, blood samples Acquisition should avoid interference physiological factors, consistent with appropriate conditions, such as can not be avoided, should indicate the factors on the specimen. 1. Peripheral:Usually select the inside of blood left ring finger, the portion should be no frostbite, inflammation, edema, damage. If the site does not meet the requirements to other parts of the fingers instead. For burn patients, optional leather Intact skin at the blood. As part of routine blood tests (eg, white blood cell count, sort, etc.) affected by physiological factors fluctuation is too large, when compared to the conditional should be consistent. It relates to the body, blood clotting function Can test items (such as platelet count, bleeding time or clotting time) testing, we must pay attention to understand whether the patient used anticoagulant, procoagulant drugs in order to reduce or avoid interfering factors influences. 2. Blood:In addition to involving a variety of projects such as hemostasis and thrombosis detector requires the use of anticoagulated blood plasma, the current analysis to detect the vast majority of projects can be directly detected using blood serum. In the serum test items , Some (such as blood sugar, blood fat) diet and circadian factors influenced, fasting blood samples were generally appropriate; some decay rapidly in the blood (serum enzyme activity assay such as ACP activity, etc.), 0 ~ 4 ℃ storage is not an activity decreased, the detection of these projects must be timely and fast; some (such as creatine kinase) influenced by exercise and other factors. Avoid hemolysis occurs when blood is also important And, more particularly potassium, LDH and other measurement. B, urine samples:With the same blood samples, urine samples affect diet, exercise, medication and other factors that are also large, especially on the diet, so the morning urine generally superior to random urine. Means getting up early morning urine After the first urine specimens, representing concentrated and acidified visible components (such as blood cells, epithelial cells, tubular) easy to observe the relative concentration. Random urine that is a random urine specimens convenient, but by diet, Sports, and even more the influence of drugs, prone to false positive and false negative results, such as diet proteinuria, glucosuria diet, vitamin C interference occult blood results and the like. Postprandial urine (patient 2 hours after lunch, collected Human Urine) suitable for urine, urine protein and urobilinogen check urine samples at this time to increase the sensitivity of the test, the detection of minor lesions. 12 hours in urine cell count is Addis count (last night 8:00 After emptying the bladder to all specimens of urine 8 o'clock the next morning), because a long time, easy to breed bacteria shall be added preservative formaldehyde. 24-hour urine (the first day of the morning after emptying the bladder specimens from 8:00 to 8:00 the next morning All urine) quantification of chemical substances, including proteins, sugars, urinary 17-one, 17-hydroxy steroids, catecholamines, Ca2 +, etc., to detect different substances, choose a different preservative preservative. clean Urine used for urine bacterial culture requires sterile specimens were taken after washing the vulva. Urine specimens should be enough to collect all, at least 12 ml, preferably 50 ml, the timing must collect all the urine of women Patients should avoid vaginal secretions, blood contamination of urine specimens. C, stool samples:Stool samples for the detection judgment digestive diseases has important reference value. Collection requirements with a clean bamboo select faecal mucus, pus and blood components and other abnormality, no abnormal appearance Droppings shall be drawn from multiple surface and deep manure end. Get parasitemia and for egg counts should be collected 24 hours feces. Dysentery amoeba trophozoites check should immediately check in after a bowel movement, and from there sepsis Softer at the drawn, insulation inspection. Charles S. japonicum eggs should take mucus, pus and blood portion 30g stool specimens from at least miracidia hatching, and to be treated as soon as possible. Check pinworm eggs must use transparent film swab Night before 12:00 or early in the morning from defecation wrinkled folds around the anus and immediately swabbing at microscopic examination. Occult blood test (chemistry), fasting before the test on the 3rd of meat and foods containing animal blood and ban clothing iron, vitamin C and so on. Should be checked in all 1 hour stool specimen collection is completed, in order to prevent damage to physical components of digestive enzymes and pH by. For clinical samples above the detection indicators. D, CSF samples:CSF samples collected immediately after submission, place too long will affect the test results: such as cell degeneration, destruction, leading to counting and classification are not allowed; some chemicals such as glucose content will decompose Save Less; bacteria occur autolysis affect bacteria detection rate. Cerebrospinal fluid extracted three general dispensing a sterile tube, the first tube for bacterial culture, a second tube for chemical analysis and immunological tests, the third tube for general Characters and microscopic examination, three of the order should be reversed. Specimen collection is difficult because all inspection and testing process should pay attention to safety. E, ascites and pleural effusion samples:CSF samples with the same attention to safety after the specimen collection, and timely submission. Generally separated into three tubes, one for routine cytology, a biochemical examination, a bacterial culture, in order CSF same is appropriate. F, prostatic fluid sample:Prostatic fluid specimen after prostate massage by the acquisition, directly drop when less liquid on a glass slide and timely submission shall be taken to prevent sample evaporation to dryness, the amount collected for a long time in a clean, dry test tube. If massage No prostatic fluid, urine sediment can be checked after the massage. G, semen samples:Abstinence before semen collection should be 3 to 7 days, drain the urine after masturbation or other available methods of semen directly into clean containers, insulation and timely submission. Due to changes in sperm production during the day and Large, generally should be checked 2 to 3 times (each time interval of 1 to 2 weeks) in order to make a diagnosis. H, samples of vaginal secretions:Vaginal samples were collected 24 hours before intercourse should be prohibited, bath, vaginal examination, vaginal lavage and local on the drug, etc., drawing instruments used need to be cleaned. Usually with brine-soaked cotton swab from the vagina deep Or rear vaginal fornix, cervical canal mouth drawn, etc., made after saline smear vaginal secretion samples observation, women with menstrual vaginal secretions were not checking. 2, do before each sample by ELISA experiment how to prepare? Before collecting the sample must have a comprehensive plan must clearly be detected component is stable enough. To be collected on the same day Sample testing, and timely backup stored at 4 ℃. For the next day re-testing samples frozen in a timely manner after dispensing -20 ℃ spare, conditional, preferably -70 ℃ cryopreservation standby. Avoid repeated freezing and thawing specimens . Liquid samples: including serum, plasma, urine, pleural effusion, cerebrospinal fluid, cell culture supernatant and the like. 1. serum: Coagulation at room temperature 10-20 mins, centrifugation 20 minutes or so (2000-3000 rev / min). Carefully collect the supernatant. If precipitation during storage, Centrifugal again. 2. Plasma: EDTA should be selected according to the requirements of the specimen, sodium citrate or heparin as an anticoagulant, mix 10-20 mins, centrifugation 20 minutes or so (2000-3000 rev / min). Carefully collect the supernatant. Save process If precipitation appeared, Centrifugal again. 3. Urine: Sterile collection tube. Centrifuged for 20 minutes or so (2000-3000 rev / min). Carefully collect the supernatant. If precipitation during storage, Centrifugal again. Pleural and peritoneal effusions, and cerebrospinal fluid Reference to this practice. 4. The cell culture supernatant: The detection of secretory component with a sterile collection tube. Centrifuged for 20 minutes or so (2000-3000 rev / min). Carefully collect the supernatant. 5. cultured cells ????When the detection of intracellular components, diluted with PBS (PH7.2-7.4) cell suspension, the cell concentration reached 1 million / ml or so. By repeated freezing and thawing or tissue protein extraction reagent was added to the cells Damage and release of intracellular components. Centrifuged for 20 minutes or so (2000-3000 rev / min). Carefully collect the supernatant. If precipitation during storage, Centrifugal again. 6. tissues ????After cutting samples, check the weight. Adding a certain amount of PBS, PH7.4. Rapidly frozen with liquid nitrogen. After thawing samples remained at 2-8 ℃. Adding a certain amount of PBS (PH7.4), or tissue protein extraction reagent, or by hand homogenizer homogenized sample. Centrifuged for 20 minutes or so (2000-3000 rev / min). Carefully collect the supernatant. A new package to be detected, which I alternate freezing. Q:Do I have to run all of my standards and samples in duplicate? A:Yes, the duplicates are run in order to monitor assay precision and increase confidence in the assay results obtained. Q:Do I have to run all of my samples at one time? A:No, each kit uses stripwell microplate. This allows the user to analyse different numbers of samples at different times. Q:What types of reproducible results are obtained with the assays? A:Each kit comes with a manual containing a graph of typical data obtained. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve. Q:Is it possible to store the reagents other than indicated? A:Storage of the kit components under conditions other than indicated is not recommended in order to assure proper performance of the test. Q:How should I store my samples? A:Samples should be stored at -20oC or lower temperature. For long-term storage, it is recommended to freeze them at -70oC -80oC. Q:Can I modify the protocol? A:BG ELISA kits have been optimized to provide the best possible results. Modifying the format or protocol may give inaccurate and wrong results. Q:Can I use a sample type that is not recommended in the kit insert? A:The kit has been validated for the sample types listed in the kit insert. Sample types other than those validated have not been tested. Contact Technical Service for further information. Q:My samples generated values that were outside the dynamic range of the assay. Can I use these values? A:It is recommended that only sample values that fall within the range of the standard curve be used. Values outside the range of the standard curve are generally non-linear, which can lead to incorrectly extrapolated values. Samples that generate values higher than the highest standard should be (further) diluted and the assay repeated. If samples fall below the range of the assay, the sample is considered to be non-detectable. Q:Do I have to run a Blank or Zero Standards every time? A:Yes, these are required for the calculations, and reflect any subtle but significant performance changes from day to day and assay to assay. They are also extremely helpful when troubleshooting the source of a particular assay problem. Q:Can I alter the volume of sample I use in the assay? A:It is not recommended that you alter the volumes since all BG kits are designed for optimal performance at the given volumes Q:Can components from different kits be used? A:Each kit contains components which have specific lot numbers to ensure that all of the components are performing optimally alone, as well as with all of the other components in the kit. QC testing is performed on these specific lots. It is never recommended to use your own components or components from other kits or vendors. Q:My standard curve looked fine, but I didn’t get a signal in my sample when I expected to, why? A:The sample may not contain the analyte. A matrix effect may be masking the detection. Ensure that the recommended dilution was followed as stated in the kit insert. If dilution was recommended, check to be sure that the dilution was performed properly. Over-dilution may cause the sample to fall below the range of the standard curve. Q:How do you recommend I wash my plate? A:If you are using an automated plate washer we recommend that the calibration be checked on a regular basis, and that the system is flushed with the Plate Washing Buffer prior to washing. The same is true for a manual washer. A repeater or a wash bottle can also be used. The user should be careful to ensure that all of the contents are aspirated and the plate tapped dry on lint-free paper. Q:Do I need to use a plate shaker? A:Reliable results can be obtained without a plate shaker, but the O.D.'s will generally be lower than those obtained using a plate shaker. Q:Why do I have to use wavelength correction between 450-570nm? A:For the ELISA assay, reading at dual wavelengths is done to correct for the optical density contributed by the plastic well, the lamp and optical fluctuations. Q:If I extract my sample, do I still need to follow the recommended dilutions given in the kit insert? A:The amount of sample dilution needed after an extraction procedure will be affected by the effects of purification and concentration in the protocol used. The amount of dilution or concentration will have to be determined by the end-user. Q:What is the expected concentration of analyte that I should expect to find? A:The amount of a given analyte may vary not only from species-to-species, but also between tissue and cellular sources. The best source of this information is the current literature that is easily accessed through the Internet at multiple scientific databases. Q:My optical densities were a little higher (or lower) than those in the manual that came with my kit. Why? A:The optical density is affected by a number of physical conditions such as time and temperature. We suggest that you shorten or lengthen the final incubation with substrate solution to compensate. Q:What are the reasons for High Background? A:1) Improper Washing: Check volume of washing buffer reservoir and make sure all recommended washing steps are performed. 2) Contaminated Substrate: Make sure there is no contamination of the substrate with metal ions or oxidizing reagents, before use. Keep the extra substrate solution separately during the ELISA substrate development time. 3) Substrate exposed to light: Exposure to light may result in a blue color of the substrate. Keep solutions in the dark (vial) until ready to dispense into the plate. 4) Wrong Incubation Times/Temperatures: Generally follow the test protocol regarding incubation times and temperatures. However, if all wells are intensely and equally colored with no intensity gradient observed in the standard dilution series, then it may be necessary to observe the substrate reaction as the color is developing, in order to stop the reaction sooner.