Bovine Ig(Total Immunoglobulin) ELISA Kit使用说明（一）
Bovine Ig(Total Immunoglobulin) ELISA Kit
Catalogue No: EB0001 Size: 48T/96T Reactivity: Bovine
Detection Range: 1.563-100ug/ml
Sensitivity: <0.938ug/ml
Application: For quantitative detection of Ig in serum, plasma, tissue homogenates and other biological fluids.
Storage: 4°C for 6 months
NOTE: FOR RESEARCH USE ONLY.
Kit Components
| Item | Specifications(48T/96T) | Storage |
| ELISA Microplate(Dismountable) | 8×6 /8×12 | 4°C/-20°C |
| Lyophilized Standard | 1 vial/2 vial | 4°C/-20°C |
| Sample / Standard Dilution Buffer | 10ml/20ml | 4°C |
| Biotin-labeled Antibody (Concentrated) | 60ul/120ul | 4°C |
| Antibody Dilution Buffer | 5ml/10ml | 4°C |
| HRP-Streptavidin Conjugate(SABC) | 60ul/120ul | 4°C(shading light) |
| SABC Dilution Buffer | 5ml/10ml | 4°C |
| TMB Substrate | 5ml/10ml | 4°C(shading light) |
| Stop Solution | 5ml/10ml | 4°C |
| Wash Buffer (25X) | 15ml/30ml | 4°C |
| Plate Sealer | 3/5pieces | |
| Product Description | 1 copy |
Principle of the Assay
This kit was based on sandwich enzyme-linked immune-sorbent assay
technology. Anti- Ig antibody was pre-coated onto 96-well plates. And
the biotin conjugated anti- Ig antibody was used as detection
antibodies. The standards, test samples and biotin conjugated detection
antibody were added to the wells subsequently, and washed with wash
buffer. HRP- Streptavidin was added and unbound conjugates were washed
away with wash buffer. TMB substrates were used to visualize HRP
enzymatic reaction. TMB was catalyzed by HRP to produce a blue color
product that changed into yellow after adding acidic stop solution. The
density of yellow is proportional to the Ig amount of sample captured in
plate. Read the O.D.
absorbance at 450nm in a microplate reader, and then the concentration of Ig can be calculated.
Precautions
To inspect the validity of experiment operation and the appropriateness of sample dilution proportion, pilot experiment using standards and a small number of samples is recommended.
After opening and before using, keep plate dry.
Before using the Kit, spin tubes and bring down all components to the bottom of tubes.
Storage TMB reagents avoid light.
Washing process is very important, not fully wash easily cause a false positive.
Duplicate well assay is recommended for both standard and sample testing.
Don’t let Micro plate dry at the assay, for dry plate will inactivate active components on plate.
Don’t reuse tips and tubes to avoid cross contamination.
Avoid using the reagents from different batches together.
Material Required but Not Supplied
Microplate reader (wavelength: 450nm)
37°C incubator
Automated plate washer
Precision single and multi-channel pipette and disposable tips
Clean tubes and Eppendorf tubes
Deionized or distilled water
Manual Washing
Discard the solution in the plate without touching the side walls. Clap
the plate on absorbent filter papers or other absorbent material. Fill
each well completely with 350ul wash buffer and soak for 1 to 2 minutes,
then aspirate contents from the plate, and clap the plate on absorbent
filter papers or other absorbent material. Repeat this procedure two
more times for a total of THREE washes.
Automatic Washing
Aspirate all wells, and then wash plate THREE times with 350ul wash
buffer. After the final wash, invert plate, and clap the plate on
absorbent filter papers or other absorbent material. It is recommended
that the washer shall be set for soaking 1 minute.
Sample Collection and Storage
Isolate test samples soon after collecting, then, analyze immediately
(within 2 hours). Or aliquot and store at -20°C for long term. Avoid
multiple freeze-thaw cycles.
Serum: Place whole blood sample at room temperature for 2 hours or put it at 4°C overnight and centrifugation for 20 minutes at approximately 1000×g, Collect the supernatant and carry out the assay immediately. Blood collection tubes should be disposable, non-pyrogenic, and non-endotoxin.
Plasma: Collect plasma using EDTA-Na2 as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8°C within 30 minutes of collection. Collect the supernatant and carry out the assay immediately. Avoid hemolysis, high cholesterol samples.
Tissue Homogenates: As hemolysis blood has relation to assay result, it is necessary to remove residual blood by washing tissue with pre-cooling PBS buffer (0.01M, pH=7.4). Mince tissue after weighing it and get it homogenized in PBS (the volume depends on the weight of the tissue. Generally speaking, 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitors are recommended to add into the PBS) with a glass homogenizer on ice. To further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifuged for 5minutes at 5000×g to get the supernate.
Cell Culture supernate: Centrifuge supernatant for 20 minutes at 1000×g at 2 - 8°C to remove insoluble impurity and cell debris. Collect the clear supernate and carry out the assay immediately.
Other Biological Fluids: Centrifuge samples for 20 minutes at 1000×g at 2-8°C. Collect supernatant and carry out the assay immediately.
Sample Preparation: Samples shall be clear and transparent and remove suspended solids by centrifugation.
