The coated well immunoenzymatic assay for the quantitative measurement of analyte utilizes a multiclonal anti‐analyte antibody and an analyte‐HRP conjugate. The assay sample and buffer are incubated together with analyte‐HRP conjugate in pre‐coated plate for one hour. After the incubation period, the wells are decanted and washed five times. The wells are then incubated with a substrate for HRP enzyme. The product of the enzyme‐substrate reaction forms a blue colored complex. Finally, a stop solution is added to stop the reaction, which will then turn the solution yellow. The intensity of color is measured spectrophotometrically at 450nm in a microplate reader.
The intensity of the color is inversely proportional to the analyte concentration since analyte from samples and analyte ‐HRP conjugate competes for the anti-analyte antibody binding site. Since the number of sites is limited, as more sites are occupied by analyanalytete from the sample, fewer sites are left to bind analyte‐HRP conjugate. Standards of known analyte concentrations are run concurrently with the samples being assayed and a standard curve is plotted relating the intensity of the color (Optical Density) to the concentration of the analyte. The analyte concentration in each sample is interpolated from this standard curve.
Manufactured and distributed by:
NovaTein Biosciences 310 West Cummings Park Woburn, MA 01801 US
|1||Microelisa Stripplate||96 well||6||Chromogenic Substrate A||6ml X 1vial|
|2||Standard||1.0 ml X 6 vials||7||Chromogenic Substrate B||6ml X 1 vial|
|3||100 X Wash Solution||10ml X 1vial||8||Stop Solution||6ml X 1 via|
|4||Lysis Buffer Solution||6ml X 1vial||9||Specification||1|
|5||5 HRP‐Conjugate Reagent||6ml X 1 vial|
Note: Standard (S1 → S6) concentration was followed by:Variable with different kit
Sample collection and storage
- Serum‐Use a serum separator tube (SST) and allow samples to clot for 30 minutes before a centrifugation for 15minutes at approximately 1000 x g. Remove serum and perform the assay immediately or aliquot and store samples at ‐20 °C or ‐80 °C.
- Plasma‐Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2‐8 °C within 30minutes of collection. Store samples at ‐20°C or ‐80°C.Avoid repeated freeze‐thaw cycles.
- Cell culture fluid and other biological fluids‐Remove particulates by centrifugation and assay immediately or aliquot and store samples at ‐20°C or ‐80°C. Avoid repeated freeze‐thaw cycles.
- NOTE: The Lysis Buffer Solution is used only when the sample is cell culture fluid & body fluid & tissue homogenate; if the sample is serum or blood plasma, then the Lysis Buffer Solution is a superfluous reagent. Serum, plasma, and cell culture fluid samples to be used within 7 days may be stored at 2‐8 °C, otherwise samples must be stored at ‐20°C(≤2months) or ‐80°C(≤6months) to avoid loss of bioactivity and contamination. Avoid freeze‐thaw cycles. When performing the assay, warm up samples to room temperature slowly. DO NOT USE HEAT‐TREATED SAMPLES.
Materialsrequired but notsupplied
- 1.37 °C incubator
- 2.Standard plate reader capable of measuring absorbance at 450 nm.
- 3.Precision pipettes and disposable pipette tips
- 4.Distilled water
- 5.Multi‐channel pipettes, manifold dispenser or automated microplate washer.
- 6.Absorbent paper
- 1.Novateinbio is only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient amount ofsamplesin advance.
- 2.Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
- 3.If the samples are not indicated in the manual, a preliminary experiment to determine the validity ofthe kitis necessary.
- 4 Owing to the possibility of mismatching between antigen from other resource and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteinsfrom other manufacturers may not be recognized by our products.
- 5.Influenced by the factorsincluding cell viability, cell number and also sampling time,samplesfrom cell culture supernatant may not be detected by the kit
- 6.Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may occurin those samples and finally lead to wrong results.