Monkey Hemoglobin (Hb) ELISA Kit

This assay employs a two-site sandwich ELISA to quantitate Hb in samples. An antibody specific for Hb has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Hb present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for Hb is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of Hb bound in the initial step. The color development is stopped and the intensity of the color is measured.

Hemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells of vertebrates, and the tissues of some invertebrates.In mammals, the protein makes up about 97% of the red blood cell’s dry content, and around 35% of the total content. Hemoglobin transports oxygen from the lungs or gills to the rest of the body where it releases the oxygen for cell use. It also has a variety of other roles of gas transport and effect-modulation which vary from species to species, and are quite diverse in some invertebrates. Hemoglobin has an oxygen binding capacity of between 1.36 and 1.37 ml O2 per gram of hemoglobin, which increases the total blood oxygen capacity seventyfold.Hemoglobin is found in nonerythroid cells including the A9 dopaminergic neurons in the substantia nigra, macrophages, alveolar cells, and mesangial cells in the kidney.

This assay has high sensitivity and excellent specificity for detection of Monkey Hb. No significant cross-reactivity or interference between Monkey Hb and analogues was observed.

Matrices listed below were spiked with certain level of recombinant Monkey Hb and the recovery rates were calculated by comparing the measured value to the expected amount of Monkey Hb in samples.

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in forty separate assays to assess inter-assay precision. CV (%) = SD/meanX100 Intra-Assay: CV<8% Inter-Assay: CV<12%

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Monkey Hb and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 4 and 7 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37°C can be considered as 6 months at 2 - 8°C, which means 7 days at 37°C equaling 12 months at 2 - 8°C).

Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 2 - 8°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.