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This immunoassay kit allows for the in vitro quantitative determination of mouse GHbA1c concentrations in cell culture supernates, serum, plasma and other biological fluids.

Mouse Glycated hemoglobin A1c (GHbA1c) ELISA Kit


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Background

Glycosylated (or glycated) hemoglobin (hemoglobin A1c, Hb1c , or HbA1c, A1C) is a form of hemoglobin used primarily to identify the average plasma glucose concentration over prolonged periods of time. It is formed in a non-enzymatic pathway by hemoglobin's normal exposure to high plasma levels of glucose. Glycosylation of hemoglobin has been implicated in nephropathy and retinopathy in diabetes mellitus. Monitoring the HbA1c in type-1 diabetic patients may improve treatment.
In the normal 120-day life span of the red blood cell, glucose molecules join hemoglobin, forming glycated hemoglobin. In individuals with poorly controlled diabetes, increases in the quantities of these glycated hemoglobins are noted. Once a hemoglobin molecule is glycated, it remains that way. A buildup of glycated hemoglobin within the red cell reflects the average level of glucose to which the cell has been exposed during its life cycle. Measuring glycated hemoglobin assesses the effectiveness of therapy by monitoring long-term serum glucose regulation. The HbA1c level is proportional to average blood glucose concentration over the previous four weeks to three months.
Higher levels of HbA1c are found in people with persistently elevated blood sugar, as in diabetes mellitus. While diabetic patient treatment goals vary, many include a target range of HbA1c values. A diabetic person with good glucose control has a HbA1c level that is close to or within the reference range. The International Diabetes Federation and American College of Endocrinology recommend HbA1c values below 6.5%, while American Diabetes Association recommends that the HbA1c be below 7.0% for most patients. A high HbA1c represents poor glucose control. However, a 'good' HbA1c in a patient with diabetes can still be riddled with a history of recent hypoglycemia, or, alternatively, spikes of hyperglycemia. Regular blood glucose monitoring is still the best method for the analysis of overall vascular health with respect to blood sugar control. Often, patients with diabetes mellitus are scolded by their doctors for having a HbA1c which is too low, because a lower A1c would indicate a likelihood of frequent hypoglycemia in the recent past. This is often assessed with blood sugar data and receptions are typically mixed. A balance of long term health (hyperglycemia prevention) versus short term health (hypoglycemia prevention) is always a constant concern for both patients and their doctors. Doctors are especially sensitive about lower level HbA1c's with patients who regularly drive, this being a prime example of a short-term motivation for preventing hypoglycemia. Many diabetics have died behind the wheel as a result of a low blood sugar, especially for the reason that frequent hypoglycemia results in a higher tolerance (ideally the patient is seized with a feeling of panic, an increased heart rate, profuse sweating, etc.) for the condition and some patients may not even consciously realize their blood sugar has dropped to dangerous levels. In addition to acquired tolerance, the use of alcohol and certain drugs (marijuana, for example) can create moderately similar symptoms to those of hypoglycemia (especially when used in combination) and for this reason the patient may not realize he/she has contracted hypoglycemia.
Persistent elevations in blood sugar (and therefore HbA1c) increase the risk for the long-term vascular complications of diabetes such as coronary disease, heart attack, stroke, heart failure, kidney failure, blindness, erectile dysfunction, neuropathy (loss of sensation, especially in the feet), gangrene, and gastroparesis (slowed emptying of the stomach). Poor blood glucose control also increases the risk of short-term complications of surgery such as poor wound healing.
Lower than expected levels of HbA1c can be seen in people with shortened red blood cell life span, such as with glucose-6-phosphate dehydrogenase deficiency, sickle-cell disease, or any other condition causing premature red blood cell death. Conversely, higher than expected levels can be seen in people with a longer red blood cell life span, such as with Vitamin B12 or folate deficiency.

Feature

<PRINCIPLE OF THE ASSAY>
The microtiter plate provided in this kit has been pre-coated with an antibody specific to GHbA1c. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for GHbA1c and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3'5, 5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain GHbA1c, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of GHbA1c in the samples is then determined by comparing the O.D. of the samples to the standard curve.

< SPECIFICITY>
This assay recognizes recombinant and natural mouse GHbA1c. No significant cross-reactivity or interference was observed.

<DETECTION RANGE>
15.6 ng/ml-1000 ng/ml.
The standard curve concentrations used for the ELISA’s were 1000ng/ml, 500 ng/ml, 250 ng/ml, 125 ng/ml, 62.5 ng/ml, 31.2 ng/ml, 15.6 ng/ml.

<SENSITIVITY>
The minimum detectable dose of mouse GHbA1c is typically less than 3.9 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.

Composition

  Reagent Quantity
1. Assay plate 1
2. Standard 2
3. Sample Diluent 1 x 20 ml
4. Biotin-antibody Diluent 1 x 10 ml
5. HRP-avidin Diluent 1 x 10 ml
6. Biotin-antibodyt 1 x 120μl
7. HRP-avidin 1 x 120μl
8. Wash Buffer 1 x 20 ml (25×concentrate)
9. TMB Substrate 1 x 10 ml
10. Stop Solution 1 x 10 ml
Product List

Product Name Cat# Quantity Price

Species : 15.6 ng/ml - 1000 ng/ml.
Assay range : 100 µL(Cell Culture Supernates, Plasma,Serum)
Assay sample volume : 6-8 hr.{3 hr


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