Insulin antibodies assay

The radioimmunoassay of insulin permits detection of insulin in picomolar quantities. The assay is based on antibodies developed in guinea pigs against bovine or pork insulin. Because of the similarities between these two insulins and human insulin, the assay successfully measures the human hormone as well. 

Spencer, R. D., Toledo, F. B., Williams, B. T., and Yoss, N. L., Design, construction, and two applications for an automated flow-cell polarization fluorometer with digital read out Enzyme-inhibitor (antitrypsin) assay and antigen-antibody (insulin-insulin antiserum) assay. Clin. Chem. 19, 838-844 (1973). 

Competitive RIA of Free Anti-Insulin Antibodies with PEG Precipitation (Semi-Quantitative Assay)... 

Two-Site Assay Protocol for the Measurement of Human a-Fetopro-tein. Purified antibodies to human a-fetoprotein (AFP) are prepared by allowing 1 ml of high titer antiserum (binding capacity approximately 125 p,g of AFP per milliliter) to react with 1 mg of AFP immunoadsorbent (protein uptake 120 p,g per milligram of cellulose) for 48 hr at 4°. Elution of high affinity antibody is carried out as described above for the preparation of purified insulin antibody. The antibody solution is diluted to 100 ml in 50 vaM sodium hydrogen carbonate buffer (pH 9.6) and used to coat 500 plastic tubes as described above. 

Indirect Two-Site Immunoradiometric Assay of Human Proinsulin. The method used is that described by Rainbow et al. Plastic tubes coated with purified guinea pig anti-insulin antibodies are prepared as described above 200-jul samples containing human proinsulin are added to these coated tubes and incubated at 4° for 24 hr. After removal of the sample, tubes are washed twice with 400 /tl of NIGP buffer. Rabbit antibody to human C-peptide is diluted to 1/1000 in 50 mM sodium phosphate buffer, pH 7.4, containing 150 mM sodium chloride, 10 g of bovine serum albumin per liter, and 100 mg of guinea pig IgG per liter 200 /til are added to each tube. After a further 24 hr of incubation at 4 the tubes are washed twice as previously and 200 /u,l of I-labeled sheep anti-rabbit IgG (10,000 cpm) are added in the same buffer as that used for diluting the C-peptide antiserum. After a final 24 hr of incubation and two further washes as above, the tubes are counted. 

Measurement of C-peptide has a number of advantages over insulin measurement. Because hepatic metabolism is negligible, C-peptide concentrations are better indicators of P-ceU ffinction than is peripheral insulin concentration. Furthermore, C-peptide assays do not measure exogenous insulin and do not cross-react with insulin antibodies, which interfere with the insulin immunoassay. 

Assays for insulin antibodies fall into three categories (1) quantitative radioimmunoelectrophoresis, which measures the binding of IgG antibody to radiolabeled insulin by rocket Immunoelectrophoresis into anti-IgG-containing agarose (2) RIAs with separation of bound and free insulin by precipitation with PEG or a second antibody and (3) solid phase immobilization of insulin to test tubes or Sepharose. These are discussed in more detail in Reeves. ... 

The measurement of C-peptide provides a fully validated means of quantifying endogenous insulin secretion, preventing influence of exogenous insulin or insulin antibody. However, most C-peptide assay kits cannot differentiate C-peptide from proinsulin and proinsulin conversion products. The influence of proinsulin may be significant in cases where serum proinsulin is elevated, as in Type 2 diabetes, familial hyperproinsulinemia and in patients with proinsulin antibody. 

Human T-cell lymphotrophic virus Immunoglobulin electrophoresis Insulin antibody Insulin assay... 

Insulin 15 Immuno-genicity of rh-insulin relative to porcine insulin 100 subjects receiving rh-insulin vs. 121 receiving porcine insulin over 12 months Species- specific binding assay -44% rh-insulin patients developed antibodies (Abs) reaching a plateau at 6 months. 60% patients receiving porcine insulin developed Abs in 12 months... 

In a protein-binding assay with fluorescence detection a microarray of biotin, HPYPP-peptide and WSHHPQFEK-peptide was screened against streptavidin-Cy3 and avidin-Cy5. By following the same principle an anti-human insulin monoclonal antibody was also screened against a set of different peptides. 

In the selected example by Lam et al. [101] many peptide libraries were prepared using the mix and split technique and tested in different on-bead screens. Incomplete libraries were tested (the population of most of them was more than a million compounds), and the positive structures were exploited through focused libraries. Some libraries were screened against an anti-insulin monoclonal antibody tagged with alkaline phosphatase, which allowed an enzyme-linked colorimetric detection. Only the beads bound to the murine MAb showed a tourquoise color, while the vast majority remained colorless (details of the technical realization of the assay can be found elsewhere [101, 102]). The chemical structure linked to the positive beads was then easily determined via Edman degradation of the peptide sequences. 

The most definitive laboratory test to distinguish type 1 from type 2 diabetes is the C-peptide assay, which is a measure of endogenous insulin production. With type 2 diabetes, proinsulin can be split into insulin and C-peptide lack of C-peptide indicates type 1 diabetes. The presence of anti-islet antibodies (to glutamic acid decarboxylase, insulinoma associated peptide-2 or insulin) or absence of insulin resistance (determined by a glucose tolerance test) is also suggestive of type 1. 

An enzyme immunoelectrode suitable for the assay of human serum albumin and insulin uses an oxygen electrode covered with an antibody-containing nylon net kept in place with an O-ring. From 1 to 25 ng/L of albumin and 5 to 100 ng/L of insulin can be assayed (306). A specific sensor for the tumor antigen a-fetoprotein (AFP) is prepared by immobilizing anti-AFP antibody covalendy on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyl octane, and GA (307). The sensor is applied to an EIA based on competitive Ab/... 

Charcoal is widely used to separate antibody-bound ligand (bound) from non-antibody-bound ligand (free) in competitive protein binding assays. Its use was originally described by Miller for vitamin Bi assay and later by Herbert et al. for B12. In 1965, Herbert suggested its use to separate bound and free in the radioimmunoassay of insulin. Since then it has been used for a large number of radioimmunoassays and radioreceptor assays. 

Fig. 1. Dose-response curve for amount of dextran-coated charcoal added versus percentage free and bound, adsorbed. These are theoretical curves drawn from generalizations derived from several assays. —, Curve showing binding of free ligand —, curve showing binding of antibody bound ligand. The exact amounts of charcoal giving a particular bound value vary with each assay (e.g., growth hormone assay differs from insulin assay). For significance of arrows A and B, see the section on selection of charcoal dose.

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