Notes

1. Phosphate buffer, with disodium EDTA as its acidic component, is widely used in RIA procedures. EDTA inhibits divalent cation-dependent enzymes that might degrade insulin during long incubations. Furthermore, it inactivates complement that might inhibit antigen-antibody binding.

2. The fatty-acid-free BSA, which is also partially purified as the free fatty acids are removed by charcoal extraction, prevents nonspecific surface binding of insulin and antibody. We found this particular BSA to have wide application in RIA procedures, presumably because the charcoal extraction removes endogenous ligands and/or impurities.

3. 125I-Human insulin (cat. no. 07-260121, ICN Pharmaceuticals, Inc.) can also be used.

4. Triton™ X-100 is a very viscous liquid and is best handled by preparing an intermediate concentrate of 10% (v/v). The presence of the nonionic detergent in the RIA buffer reduces the nonspecifically bound labeled insulin fraction [N] to <1% of the total labeled insulin [T], without significantly reducing the antibody-bound fraction of labeled insulin [R]. With Triton X-100 in the RIA buffer, the ratio [R] : [N] increases to 28 :1 from 7:1 in the BSA-RIA buffer. However, Triton X-100 should not be added to any RIA buffer without first comparing [R]: [N] ratios, in the presence of graded amounts of the detergent, to the [R]: [ N] ratio obtained with protein-supplemented RIA buffer.

5. Aprotinin, a competitive inhibitor of proteolytic activity, helps to preserve insulin during long incubations.

Fig. 6. Plasma insulin levels in a NOD-SCID mouse bearing a human islet xenograft under the renal capsule. Glucose, 2 g/kg body weight, was given intraperitoneally on nine occasions, at 6- to 21-d intervals, during a 90-d period. Blood was collected from the saphenous vein without anesthesia before (open bars) and 30 min after glucose administration (solid bars) for the measurement of total and human plasma insulin by the mouse plasma insulin RIA and the human insulin RIA, respectively. The level of endogenous mouse plasma insulin was derived by subtracting the amount of human insulin from the total amount of insulin found in the sample. Each bar represents the mean ± S.E.M. of the nine insulin levels. (The data are courtesy of Michael J. Fowler, Vanderbilt University Medical Center, Nashville, TN).

Fig. 6. Plasma insulin levels in a NOD-SCID mouse bearing a human islet xenograft under the renal capsule. Glucose, 2 g/kg body weight, was given intraperitoneally on nine occasions, at 6- to 21-d intervals, during a 90-d period. Blood was collected from the saphenous vein without anesthesia before (open bars) and 30 min after glucose administration (solid bars) for the measurement of total and human plasma insulin by the mouse plasma insulin RIA and the human insulin RIA, respectively. The level of endogenous mouse plasma insulin was derived by subtracting the amount of human insulin from the total amount of insulin found in the sample. Each bar represents the mean ± S.E.M. of the nine insulin levels. (The data are courtesy of Michael J. Fowler, Vanderbilt University Medical Center, Nashville, TN).

6. Initially, NGPS serves as a protein carrier for the primary insulin antibody. Later, the gamma globulin component of NGPS and the primary insulin antibody coreact with the secondary antibody to guinea pig gamma globulin to form a precipitable complex.

7. The dilution factor of the 125I-insulin preparation should be adjusted initially to deliver 5000-6000 cpm per 0.1 mL and readjusted as the isotope decays.

8. Some batches of BSA cause the pancreas to swell during perfusion resulting in a dramatic drop in the flow rate. We recommend testing BSA quality in the perfusion system using wild-type mice before attempting pancreatic perfusion of more valuable experimental animals.

9. A linear glucose gradient is frequently used to test pancreatic insulin secretion in the transgenic animals (9). This can be accomplished by using Amersham's Gradient Mixer GM-1 (cat. no. 19-0495-01, Amersham Biosciences) in the pancreatic perfusion system.

10. When the pancreas is being perfused initially with the basal medium, the effluent is usually contaminated with blood during the first 15 min but clears up completely by 30 min.

11. Sealing the rack of tubes in a polyethylene bag to prevent evaporation during long incubations is an alternative to capping each tube.

12. Transferring the tubes to foam racks for decanting and blotting is an alternative to either decanting the tubes while they stand in the incubation racks or decanting or aspirating individual tubes.

13. In this solid-phase RIA, the rat insulin standard is indistinguishable from the First International Standard for Human Insulin, coded 83/500 and established in 1986 by The World Health Organization's Expert Committee on Biological Standardization. The ED10, ED50, and ED90, the concentration of either rat or human insulin reference standard required to reduce reference binding by 10%, 50%, and 90%, was 0.22, 2.5, and 29 ng/mL, respectively.

14. Immunoassay is the method of choice for the measurement of insulin from any source in any species. During the past 4.5 decades, numerous commercial immunoassays for insulin have been developed. Especially plentiful are the het-erospecies-specific competitive immunoassays employing antibodies made to either human, porcine, or bovine insulin, which tend to recognize the hormone from several species. Competitive immunoassays are those in which a fixed amount of labeled insulin and a variable amount of reference standard or sample insulin are allowed to compete for a limited number of insulin antibody-binding sites. The amount of antibody-bound labeled insulin, found at the end of a specific interval, is inversely proportional to the amount of unlabeled insulin present. The high degree of sequence homology between these vertebrate insulins, 2-4 amino acid substitutions out of 51, suggests that some of the established heterospecies-specific large-animal insulin immunoassays might recognize mouse insulin to the extent that they could be used to measure the mouse hormone. When the concentration of mouse insulin is expected to be >0.2 ng/mL (3.4 X 10-11 M) and sample size >0.25 mL (e.g., insulin secretion by the perfused mouse pancreas in situ or by isolated mouse islets and b-cell lines during perifusion or static culture), the antibody component of a pre-existing large-animal competitive immunoassay, with an affinity for mouse or rat insulin of at least 2 X 1010 L/mol, can become a basis of a customized immunoassay for the mouse hormone. To study mouse insulin released under these conditions, we selected the antibody component from a commercial human insulin solid-phase RIA kit with a Ka of 2.3 X 1010 L/mol (Kd = 4.3 X 10-11 mol/L or 0.25

ng/mL) and 85% crossreactivity with rat insulin as the basis for our customized mouse insulin RIA. Currently, immunoassays for mouse or rat insulin reflect an estimate of the sum of insulin I and II in the sample plus any proinsulin or partially hydrolyzed proinsulin detected by the assay. C-Peptide is not detected by insulin immunoassays.

15. An alternative way of performing glucose tolerance testing is to use a conscious mouse. In this case, the mouse can be restrained in a 50-mL Falcon centrifuge tube and blood is sampled from the saphenous vein using a Microvette system (cat. no. 16-443-300, Sarstedt, Newton, NC).

16. Extra precautions should be taken when handling blood samples. Blood from the heparinized capillary tube should be expelled immediately into a chilled Eppendorf tube to facilitate anticoagulation and minimize hemolysis. Red blood cells contain an insulin-degrading enzyme or insulinase that can cleave insulin molecules and have a devastating impact on insulin measurement by immunoassay (10,11). Sapin et al. showed that insulin degradation in hemolyzed plasma could be prevented much more effectively by cold storage of samples and 4°C incubations (during insulin assay) than by using the chelating agent EDTA (10 mM/L) (10). If samples cannot be handled on ice, these authors recommended the use of 1 mM/L j?-chloromercuriphenyl sulfonic acid (CPMS) inhibitor to protect insulin from degradation because of hemolysis (10).

17. With the help of the second person, it is possible to test up to 12 mice in one series by dividing them into two groups and injecting them with glucose in 2-min intervals.

18. Effective decanting and blotting of the assay tubes is facilitated by the handling, volume, and composition of the separation buffer. The immediate ice-cold, 30-min, 6000g spin helps to solidify the precipitate. The volume of buffer dilutes the counts and renders any hanging drop(s) less radioactive. The BSA binds any excess detergent, thereby reducing pellet slippage. A less pure and more economical preparation of BSA is recommended for this purpose, as it is in contact with the reactants for a short period of time. The PEG enhances precipitation of the secondary antibody-first antibody complex as PEG, in higher concentrations, can completely precipitate gamma globulin.

19. When the concentration of mouse insulin is expected to be as low as 60 pg/mL (1011 M) and sample size as small as 0.01 mL (e.g., plasma insulin in wild-type or transgenic mice undergoing glucose tolerance testing), the antibody component of a preexisting heterospecies-specific competitive immunoassay, with an affinity for mouse or rat insulin of approx 1011 L/mol, can become the basis of an immunoassay with the required sensitivity to measure the hormone in mouse plasma. To study mouse plasma insulin, we selected a rat insulin antibody with a Ka of 5 X 1010 L/mol (Kd = 2 X 10-11 mol/L or 115 pg/mL) and 100% crossreactivity with mouse, human, porcine, hamster, or sheep insulin, as the basis for our mouse plasma insulin RIA.

20. Delayed addition of the labeled antigen, shown in some instances to improve competitive immunoassay sensitivity, results in a more than 10-fold increase in sensitivity as the ED10, ED50, and ED90, of this insulin RIA shifted from 95, 700, and

5400 (manufacturer's data) to 7.5, 40, and 200 pg insulin/mL, respectively. This converts to a sensitivity of 75 pg/mL when using a 0.01-mL plasma/assay tube.

21. The First International Standard for Human Insulin is indistinguishable from the rat insulin reference standard in this insulin RIA.

22. Immunoassay of insulin released by insulin-secreting xenografts in mice requires an antibody with an affinity for the graft insulin of approx 1011 L/mol and virtually no affinity for mouse insulin. We selected a human insulin antibody with a Ka of 5.6 X 1010 L/mol (Kd = 1.8 X 10-11 mol/L or 105 pg/mL) and <0.1% crossreac-tivity with rat insulin, as the basis for our RIA of human insulin secreted by islet grafts in mice.

23. Delayed addition of the labeled antigen results in a more than 10-fold increase in sensitivity as the ED10, ED50, and ED90 of this antiserum shifted from 100, 1050, and 9500 pg/ mL (manufacturer's data) to 5, 70, and 1000 pg human insulin/mL, respectively. This converts to a level of detectability of 50 pg/mL when using 0.01 mL of plasma/assay tube.

24. Rat insulin crossreacts 0.4% under the conditions of this assay. Endogenous mouse insulin levels of <12,500 pg/mL would not be detected (12,500 pg/mL X 0.004 = 50 pg/mL) and would, therefore, not contribute to human insulin measured in the host animal.

25. The recent development of two-antibody-site immunoassays for insulin has provided an alternative method for measurement of this hormone in the mouse. These immunoassays are operated by employing two antibodies raised to separate insulin epitopes. One antibody is tagged for detection and the other is immobilized to facilitate separation of the tagged antibody-insulin-immobilized antibody complex from the tagged antibody not bound to insulin. The amount of tagged antibody-insulin-immobilized antibody complex formed is directly proportional to the amount of insulin present. Manufacturers of these immunoassays have elected to tag the detection antibody by coupling it to either (1) a specific enzyme that is allowed to react with an appropriate substrate, yielding a spectrophotometricly detectable chromogen (the enzyme-linked immunosorbent assay [ELISA]), or (2) an appropriate chemiluminescent substrate that can be induced to emit light (the immunochemiluminiscent assay [ICLA]). At least seven of these immunoassays are commercially available as possible alternatives to the RIAs we have described for measurement of insulin in the mouse (see Table 1). Only the four ELISAs designed for measurement of mouse (rat) insulin should be considered as immediate alternatives to the present or other competitive mouse insulin immunoassays (see Table 1). The species specificity of these ELISA and ICLA assays is either unknown or so wide that only one combination of any two of them could be used immediately, in place of the present mouse and human RIAs, for assessing insulin secretion in mice bearing human xenografts. This combination of insulin ELISAs (EIA-2048 and EIA-2337) was used to monitor host and human insulin in the plasma of rats following in vivo transfection of hind limb soleus muscle with a single intramuscular injection of a wild-type human preproinsulin plasmid and a mutant construct engineered to facilitate furin cleavage (24).

Table 1

Insulin Immunoassays

Range6

Cross-reaetivityc

Species"

Format

(pg/mL)

Mouse

Human

Source

Ref

Mouse

ELISA

125-3750

100

N/A

Cat. no. 008-10-1150-01 ALPCO Diagnostics, Windham, NH

12,13

Mouse

ELISA

78-5000

100

200

Cat. no. INSKR020 Crystal Chem, Inc., Chicago, IL

Mouse

ELISA

50-3200

100

545

Cat. no. 90060 Crystal Chem, Inc.

14-22

Human

ELISA

15-1000

N/A

100

Cat. no. 008-10-1132-01 ALPCO Diagnostics

Human

ICLA

80-24,000

N/A

100

Cat. no. 33410 Beckman Coulter, Inc., Chaska, MN

23

Rat

ELISA

375-13,750

100

120

Cat. no. EIA - 2048 DRG International, Inc., Mountainside, NJ

24

Human

ELISA

15-2000

0.7

100

Cat. no. EIA - 2337 DRG International

24

Rat

RIA

50-2000

100

100

present chapter

9

Human

RIA

50-10,000

0.4

100

present chapter

"Species for which insulin assay was designed.

ftRange of standard insulin concentrations tested and based on 0.01 mL of standard or sample/well or tube.

^Percent as related to species for which the insulin assay was designed. ^N/A: not available.

"Species for which insulin assay was designed.

ftRange of standard insulin concentrations tested and based on 0.01 mL of standard or sample/well or tube.

^Percent as related to species for which the insulin assay was designed. ^N/A: not available.

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