Abstract
We describean amplifiedenzyme-linked immunoassay of humanproinsulinin serumthat detectsintactproinsulin and both the 32/33 and 65/66 split forms.The method usesthe lgG fractionof a polyclonal antibodyraisedin a guineapigagainstintactproinsulin, whichwe usedtocoat plastic microtiterplates. A sandwichwas formed with proinsulin by usinga monoclonal antibodyagainstC-peptide labeledwithalkalinephosphatase. We quantifiedthe reactionby usingthe enzyme amplificationprocedure, whichdetectedas littleintactproinsulin as 0.1 pmol/L. We foundnocross-reactivity withC-peptideinthe assay,and decreasedrecoveryattributable tothe presenceof insulin couldbe demonstrated onlywitha 30-foldexcessof this hormoneover proinsulin.
Abstract
AddItional Keyphrases: enzyme amplification assay C-peptlde
Abstract
Proinsulin, the precursor of insulin, is synthesized in the cells of the islet of Langerhans (1). It is converted to insulin and C-peptide by proteolytic cleavage at amino acids 32/33 and 65/66. Thus, insulin and C-peptide are released into the circulation with small amounts of both intact and partially cleaved proinsulin molecules (2).
Abstract
Human proinsulin, not surprisingly, cross-reacts with human insulin and C-peptide in many assays, and Heding (3) drew attention to the problem of specificity in such assays. The presence of the fragments might lead to an overestimation in competitive assays and an underestimation in sandwich assays. In normal individuals the contribution of proinsulin to insulin and C-peptide assays is fortunately relatively low. However, the high concentrations of proinsulin in some diabetic subjects (4) may lead to an overestimation of their concentrations of active insulin. A similar situation exists in patients with insulinomas, in which large amounts of proinsulin may be released into the circulation. To assess and understand the importance of proinsulin and insulin in metabolic regulation and in pathogenesis of disease, one must therefore have specific and sensitive assays of each of these substances.
Abstract
Since Steiner and Oyer (1) discovered proinsulin, many have attempted to measure it in blood. However, accurate measurement has been difficult because of the lack of pure intact human proinsulin for standardiza- tion and the problems of cross-reactivity with insulin and C-peptide with available antisera. Also, because the normal concentration of circulating human proinsulin is very low, a highly sensitive and specific assay is required.
Abstract
The problem of a pure standard preparation has been overcome by the development of recombinant DNA technology and its use in the synthesis of human proinsum (5). As for assay specificity, early attempts involved gel filtration to separate immunoreactive components before assay (6) or used an "insulin-specific protease" to selectively degrade insulin (7). However, competitive RIAs were largely unsuccessful because of their limited sensitivity and specificity. Nonetheless, Deacon and Conlon (8) reported such an assay involving a proinsulin-specific antiserum raised against biosynthetic human proinsulin, in a single guinea pig, yielding a batch that provided only a minimal quantity of specific reagent. This specific assay had a sensitivity of 4 pmol/L after an extraction step and a twofold concentration step. The development of assays with sufficient specificity and sensitivity to permit the detection of basal concentrations of proinsulin in human blood has had to wait until the development of the two-site immunometnc approach.
Abstract
have also been described by others (9-14) .
Abstract
Recently After a simple washing step, an enzyme-labeled monoclonal antibody against human C-peptide is added and incubated. After further washing to remove unbound enzyme-

Received June 10, 1991; accepted November 27, 1991.

tion and the problems of cross-reactivity with insulin and C-peptide with available antisera. Also, because the normal concentration of circulating human proinsulin is very low, a highly sensitive and specific assay is required.

The problem of a pure standard preparation has been overcome by the development of recombinant DNA technology and its use in the synthesis of human proinsum (5) . As for assay specificity, early attempts involved gel filtration to separate immunoreactive components before assay (6) or used an "insulin-specific protease" to selectively degrade insulin (7) . However, competitive RIAs were largely unsuccessful because of their limited sensitivity and specificity. Nonetheless, Deacon and Conlon (8) reported such an assay involving a proinsulin-specific antiserum raised against biosynthetic human proinsulin, in a single guinea pig, yielding a batch that provided only a minimal quantity of specific reagent. This specific assay had a sensitivity of 4 pmol/L after an extraction step and a twofold concentration step. The development of assays with sufficient specificity and sensitivity to permit the detection of basal concentrations of proinsulin in human blood has had to wait until the development of the two-site immunometnc approach.
Specific inununoassays
have also been described by others (9-14).
Specific inununoassays
Recently, Sobey et al. (15) have reported major progress in the production of a panel of monoclonal antibodies with different specificities for insulin-related molecules. They evaluated the utility of these antibodies in two-site immunoradiometric assays of human insulin, proinsulin, and proinsulins split at amino acids 32/33 and 65/66. All guinea pigs treated in this way produced antisera,, with the maximum titer being reached by six months, after which injections were discontinued.
Titer was determined
by reaction with lssIlabeled insulin, because all of the antisera raised against proinsulin crossreacted with human insulin.
Titer was determined
IgG fractions of the antisera were prepared by precipitation with ammonium sulfate. and synthetic human C-peptide were obtained from Novo Labs.
Reagents and Buffers
We observed a marked effect of water purity on C-peptide and proinsulin immunoassays in preliminary work. Thus, for all reagents, buffers, and washing solutions we used de-ionized water produced from a Millipore Milli-Q system (Waters Associates, Milford, MA).
Reagents and Buffers
The water obtained from this purification system was of a resistivity of 18. Comparison Method For comparison, we used an immunoradiometric assay of proinsulin. We iodinated 10 ,ig of antibody with 0.4 pCi of sodium I (ICN Biomedicals, Irvine, CA) by the Chioramine T method (20) . lodinated antibody was purified by gel filtration on a column of G-75 Sephadex eluted with phosphate buffer. The assay procedure was identical to the enzyme method except that the capture antibody was coated onto Immulon microwell strips (Dynatech, Chantilly, VA), which can be made up in a frame into a microtiter plate form. After incubation in the assay with the labeled antibody, we washed the strips three times with wash buffer. They are designed so that they can then be easily broken apart so the radioactivity of the individual wells can be counted with a gamma counter for quantifying the results.
Results
Assessment ofthePEP-001--alkalinephosphatase con-
Results
We incubated 100 pL of different dilutions of the nine HPLC fractions of the conjugate with 100 pL of proinsulin standards (0 and 20 pmol/L) on an antibodycoated plate for 24 h at 4#{176}C, and then treated the plate as described in the assay procedure. The color development is shown in Table 1 , which shows that fractions numbered 5-7 gave the greatest color reaction and were used for further work. Fractions 1-3 and 9 did not give significant color.
Results
Assay characteristics. A standard curve for human proinsulin constructed in these conditions with the kinetic estimation is shown in Figure 1 . The detection limit of the assay was 0.02 fmol of proinsulin per well, corresponding to 0.1 pmol/L in serum, as assessed by the response representing 2.5 SD greater than the mean zero response. Anti-prolnsullnantibody-coatedwells were incubated wIth 100 4. of promsulinstandard(20 pmoVL)or withzero standard(forassessment of nonspechic binding) and 100 1zLof conjugated antibody fraction at appropilate dilution. The rest of the procedure was as desaibed in the text. it has been difficult to assess the point at which a high-dose thook effect" occurs. However, when we used these same antibodies in an immunoradiometric assay, no hook effect was evident at proinsulin concentrations <1000 pmol/L. We determined the interassay CV from the mean of duplicate estimations in a limited number of assays (n=6). hnterassay CV was 16.1% at a mean serum concentration of 1 Serial dilution of a serum sample from a healthy subject gave results linearly related to the dilution factor from two-to 24-fold dilution in bovine serum (Table 2) . In healthy fasting subjects (n = 16) the mean promsulin concentration was determined as 4.73 pmol/L (± 2.92 SD). This is in good agreement with results from previous studies (8-11, 13-15,21) .
Results
The proinsulin response during a standard glucose tolerance test in a single healthy adult subject is shown in Figure 2 . Blood samples were withdrawn from an in-dwelling cannula while the subject was resting in an armchair. Glucose and proinsulin were estimated both by an immunoradiometric procedure and by the amplified enzyme method. The samples were taken before the subject ingested 75 g of glucose and then at 15, 30, 45, 60, 90, and 120 mm after the glucose ingestion. Cross -reactivity with structurally related peptides. Human C-peptide and porcine and bovine proinsulin did not cross-react at concentrations 10 000 pmoIJL. Given the lack of cross-reactivity of bovine proinsulin, it was convenient to use bovine serum for dilution of proinsulin standards. Standard curves were not substantially different whether prepared in bovine serum or human serum free of proinsulin.
Results
Interference by human insulin. We assessed the effect of insulin, which is normally present in much greater concentration than proinsulin (Figure 3) . Insulin concentrations as great as 500 pmol/L did not substantially interfere. This low interference with 30-fold excess insulin reflects the high binding capacity of the antibodycoated solid phase. At much greater excess of insulin, the analytical recovery of proinsulin may be expected to decrease, owing to insulin occupation of the antibody- at very high concentrations of proinsulin a similar effect might be expected.
DiscussIon
The assay described, an application of the amplified enzyme-linked immunoassay detection system (16-19) , represents an improvement of sensitivity for the assay of human proinsulin by 10-fold over previous techniques (10, 15) . This method enables the estimation of the low concentrations of proinsulin expected in fasting normal individuals and in some diabetics, in addition to its capability of measuring proinsulin concentrations in normal fasting subjects and in other pathological conditions. body specific for the C-peptide, such that the only 103.7 possibility for the sandwich formation is when the 94.4 pro#{252}in molecule is present. This approach was described by Rainbow et al. (14) in an indirect immunora-
988
diometric assay in which the signal was provided by an iodinated sheep anti-rabbit IgG and a rabbit antibody to human C-peptide. The capture antibody was a guinea pig anti-human insulin antiserum coated on a tube. For this purpose, it is convenient to use a polyclonal antibody recognizing insulin, which may be raised against insulin or proinsulin. Theoretically, the use of a polyclonal antiserum in a sequentially incubated twosite system may give rise to problems caused by recognition of the second site on the antigen by minor components of the mixed population of antibodies in the antiserum. This is precluded in a system for proinsulin assay when the capture antibody recognizes the insulin moiety. The second site, being in the entirely separate C-peptide sequence, is thus not affected by incubation with insulin antisera.
988
By incubating the serum sample in the coated well, and then removing it and washing the well before adding the labeled anti-C-peptide, interference by binding of C-peptide in the serum sample to the labeled antibody is precluded.
988
The 0.1 pmol/L sensitivity for proinsulin is combined
988
with a wide dynamic range by applying a kinetic technique to the measurement of color development. Human C-peptide and porcine and bovine proinsulins did not cross-react, even at concentrations of 10000 pmol/L. However, Sobey et al. (15) showed that nearly all of the partially processed proinsulin in humpn serum is split at amino acids 32/33 and that the concentration ofthis material may be as high as, or higher than, that of intact proinsulin in normal subjects, especially after glucose.The proinsulin split at amino acids 65/66 is only about 10% as frequent as the 32/33 split proinsulin.
988
Sobey et al. overcame the problem of specificity by developing a panel of monoclonal antibodies and combining them to produce assays for the intact form and the 32/33 and 65/66 split forms. Because an intact proinsulin assay cross-reacted only with the 65/66 split form, which circulates at such low concentrations that it could be ignored for all practical purposes, and because the degree of cross-reaction of intact proinsulin in an assay for the 32/33 split proinsulin was determined, they could correct the 32/33 split proinsulin estimations for the presence of known amounts of intact proinsulin. Such antibodies would be of great value in amplified enzyme procedures to achieve both sensitivity and specificity for all three peptides.
988
The assay we describe was responsive to intact promsulin and to both of the split forms and therefore should reflect the total circulating proinsulin. The concentrations observed in human serum are in line with previous reports, though the antibody pairing we used actually detects the 32/33 split form better than it does intact proinsulin. Because the 32/33 split proinsulin is the predominant circulating form, especially after glucose ingestion, this implies an overestimation of the actual proinsulin concentration if intact proinsulin is used as standard Detection of the very low concentrations of the 65/66 split proinsulin alone would require even greater sensitivity. Attaining this is within the scopeof the general method ofenzyme amplification, however, by increasing the "gain" of the amplification step (22) In such situations, the proinsulin mea- However, the simplest way to circumvent the problem would be merely to dilute the sample with bovine serum, which can be done to at least 24-fold dilution without concern about assay performance.
988
Concentrations of insulin and proinsulin would thereby be brought well within the concentration suitable for the amount of antibody coated to the wells.
988
In serum from patients with longstanding insulindependent diabetes mellitus, which might contain insulin antibodies, we have observed poor recovery of promsulin, presumably owing to competition in the system by the endogenous antibodies. In such cases, precipitation of the IgG would be essential before analysis. This phenomenon also precludes the use of such serum for preparing proinsulin standards.
988
The amplified enzyme reagents we used were those in the standard Ampak kit supplied by Novo Nordisk Diagnostics; we followed the manufacturer's instructions strictly. It is vital to avoid any possible crosscontaminationof reagents because the amplification procedure also amplifies any nonspecific signal-which is of special importance in determining low concentrations of analytes.
988
The amplified enzyme procedure represents a rapid and technically simple procedure for estimating proin- 

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