Sheep antiserum developed against the human glycohemoglobin, Hb A.sub.1c, distinguishes this fraction from the major component Hb A.sub.o. Partial cross relativity is observed with Hb A.sub.1a and Hb A.sub.1b, as well as with analogous glycohemoglobins from mouse and dog hemolysates. The reactivity of the hemoglobins with this specific antiserum is abolished by the reduction of the keto group of the sugar ligand. The immunological specificity displayed provides the basis of a quantitative assay for Hb A.sub.1c, which is suitable for studies of clinical and experimental diabetes.

Hemoglobin (Hb) A.sub.lc is a glycohemoglobin with an amino acid structure 
which is identical to that of Hb A; the only detectable difference is the 
presence of 1-amino-1-deoxy-fructose attached in the 
2,3-diphosphoglycerate pocket to the N-terminal valine in the beta-chain 
of Hb A.sub.lc. The modification of Hb A to form Hb A.sub.lc is a 
continuous post-translational process, the rate of which is a function of 
the blood glucose concentration. The level of Hb A.sub.lc therefor 
reflects the status of the individual's carbohydrate metabolism. Normal 
adults have about 90% of their total hemoglobin as A.sub.o, 2-3% as 
A.sub.1a and A.sub.1b and 3-6% of their total hemoglobin as Hb A.sub.1c 
whereas the range in juvenile and maturity onset diabetics is 6-15% as Hb 
A.sub.1c. A similar increase in Hb A.sub.1c concentration has been noted 
in mice with genetic and chemically induced diabetes and in 
pancreatectomized dogs. 
Diabetes mellitus has been found to occur naturally or can be induced in 
virtually every species in the animal kingdom. Greater insight into human 
diabetes can be gained by studying this disease process in animals whose 
diabetes closely resembles the human condition. Such animal models include 
the diabetic dog and mouse; see R. Engerman et al. Diabetes 26: 760-769; 
K. P. Hummel et al. in Science 153: 1127-1128 (1966); and A. A. Like et 
al. in Am. J. Pathol. 66: 193-204 (1927). Both of these species 
demonstrates increased levels of Hb Al.sub.1c in the diabetic state: 
(Koenig and Cerami: Proc. Natl. Acad. Sci. USA 72: 3687-91 (1975) and 
Koenig, unpublished data.) 
These animal hemoglobins A.sub.1c cross-react sufficiently with the human 
Hb A.sub.1c antibody described herein so that their concentrations may be 
determined accurately by this RIA method. An RIA for animal Hb A.sub.1c is 
very useful because this technique requires only microgram quantities of 
hemoglobin, compared to milligrams for the widely used prior art column 
chromatographic method of Trivelli et al. described in New Engl. J. Med. 
284: 353-7 (1971). RIA techniques for animal Hb A.sub.1c are thus useful 
in facilitating the evaluation of new drugs and other forms of therapy 
designed to treat human diabetes and accordingly should facilitate 
research into the basic pathophysiology of this disease. 
Recent studies have indicated that the quantification of Hb A.sub.1c 
concentration is a useful means of assessing carbohydrate intolerance as 
well as adequacy of control in patients with diabetes: see Koenig et al. 
in New England J. Med. 295: 417-420 (1976). One of the difficulties in 
applying this measurement to clinical studies has been the technical 
problem of assaying Hb A.sub.1c in a large number of samples with the 
column chromatography method currently available which has been described 
by Trivelli et al. in New England J. Med. 284: 353-357 (1971). In order to 
circumvent this obstacle, we have studied the immunological properties of 
Hb A.sub.1c with a specific antiserum. The present invention applies these 
results to the radioimmunoassay of glycohemoglobins. 
OBJECTS OF THE INVENTION 
Accordingly, it is a general object of the present invention to provide an 
improved method for assaying hemoglobin A.sub.1c. 
Another object of the present invention is to provide anitbodies which are 
highly specific to hemoglobin A.sub.1c as an antigen, even in the presence 
of other, very similar antigens. 
Another object of the present invention is to provide an improved test for 
hemoglobin A.sub.1c which enables a single person in one day to conduct 
30-40 tests more readily than he could run the 2 or 3 tests per day in 
accordance with prior art methods. 
Yet another object of the present invention is to provide immunological 
assay for hemoglobin A.sub.1c which exhibits high specificity with respect 
to the particular hemoglobin, yet has sufficient species cross-reactivity 
to facilitate its use for the study of diabetes in experimental animals. 
Other objects, features and advantages of the present invention will become 
more fully apparent to those skilled in the art to which the invention 
pertains from the following specification and claims. 
SUMMARY OF THE INVENTION 
Briefly, the above and other objects, features and advantages of the 
present invention are attained in one aspect thereof by providing 
antibodies against human hemoglobin A.sub.1c which are substantially free 
of cross-reactivity against the human hemoglobin A.sub.o, A.sub.1a and 
A.sub.1b. 
In a second aspect, the present invention provides a radioimmunoassay 
method for detecting hemoglobin A.sub.1c which comprises reacting a blood 
or other body fluid sample with said antibodies and determining the 
presence of an antigen-antibody complex by radioimmunoassay. 
In a third aspect, the present invention provides an improved technique for 
the diagnosis of diabetes and other related diseases associated with 
elevated blood glucose levels by correlating the levels of hemoglobin 
A.sub.1c ; the method is especially suited for radioimmunoassay and 
sequentially reacts the antibodies with the test antigen alone, followed 
by reaction with the index antigen for a short period of time, e.g. about 
0.5 hour at incubation temperatures from room temperature to about 
37.degree. C. 
In a fourth aspect, the present invention provides a method for preparing 
such antibodies which comprises administering an antigenically effective 
amount of hemoglobin A.sub.1c antigen to an animal capable of producing 
antibodies thereto, preferably to an animal whose metabolism does not 
naturally form hemoglobin A.sub.1c, and collecting said antibodies from 
the animal. 
DETAILED DISCUSSION 
Because the transformation of hemoglobin A into hemoglobin A.sub.1c is a 
function of glucose concentration in the blood, there is a great need for 
a quantitative assay method which is rapid and easily conducted without 
prohibitively expensive equipment. By virtue of certain species 
cross-reactivity discussed hereinafter, the provision of such a test in 
accordance with the present invention now provides an additional tool for 
the study of diabetes and similar diseases causing an elevation in blood 
glucose concentration in standard laboratory animals. 
Highly specific antisera have been prepared that can recognize single amino 
acid substitutions in human hemoglobin by the radioimmunoassay (RIA) 
method, e.g. see Javid and Pettis in J. Lab. Clin. Med. 88: 621-626 
(1976); Rowley et al. in Blood 43: 607-611 (1974); and Garver et al. in 
Science 196: 1334-36 (1977). Differences between the structure of the 
hemoglobin used for immunization and that of the immunized animal's own 
hemoglobin largely determine the specificities of the antibodies produced. 
While a wide variety of different species can be employed as the immunized 
animal for preparing antibodies to hemoglobin A.sub.1c, it is presently 
preferred to immunize an animal whose metabolism does not naturally form 
hemoglobin A.sub.1c, e.g. the cat, goat, sheep, etc. Sheep hemoglobin does 
not react with 2,3-diphosphoglycerate and may lack the configuration of 
the "DPG pocket" which permits the glycosylation of the beta-chain 
N-terminus. Indeed, glycosylated hemoglobin cannot be demonstrated in 
sheep red cell hemolysates. The sheep, then, apparently recognizes the 
N-terminus of the human Hb A.sub.1c as antigenic. The immunodominant 
feature of this antigenic determinant must require the spatial 
conformation provided by 1-amino-1-deoxyfructose. Reduction of the keto 
group strikingly and selectively reduces the reactivity with 
anti-A.sub.1c, while leaving the affinity of other determinants for anti-A 
unaffected. The ability of the antibody to distinguish between a ketone 
and an alcohol might in fact reflect its ability to recognize a ring 
structure, since the keto moiety could readily form a hemiketal with the 
hydroxyl group of either carbon 5 or 6. The cross reaction of Hb A.sub.1a 
and Hb A.sub.1b with the antibody made to human Hb A.sub.1c is of interest 
since both molecules are probabaly glycohemoglobins. An exact structure 
has not been assigned to these hemoglobins. 
Dog and mouse Hb A.sub.1c react less well than their human counterparts. 
This observation suggests that the steric fit of the antibody includes 
more than the sugar molecule and probably extends to surface features of 
the protein adjacent to it. 
The specific immunologic recognition of Hb A.sub.1c has an immediate 
practical value. The quantification of this minor hemoglobin fraction is 
valuable in monitoring the control of diabetic patients, as has been noted 
in Koenig et al. in New England Journal of Medicine 295: 417-20 (1976). 
Currently available assays call for the column chromatographic separation 
of Hb A.sub.1c from the other hemoglobin components of the hemolysate. 
This is a tedious and not entirely accurate semi-quantitative method since 
only some 70-80% of the hemoglobin applied to the column is recovered. 
The level of Hb A.sub.1a +Hb A.sub.1b in hemolysates is generally less than 
50% that of Hb A.sub.1c and, under assay conditions, their contribution to 
the blocking of anti-A.sub.1c is less than 10% of a comparable amount of 
Hb A.sub.1c. It is therefore unlikely that these two minor 
glycohemoglobins contribute significantly to the radioimmunoassay value of 
Hb A.sub.1c. In 14 hemolysates in which Hb A.sub.1c was assayed by RIA and 
all three glycohemoglobins were measured by chromatography, correction of 
the RIA values for the contribution by Hb A.sub.1a+b did not significantly 
affect the correlation between the two methods. 
The RIA can be standardized with purified Hb A.sub.1c and Hb A.sub.o and is 
not subject to error due to the selective loss of one or another 
hemoglobin fractions from the samples. This method offers a number of 
advantages over available techniques. The assay is specific and 
reproducible, yet simple enough to permit the processing of 30 or more 
samples under identical conditions within a working day. It should find 
ready application in the study of diabetes in man, as well as in 
experimental animals. 
Hb A.sub.o and Hb A.sub.1c are immunologically identical except for a 
single antigenic determinant. Therefore, following the adsorption of 
anti-Hb A.sub.1c with Hb A.sub.o, the residual specific antibody has a low 
titer and affinity. This inherent problem has been overcome in the present 
system by two modifications of the usual RIA method: First, the antibody 
is incubated sequentially with the test and index antigens, rather than 
with a mixture of the two, thus maximizing the blocking of the antibody by 
the test antigen. Second, the incubation with the index antigen is 
generally limited to about 0.5 hour, rather than overnight, so that the 
displacement of antigen from the low-affinity antibody is minimized. 
Without further elaboration, it is believed that one skilled in the art 
can, using the preceding description, utilize the present invention to its 
fullest extent. The following preferred specific embodiments are, 
therefore, to be construed as merely illustrative and not limitative of 
the remainder of the disclosure in any way whatsoever. All temperatures 
are set forth uncorrected in degrees Celsius; unless otherwise indicated, 
all pressures are ambient and all parts and percentages are by weight.

EXAMPLE 1 
Preparation of Hemoglobin Samples 
Blood samples were obtained from normal individuals and from patients with 
diabetes mellitus. Hemolysates were made from saline washed red cells by 
hypotonic lysis. Stroma was removed by centrifugation. Total hemoglobin 
concentration was measured as cyanmethemoglobin, using the method of 
Cartwright described with "Diagnostic Laboratory Hematology", 4th edition, 
Grune and Stratton, New York (1972). Assay of the Hb A.sub.1c fraction was 
performed as previously described by Trivelli et al. 
The main hemoglobin fraction A.sub.o and the minor hemoglobins A.sub.1a, 
A.sub.1b and A.sub.1c were isolated and purified by column chromatography 
from the erythrocytes of normal volunteers following the procedures 
described in New England J. Med. 284: 353-357 (1971) and Proc. National 
Acad. Sci. USA 72: 3687-91 (1975). The Hb A.sub.o fraction thus isolated 
was then stripped of ionically-bound organic phosphates by dialysis in 
vacuum-expanded bags against 0.5 M NaCl--0.01 M sodium phosphate buffer, 
pH 7.0. The stripped Hb A.sub.o was then repurified by the above column 
chromatographic procedure. This process is necessary to prevent small 
amounts of Hb A.sub.1b --like species from forming in the Hb A.sub.o 
fraction; see V. J. Stevens et al. in J. Biol. Chem. 252: 2998-3002 
(1977). A similar chromatographic method was used for the separation of 
mouse and dog glycohemoglobins except that the equilibrating and eluting 
buffers were 0.05 M sodium phosphate, 0.01 M KCN, with pH 6.68 for the dog 
and pH 6.78 for the mouse hemolysates. The components A.sub.1a and 
A.sub.1b were eluted in one peak. The glycoproteins were concentrated by 
vacuum dialysis. All purified hemoglobins were stored at -20.degree. C. in 
the PCG buffer described below. 
For reduction with NaBH.sub.4, each glycohemoglobin was dialyzed against 
0.1 M sodium phosphate buffer, pH 7, and was subsequently reacted with a 
200-fold molar excess of NaBH.sub.4 at room temperature for one hour. The 
unreacted sodium borohydride was removed by dialysis. The synthesis of 
glycosylated dipeptides has been previously described by Koenig et al. in 
J. Biol. Chem. 252: 2992-97 (1977). 
EXAMPLE 2 
Preparation of Antibodies 
A cheviot sheep was injected biweekly with 10 mg of purified human Hb 
A.sub.1c in 3 ml water and 2 ml Fruend's adjuvant. The first 8 injections 
utilized complete adjuvant and the five subsequent injections were with 
incomplete adjuvant. Each 5 ml injection was given into 25 intradermal 
sites. Following the 8th injection, 500 ml blood was removed from the 
sheep on each non-immunization week. The blood was left overnight at 
4.degree. C. and the serum was collected by centrifugation and stored at 
-85.degree. C. This is referred to herein as the parent antiserum. This 
antiserum showed minimal difference in its reactivity with Hemoglobin 
A.sub.1c and Hemoglobin A.sub.o. This difference was amplified with 
progressive absorption of the antiserum with Hb A.sub.o at the expense of 
a considerable fall in antibody titer. Approximately 10% of the initial 
titer was retained as specific anti-A.sub.1c. Anti-A, the fraction of the 
parent antiserum that was retained on and eluted with the Hb A.sub.o 
immunoabsorbent, did not discriminate between Hb A.sub.1c and Hb A.sub.o. 
In the reaction of human hemoglobin A fractions with anti-A.sub.1c, the 
absorbed antiserum clearly distinguishes Hb A.sub.o from its glycosylated 
derivatives. Of the latter, Hb A.sub.1c is most effective in blocking the 
antiserum; Hb A.sub.1a and Hb A.sub.1b show about 5% and 10% of its 
reactivity, respectively. The four hemoglobin A fractions are equally 
effective in blocking anti-A, which recognizes only the determinants 
unrelated to the carbohydrate ligand. 
Treatment of the hemoglobins with NaBH.sub.4 had no effect on their 
reaction with the non-specific anti-A, but it led to a striking reduction 
of reactivity with anti-A.sub.1c, as shown in the following Table 1: 
TABLE 1 
______________________________________ 
THE SPECIFICITY OF BOROHYDRIDE REDUCTION 
FOR THE IMMUNODOMINANT DETERMINANT 
OF GLYCOHEMOGLOBINS 
Maximal blocking of 
Anti-A.sub.lc 
Anti-A 
______________________________________ 
Hb A.sub.1a, unmodified 
78% 97% 
Hb A.sub.1b, unmodified 
90% 94% 
Hb A.sub.1c, unmodified 
98% 91% 
Hb A.sub.1a, reduced 
18% 94% 
Hb A.sub.1b, reduced 
24% 89% 
Hb A.sub.1c, reduced 
19% 94% 
______________________________________ 
The common antigenic determinants of hemoglobin (Column 2) are present in 
all three glycohemoglobins and are not affected by borohydride reduction. 
The specific glycohemoglobin determinant (Column 1) is most reactive in H 
A.sub.1c, least so in Hb A.sub.1a, and is significantly altered by 
borohydride reduction. 
EXAMPLE 3 
Preparation of Radioimmunoassay Reagents 
The following reagents were prepared: 
TBS: (tris-buffered saline) 0.05 tris-hydroxymethylaminomethane, 0.1 M 
NaCl, pH 8.3. 
PBS: (phosphate buffered saline) 0.05 M sodium phosphate, 0.1 M NaCl, pH 
7.0. 
BGG: 2% bovine gamma globulin in TBS. This was used for the final dilution 
of all antisera for the radioimmunoassay. 
PCG: (phosphate-cyanide-glycerol) 0.1 M sodium phosphate, 1.5 mM KCN in 40% 
glycerol, pH 7.0. This buffer was used for storing hemoglobin samples at 
-20.degree. C., at which temperature they did not freeze. The storage 
stability of the samples was considerably enhanced by this means. 
Ammonium sulfate: Saturated solution, adjusted to pH 7.0 with 5 M NaOH. 
Antisera: An aliquot of the parent antiserum was repeatedly absorbed with 
agarose-linked Hb A.sub.o according to the method of Javid and Liang, 
described in J. Lab. Clin. Med. 82: 991-1002 (1973) and the specificity of 
the residual antibodies for Hb A.sub.1c was monitored as described below. 
The final preparation is designated anti-A.sub.1c. The cross-reacting 
antibodies which bound to the immunoabsorbent were eluted with 0.2 M 
glycine, pH 2.8, and dialyzed against PBS. This preparation is referred to 
as anti-A since it is directed against those antigenic determinants common 
to Hb A.sub.1c and Hb A.sub.o. 
Each antiserum was calibrated against the index antigen following the 
procedure of Javid and Yingling described in J. Clin. Invest. 47: 
2290-2296 (1968). For the parent antiserum and anti-A, the equivalences 
(micrograms Hb A.sub.1c bound/ml antiserum) were 1120 micrograms/ml and 
185 micrograms/ml, respectively. These antisera were diluted in BGG to a 
final titer of 0.375 micrograms/ml. Equivalent amounts of index antigen 
were used for these antisera. The calibration curve for anti-A.sub.1c did 
not show a sharp end point because of the low affinity of the antiserum; 
the equivalence was estimated at 100 micrograms/ml. This antiserum was 
diluted to a titer of 2 micrograms/ml and used in three-fold excess over 
the index antigen. This yielded lower values for "Antibody Control" (see 
below) and more reproducible results but did not alter the values 
obtained. 
For an index antigen, purified Hb A.sub.1c was iodinated with 125.sub.I by 
the chloramine-T method of McConahey and Dixon, described in the 
International Archives of Allergy and Immunology 29: 185-189 (1966). The 
hemoglobin, as obtained by chromatography, is in a buffer containing 
cyanide. This ion interferes with the oxidation of iodide to iodine. Prior 
to iodination, the cyanide must be removed from the sample by gel 
filtration or by dialysis. The specific activity of the preparations was 
about 1 mCi/mg. The antigen was stored as a 0.3 mg/ml solution in PCG 
buffer. Immediately prior to use, the antigen was diluted 100-fold in 
sheep hemoglobin (1 mg/ml). 
Test hemoglobins: For studies of the primary inhibition of antisera, 
purified hemoglobins were used in amounts ranging from 0.025 to 25 
microgram. A set of mixtures of purified Hb A.sub.1c and Hb A.sub.o were 
used as primary standards for the quantitative assay. These contained 0.0, 
1.5, 3.6, 7.5, 9.0, 10.0, 12.0 and 15.0% Hb A.sub.1c in Hb A.sub.o. Stock 
solutions, 2 mg/ml, were diluted 1:20 in sheep hemoglobin (0.5 mg/ml) and 
50 microliters of each mixture was used to construct the standard curve. 
Assay unknowns were hemolysates from normal and diabetic donors. These 
were diluted from stock solutions as described for the standards. 
EXAMPLE 4 
Radioimmunoassay (RIA) Testing: 
All antisera and hemoglobins were diluted as outlined in the preceding 
section. Reactions were carried out in triplicate for the standards, and 
in duplicate for other test hemoglobins. The assay consists of three 
stages 
Stage 1: The reaction mixture which contained 200 microliters antiserum and 
50 microliters test hemoglobin was incubated at room temperature for 30 
minutes. Each experimient included two controls. In the "Antibody Control" 
the test hemoglobin was replaced by the sheep hemoglobin diluent, 
permitting full reaction between antibody and index antigen in the 
subsequent stage. In the "Antigen Control" the antibody was omitted (only 
the BGG diluent was used) to establish the inherent solubility of the free 
index antigen under the assay conditions. 
Stage 2: Freshly diluted index antigen was added to each reaction mixture 
and further incubated at room temperature for 30 minutes. 
Stage 3: TBS, 1.45 ml and ammonium sulfate, 1 ml, were added sequentially 
to each tube with thorough vortex mixing. After 1/2 hours at 4.degree. C. 
the precipitated immune complexes were sedimented at 1,000.times.G for 10 
minutes. Supernatant radioactivity was calculated from the counts in 1 ml 
and was expressed as the percent of total counts. 
Percent Blocking of the Antiserum is defined as: 
EQU (T-B)/(A-B).times.100 
wherein the symbols are the percent supernatant counts for test hemoglobins 
(T), antigen control (A), and antibody control (B). 
For assay purposes, a standard curve was constructed by plotting the 
percent blocking for each of the primary standards against its known 
fractional content of Hb A.sub.1c and Hb A.sub.o. 
EXAMPLE 5 
Reaction with glycosyl dipeptides 
A number of glycosylated derivatives of the N-terminus of the human 
hemoglobin beta-chain were tested for their ability to inhibit the primary 
reaction between Hb A.sub.1c and its specific antibody. None of the 
compounds tested showed significant blocking activity, even in up to 
1,000-fold molar excess, as shown in Table 2: 
TABLE 2 
______________________________________ 
MAXIMAL BLOCKING OF ANTI-A.sub.1c ANTISERUM 
BY SYNTHETIC GLYCOPEPTIDES 
Substance Moles Added % Blocking 
______________________________________ 
Hb A.sub.1c 7 .times. 10.sup.-11 
95% 
Hb A.sub.1c, reduced 
7 .times. 10.sup.-11 
20% 
1-deoxy 
Mannosyl-valine, reduced 
2 .times. 10.sup.-9 
6% 
1-deoxy 
Mannosyl-valine, reduced 
1 .times. 10.sup.-9 
6% 
1-deoxy 
Galactosyl-valine, reduced 
2 .times. 10.sup.-9 
6% 
1-deoxy 
Galactosyl-valine, reduced 
2 .times. 10.sup.-7 
8% 
1-deoxy 
Glucosyl-valyl-histidine, reduced 
2 .times. 10.sup.-9 
9% 
1-deoxy 
Glucosyl-valyl-histidine, reduced 
2 .times. 10.sup.-8 
9% 
______________________________________ 
There is no significant blocking of the antiserum by glycopeptides in up to 
1,000-fold molar excess over totally blocking amounts of Hb A.sub.1c or 
partially reactive reduced Hb A.sub.1c. 
EXAMPLE 6 
Species Cross-Reactivity 
The Hb A.sub.o, A.sub.1a+b and A.sub.1c components of dog and mouse 
hemoglobin were tested for cross reactivity in the RIA system. The 
reaction of these hemoglobins with anti-A.sub.1c is qualitatively 
analogous to that of their human counterparts. The mouse and dog Hb 
A.sub.1c have about 15% of the reactivity of the human fraction for 
half-maximal blocking of anti-A.sub.1c. The A.sub.o component in each 
species is inactive, while the hemoglobins A.sub.1a+b have intermediate 
reactivities. 
EXAMPLE 7 
Quantitative assay of Hb A.sub.1c by RIA: 
The relation between the percent Hb A.sub.1c in the standard mixtures, and 
the percent blocking of anti-A.sub.1c was plotted as a standard curve. 
Mean and standard deviation for 9 consecutive experiments performed over a 
40-day period with the same index antigen were included. There was no 
progressive shift of the curve in any direction; the range of values for 
each sample represents the inherent limit of reproducibility for the 
method. The y-intercept of the curve shows residual cross reactivity of 
the anti-A.sub.1c with Hb A.sub.o. 
Assay values for hemolysates with more than 9% Hb A.sub.1c fell on the 
upper, shallower slope of the standard curve. In these instances the 
hemolysates were diluted with an equal amount of Hb A.sub.o ; the percent 
blocking then fell on the steeper portion of the curve. The corresponding 
percent Hb A.sub.1c was doubled to correct for the two-fold dilution of 
the hemolysate and more reproducible results were thus obtained. It was 
found more convenient to include one such dilution with every hemolysate 
assayed, rather than perform a second assay for those specimens with high 
Hb A.sub.1c. 
Thirty-three hemolysates were assayed for Hb A.sub.1c by both the RIA and 
the column method. In general, higher values are obtained by RIA. The 
regression line for these data is y=1.16.times.0.15 with a coefficient of 
determination r.sup.2 =0.72. In order to evaluate the source of the 
discrepancy between the two methods, the following experiment was 
performed: Two hemolysates, assayed by RIA to have 4.5 and 12.4% Hb 
A.sub.1c, respectively, were mixed in 10 different proportions. The 
original hemolysates and the mixtures were assayed in a blinded fashion by 
both the RIA and the column methods, and the results were compared with 
the values of the A.sub.1c calculated from the composition of the 
mixtures. An excellent correlation was observed for the RIA with the 
regression formula Y=.times.+0.06, and r.sup.2 =0.99. By contrast, the 
regression formula for the chromatographic analysis of the same mixtures, 
using the column values for the original hemolysates for the calculations, 
was y=1.025.times.-0.81, and r.sup.2 =0.87. Thus, the RIA method has a 
high degree of internal consistency and is linear throughout the range 
examined. 
The preceding examples can be repeated with similar success by substituting 
the generically or specifically described reactants and/or operating 
conditions of this invention for those specifically used in the examples. 
From the foregoing description, one skilled in the art to which this 
invention pertains can easily ascertain the essential characteristics 
thereof and, without departing from the spirit and scope of the present 
invention, can make various changes and modifications to adapt it to 
various usages and conditions.