340 The Immunoassay Handbook
straddling the turn of the century, several workers noted
that the serum of the immunized animal could render the
broth from the bacterial culture turbid. This also was spe-
ciﬁc to the species used, but the ﬁrst suspicion that there
were nonspeciﬁc mechanisms at work also evolved. Should
the culture broth from a related organism be used, turbid-
ity developed, but to a much lesser degree, and often
agglutination of the related organism did not occur. Bordet
also noted that antibody to rabbit red cells hemolyzed
these cells, as did the serum from a naïve animal, unless the
serum was heated to 55°C. Perhaps this was ﬁrst observa-
tion of the action of complement. During this time, Paul
Ehrlich, working at the new Frankfurt Serum Institute, was
developing what was to become his “side-chain theory.”
An illustration shown from Ehrlich’s summary (Ehrlich,
1901) expanding his theoretical plan (Fig. 1) was an attempt
to organize what was known about the body’s reaction to
toxic (immunological) insult. While the nomenclature in
the diagram is somewhat different from today’s, the con-
cept is easily recognized from the diagram. The cell and its
surface structures, membrane proteins (antigens), are tar-
geted by “toxins” stimulating the cell to produce more
receptors, if it survives. These receptors were believed to
be shed into the serum to act as speciﬁc targets for free
toxin and bind to it, i.e., an antitoxin. In Ehrlich’s theory,
most of the terms appear that we associate with the immune
reaction today. Paul Ehrlich received the Nobel Prize in
Physiology or Medicine in 1908 shared with Ilya Ilyich
Mechnikov from the Institute Pasteur (Ehrlich, 2004).
Further insight was provided by Uhlenhuth (1903) who
mixed reactants, antiserum, and culture broth, layered so as
to create an interface at which precipitation, the ring test,
developed. Subsequently, ﬂocculation was observed with
the speciﬁc bacteria or at least species closely related to that
originally injected into the animal (Calmette and Massol,
1909). The use of a gelatin matrix as a medium for one of
the reactants was introduced soon after (Nicolle et al., 1920).
The importance of time as a variable in the development of
immunoprecipitates was appreciated when it was observed
that with proper concentrations of both reactants, the
development of precipitates was most rapid; the concept of
“equivalence” evolved (Ramon, 1922). Additionally, at high
antibody concentration, reaction time diminished inversely
with the concentration of antigen (Hooker and Boyd, 1935).
Inhibition of reaction in situations of excess antibody and
more so with excess antigen had been previously noted. The
latter, however, was not to be fully appreciated for several
decades as antigen excess. Further understanding (Dean
and Webb, 1926) came when time was measured against the
concentration of the two reactants. The shortest time for a
given amount of precipitate constituted the optimal propor-
tion, an early assessment of immunological potency.
FIGURE 1 Ehrlich’s “side-chain” theory (1901). The terms Ehrlich used are different from today, however, the concept is clear. Cellular antigens,
toxins, antitoxins (antibody), and complement are all represented.
341CHAPTER 4.1 The Foundations of Immunochemistry
Temperature, ionic strength, pH, and agitation also had
been observed to affect the degree of immune precipitation.
Detailed work by Heidelberger and Kendall (1935a,b,c) and
Goettsch and Kendall (1935), shown in Fig. 2, ampliﬁed
previous publications. These workers used a variety of anti-
gen–antibody systems including a highly colored antigen
(azo-albumin) that allowed careful observation of the anti-
gen–antibody reaction under a variety of reactant-pair con-
centrations. They also gave credence to the theory that
antigen–antibody reactions obeyed the “laws of classical
chemistry.” Also demonstrated was that as an animal is
immunized, its antiserum changes in precipitating charac-
teristics, believed to be due to an expansion in the number
of antigenic sites on the antigen recognized by the animal’s
immune cells. Furthermore, they showed that there were
several levels of what would now be termed avidity. It
should be pointed out that at that time and until the early
1970s, immunization was carried out by intramuscular
injection of enormous quantities of antigen. The result
was antisera of lower than expected afﬁnity and from what
was believed to be pure antigen any number of ancillary
antigen–antibody pairs developed (see below). As a result,
this careful work supplied much of the theoretical informa-
tion leading to today’s quantitative analysis of serum pro-
teins. These experiments used the laborious analysis of total
nitrogen in a precipitate, the Kjeldahl reaction. Instrument
analysis up to this point was used to indirectly measure the
product of the reaction in milligrams of nitrogen.
Critical to what was to occur in the next few years, work
in other disciplines was progressing. An early worker
(McFarland, 1907) made use of a homemade comparator
or “nephelometer” (εϕελÑ or “the mist”) as it was called, to
measure the numbers of bacteria in a sample to measure
the “opsonic index” of vaccines. Perhaps this was the ﬁrst
effort to measure the quality of immune reagents.
The measurement of fat in blood was a laborious process
in the early 1900s. A worker (Bloor, 1914, 1928) published a
turbidimetric technique for measuring lipids in tissue and
blood. After extracting tissue fats into an ether–alcohol mix-
ture, the resultant extract was evaporated to dryness and then
dissolved in water. Measured in a Richards comparator, the
turbid solution gave, for then, acceptable results. The
FIGURE 2 The original Heidelberger curve as published in 1935. Milligrams of pneumococcal polysaccharide precipitated by homologous antibody
on the ordinate and milligrams of nitrogen (N) on the abscissa; both per cubic centimeter.
342 The Immunoassay Handbook
drawbacks of nephelometers of the time were recognized
(nephelometric results were not proportional to the mass of
the suspended precipitate), and a turbidimetric instrument
(Duboscq colorimeter—a comparator) was substituted
(Denis, 1921). These devices had all been used exclusively
within the biochemical community. A new modality was
introduced (Libby, 1938a,b): the numerical measurement of
light scattered from an immunoprecipitate.
During this early period in the evolution of immunochem-
istry, several major discoveries had been made
G Animals react by producing serum factors (antibodies)
when injected with either the whole noxious agent or
extracts thereof (antigens).
G The animals producing these proteins can protect
themselves against most of the effects of these agents.
G Serum from the injected animals causes ﬂocculation of
the agents in culture media.
G Serum produces turbidity when the ﬁltered broth in
which the agents have been grown is mixed with it.
G Related species of the immunizing agent also induce all
of the above reactions to a lesser degree.
G The precipitate produced in vitro, under controlled cir-
cumstances, can be quantiﬁed.
G The reaction of antibody and antigen produces a pre-
cipitate in a gel matrix that provides some information
about the physical characteristics of the constituents.
G Simple instruments can be used to give numerical
results for a reaction between antiserum and antigen.
Qualification by Diffusion
in Gel (1861–1977)
The transition from reaction in free liquid to a semisolid
matrix, gelatin, spawned new investigation that made use
of considerable work that hearkened back 50 years. As
early as the ﬁrst decade of the twentieth century, Bechhold
(1905) and Bechhold and Ziegler (1906) described chemi-
cal reactions forming in a gelatin matrix between silver
nitrate and NH4O2 including an image of multiple pre-
cipitin rings from the single chemical pair. Initially, it was
assumed that the liquid phase of a gel presented no diffu-
sion limitations. However, it soon was appreciated that
even small molecules were inhibited during diffusion.
This, therefore, presented complications to the quantita-
tive measurements being carried out at the time. Complex
mathematical formulas (Friedman, 1930) incorporating all
the characteristics of the molecules, medium and physical
constituents of the test were used to resolve these issues.
Thus, the concept of a “diffusion coefﬁcient” was created
(Stiles, 1920, 1923). Awareness that macromolecules such
as proteins were affected by diffusion in gels had been pre-
viously described from a different point of view as far back
as the mid-nineteenth century (Graham, 1861). A curious
phenomenon, which dogs workers in this ﬁeld to this day,
was described by Liesegang (1929) and bears his name.
Liesegang rings are observed during precipitation in antigen–
antibody systems forming in gels and also in certain geo-
logical formations. These bands are described as rhythmic
precipitates formed from the reactants, which appear to
become partially soluble only to form again at some distance
further on. Certain geological formations present in the
same manner, from chemical interactions as liquid solvents
migrate through rock mobilizing solute. The observation of
these multiple bands confused workers trying to dissect the
protein constituents of animal serum. Were these bands evi-
dence of related, but somehow different forms of a single
species or were they several completely different species?
Work over 2 decades later was to provide an explanation.
A visible ring of precipitate was observed (Petrie, 1932)
when bacterial colonies on a culture medium reacted with
incorporated antiserum (Fig. 3). Petrie theorized that the
precipitin rings or “halos” were produced by soluble con-
stituents (polysaccharides) of the bacterial colony (Pneumo-
precipitates or “Liesegang rings” under a microscope. The
possibility that this ring phenomenon could be exploited to
type certain bacteria proved to be useful (Sia and Chung,
1932). The precipitin reaction in agar gel format was com-
bined with the tube method (“ring test”) of Uhlenhuth by
Oudin (1946, 1948, 1949). In this development, agar con-
taining immune serum (at low concentration) ﬁlls the lower
portion of a thin glass tube. Over this is layered an aliquot
of soluble antigen. After incubation at constant tempera-
ture, precipitin bands develop that migrate from the gel–
liquid interface into the gel column. The result is a complex
system of antigen–antibody precipitates developing as they
meet. Each pair acts independently of all others unless the
antigens are related immunologically. After several hours of
diffusion, a series of bands develop. Many features can both
complicate and enhance the results. These include
G many overlapping bands,
G the temperature at which the reaction occurs,
G the mass of the antigen molecules (some types having
several molecular sizes),
G the makeup of the solvent,
G the characteristics of the gel.
FIGURE 3 The observation of Petrie (1932) that brought attention to the diffusion of antigen into an antibody-containing gel producing a “halo” or ring.
343CHAPTER 4.1 The Foundations of Immunochemistry
For single antigen solutions, semiquantitation is possible.
Beyond this, complexities make only crude quantiﬁcation
possible. Band migration occurred as the result of con-
tinuous immunoprecipitation and dissolution. The belief
that this was the Liesegang phenomenon was dispelled
when Oudin used a multiple antigen–antibody system,
which produced several bands that were believed to be
the result of several superimposed and speciﬁc precipi-
tates migrating independently. This was not to dispel the
true Liesegang phenomenon, which can develop with a
The Oudin method of assessing the concentrations of
antigen and antibody became well recognized. Ouchter-
lony applied the one-dimensional, antigen from one well
and antibody from the other (double diffusion tech-
nique) to double diffusion in two dimensions, when he
allowed bacteria to grow in a gel matrix containing anti-
serum (Ouchterlony, 1948, 1949a,b). In this model, a
sharp ring of precipitate developed around those colo-
nies that produced toxic antigen (Fig. 3). Thus, this sim-
ple technique became a quantitative means of assessing
antiserum potency. In the ensuing years, many publica-
tions appeared that explored permutations of this
uncomplicated yet elegant means of using the exquisite
sensitivity and speciﬁcity of the animal’s immune system
to explore the evolving topics of protein chemistry.
Ouchterlony (1958) summed up the classiﬁcation of
serological reactions thus
“… simple, complex and multiple systems. A simple
precipitation system is one in which an antigen or hapten
reacts with the corresponding antibody. A complex system
is one in which several closely related antigens of varying
speciﬁcity react with a single antibody, originated by, and
corresponding to, one of the antigens. Finally, a multiple
system is one in which several different antigens react with
This classiﬁcation still stands although the physical dimension
of the three types is lost, sometimes to our loss, in modern
automated, one-dimensional, or “Black Box” instruments.
An early experiment that was to become more detailed
in work in thin gel ﬁlms was described (Surgalla et al.,
1952). A four part upper chamber in an Oudin tube format,
each containing a different antigen, is sealed to the surface
of a single gel containing antiserum. As the antigens dif-
fuse downward, out of the compartments into the gel, they
come into intimate contact as they diffuse laterally, in the
presence of antibody. In this manner, the relationships of
one antigen to another can be assessed easily. The concept
of identity/nonidentity had begun.
What was soon to become a powerful new tool began as
“the simple diffusion in two dimensions” (Petrie, 1932). In
this approach, the familiar halo precipitates were visualized
by indirect light, as we do today. It was believed that each
ring corresponded to one antigen–antibody pair. The
exception is the possible rhythmic precipitation–resolution
representing Liesegang rings mentioned above, yet are pro-
duced by a single antigen–antibody pair. Perhaps the ﬁrst
use of the term “radial diffusion” appeared in this work.
Important to these workers was the requirement that there
be a balance of concentration between the reactants with
the potential of no visible reaction at either extreme of anti-
gen or antibody excess. This situation was to become a
signiﬁcant concern 3 decades later when automated instru-
ments came into use.
Another permutation of the format in which reactants
were introduced to each other came in the “Basin Plate
Technique” again introduced by Ouchterlony (1958). An
inﬁnite number of physical arrangements were possible
and used to enhance the relationships between develop-
ing precipitin lines. In this way, complex or multiple sys-
tems could be teased apart to fully describe the
relationships in the reactant system (Fig. 4). Elegant dif-
fusion patterns were shown in this work enhanced by the
physical arrangement and shapes of the wells. In this way,
diffusion time could be used to overcome overlapping
precipitates with the result that the individual constitu-
ents could be visualized. Once again, new terms were
introduced as a result: “reaction type 1—identity,” “reac-
tion type 2—nonidentity,” and “reaction type 3—partial
identity” (Fig. 5). Manipulating the positions of the wells
allowed a plethora of subcategories for each. By placing a
FIGURE 4 (a) Actual image of a Type 1a precipitin pattern with
precipitin arcs fusing indicating immunological “Identity” (Ouchter-
lony, 1958, Fig. 23A). (b) Actual image of Type 2 precipitin pattern with
individual arcs having no common precipitate, i.e., no fusion, indicating
“nonidentity” (Ouchterlony, 1958, Fig. 28). (c) Pattern of immunodiffu-
sion with upper left square well containing many different antigens, the
center well containing a polyspeciﬁc antibody and the upper right well
containing two antigens, only one of which is present in the upper right-
hand well (Ouchterlony, 1958, Fig. 30F).
344 The Immunoassay Handbook
series of wells around a central well, Wadsworth (1963)
described a microtechnique to study antigen–antibody
relationships and do so with much smaller amounts of
reactants and also to reduce the time to completion.
Crowle published many papers describing how the physi-
cal arrangement of wells could be used better to tease
apart the immunochemical characteristics of protein
populations (Crowle and Atkins, 1974; Crowle, 1977;
Crowle and Cline, 1977; Emmett and Crowle, 1982). He
published informative texts on the topic of immunodiffu-
sion in gel (Crowle, 1980). This uncomplicated proce-
dure found use in the screening of rheumatic patients for
speciﬁc antinuclear antibodies where different patterns of
reactivity yielded valuable diagnostic information.
Another step improving interpretation of immunopre-
cipitates was the introduction of staining materials (basic
fuchsin) and ﬁxation (acid alcohol) by Björklund (1954), to
enhance the visibility of immunoprecipitates. Previously,
dark-ﬁeld illumination for interpretation or photography
was used. A year later, Sudan Black and Oil Red O were
used to demonstrate certain precipitates, presumably lipo-
proteins (Burtin and Grabar, 1967). In 1956, a review of
staining techniques was published (Uriel and Grabar, 1956;
During this time interval, visual assessment of the reaction
products of immunochemistry was the only means avail-
able. As a result, immunochemistry was still something of
an art form.
G Reactants allowed to come into contact by diffusion in
a transparent matrix gave information about the nature
of the constituents by forming visible precipitates.
G Antigen and antibody reacting in this format produced
inﬁnitely variable patterns, and the physical position-
ing of reactants could be adjusted to resolve
G Analysis of these patterns could be used to understand
what had previously only been seen as turbidity in the
Qualitative Analysis by
Immunoprecipitation in agar gel after ﬁrst being separated
by electrophoresis resulted in complex precipitates pro-
duced by reaction with polyspeciﬁc antiserum. The ﬁrst
publication of the technique was by Poulik (1952) where
the combination of electrophoresis of protein was followed
by diffusion of the appropriate antiserum. Traditionally,
the method has been ascribed to Grabar and Williams,
who published their paper a year later in 1953 (Fig. 6)
without recognizing the prior publication. This paper also
did not receive recognition in the excellent and encyclope-
dic review by Verbruggen (1975).
The new technique was called immunoelectrophoresis and
was immediately used to study a wide variety of protein
mixtures (Williams and Grabar, 1955) including allergens
(Feinberg, 1957; Lǿwenstein, 1978), plant and microbio-
logical proteins, and tissue extracts. The vast literature on
the subject was reviewed, in extensa, by Verbruggen (1975).
Immunoelectrophoresis became the standard for the clini-
cal study of monoclonal immunoglobulins in human blood
serum and remained so for over 2 decades in the micro-
scope slide format proposed by Scheidegger (1955). Sev-
eral workers attempted to extract some form of quantitative
information for immunoelectrophoresis without success
(Clark and Freeman, 1967). Weime (1965) examined the
use of agar and discussed the strong endosmotic ﬂow of the
material. This characteristic interfered with the movement
of proteins through the matrix despite the gel’s large pore
size. Very large molecules could move as though in a free
medium, however, interaction with the chemical matrix
itself caused problems that would not be resolved for 2
The introduction of agarose, a fraction of traditional
agar, followed Hjerten’s work in 1961 and that of
Brishammar and coworkers in the same year (1961). A
miniaturized electrophoretic technique appeared 2 years
later (Vanarkel et al., 1963), and zone electrophoresis in
FIGURE 5 Diagrammatic examples of double diffusion with: identity
(a,b), nonidentity (c), and partial identity (d,e) (Crowle and Cline, 1977,
FIGURE 6 The ﬁrst published image of immunoelectrophoresis
(Grabar and Williams, 1953). A polyspeciﬁc antibody has been placed in
the central horizontal trough and two samples of human serum in each
of the two wells above and below the center of the arcs. IgG is at the far
left and albumin at the right.
345CHAPTER 4.1 The Foundations of Immunochemistry
agarose appeared about the same time (Hjerten, 1963).
Further use of agarose appeared (Uriel and Avrameas,
1964) and soon after its use for immunoelectrophoresis
was published (Suellmann, 1964; Leise and Evans, 1965).
In 1966, Uriel proposed the use of an acrylamide–agarose
mixture for electrophoresis laying the groundwork for
sophisticated and high-resolution isoelectric techniques to
follow later (Arnaud et al., 1977). Cellulose acetate mem-
branes were used (Marengo-Rowe and Levenson, 1972),
however, the modiﬁcation did not catch on.
Immunoelectrophoresis was used for close to 2 decades
as the main method to identify and characterize mono-
clonal immunoglobulins for clinicians and to investigate
new proteins in research. The technique, however, did
require a considerable amount of experience to appreci-
ate the nuances of the beautiful patterns. A modiﬁcation
of the procedure by which antiserum was layered over the
electrophoretic pattern produced from a single well simi-
lar to immunoelectrophoresis, began a new and more
practical application (Wilson, 1964; Afonso, 1964, 1968).
This technique was modiﬁed by applying antiserum over
a conventional electrophoretic pattern using Laurell’s
technique in agarose (Jeppsson et al., 1979; Alper and
Johnson, 1969). After exposure to monospeciﬁc antisera,
serum proteins that occurred in phenotypic patterns, and
serum proteins that degraded, produced patterns that
resembled a standard stained electrophoretic, except that
only bands of the protein in question appeared, all others
having been washed away. They coined the word immu-
noﬁxation, which has remained in use. Our group
(Ritchie and Smith, 1976a,b,c,) described a modiﬁcation
of Alper’s method that improved resolution and decreased
the volume of antiserum required. Antiserum was applied
with a strip of cellulose acetate saturated with whichever
reagent was indicated. In this way, several antisera could
be applied to one sample electrophoresed in multiple
lanes or one antiserum applied to multiple samples in
adjacent lanes. The ability to interpret patterns became
unambiguous since the immunoﬁxation pattern had
exactly the same proportions and conﬁguration as the
standard stained electrophoresis pattern except that the
protein(s) of interest was the only band(s) visible. Some
workers added the step of excising one lane for standard
Amido Black staining. Subsequently, immunoﬁxation was
adopted by several commercial suppliers, and immuno-
electrophoresis was relegated to the archives or used only
for specialized research purposes by experienced workers.
Immunoelectrofocusing (Catsimpoolas, 1969) was a fur-
ther step toward improving resolution and characteriza-
tion, and the use of antiserum overlays on polyacrylamide
gels followed. Performed in plastic membranes, antigens
were adsorbed in one dimension, in a slotted chamber,
rotated by 90°, and exposed to antibody in the same
chamber, providing an efﬁcient means of analyzing a
number of antigens vs any number of sera (Kazemi and
Finkelstein, 1990; Ritchie et al., 1992). The technique
was called checkerboard immunoassay.
Interest in improving the visibility of immune precipitates
in gel led to the introduction of several additives to the gels.
Renn and Evans (1975) had been instrumental in developing
several modiﬁed agaroses with tailored isoelectric points.
During this work, brighteners were explored, such as
tannic, picric, and acetic acids. Other precipitants were tested
including phosphotungstic and phosphomolybdic acids,
sodium vanadate, sodium tungstate, and riboﬂavin. Phos-
phomolybdic acid was selected as being the most effective in
differentiating immunoprecipitates from the background.
Coincidentally with the development of more modern
quantitative immunochemistry, qualitative techniques
appeared aided by the application of an electric ﬁeld.
G By simply applying a monospeciﬁc antiserum to an
unﬁxed good quality serum protein electrophoresis, the
immunochemical characteristics could be used to ﬁx
the reactants in place in an identical pattern. Immuno-
electrophoresis was discovered.
G By using an electric ﬁeld, antigen and antibody could be
driven into contact thus reducing the time to test
G Protein constituents of serum, tissues, and allergens
could thus be separated and characterized by reaction
with speciﬁc antisera.
G Staining agents enhanced the visualization of proteins.
Quantification of Antigens
by In-Gel Immunochemistry
When precipitin analysis became available to clinical med-
icine, the focus was altered toward a more practical, and as
a result, more widely used procedure, often by less skilled
workers. Of paramount interest was the need for numerical
rather than qualitative answers. Understanding the import
of these words took several decades to be fully appreciated.
To quantitate or to quantify incorporates the concept of
measurement of a feature of an object (mass, length, hard-
ness, color, electrophoretic mobility) or physical force
(temperature, intensity, lumens, frequency, speed) familiar
to the laboratorian. This is in contrast to nonnumerical
estimation (gender, phenotypic identity, race, taste, spe-
cies). The former requires numerical representation while
the latter speaks to other more imprecise descriptors that
shade into judgment or artful interpretation, common-
place in clinical medicine. The requirement to derive a
measurement for a parameter can only be expressed
numerically, thus requiring several truths. A measurement
must relate to a standard unit and device to derive that
number precisely and accurately. Immunochemistry was
for years denied access to measuring instruments; not
because they were not available, but because they existed
in separate and remote disciplines.
The observation that diffusion of some chemical reac-
tions in gelatin produced rings of precipitate dates back to
the work of Bechhold (1905) and Bechhold and Ziegler
(1906). See comments above under “Qualitative diffusion
in gel.” In 1932, Petrie also noted rings of precipitate when
colonies of bacteria were grown on agar that contained
antiserum to that species (Fig. 3). However, these workers
did not propose a quantitative relationship between the
346 The Immunoassay Handbook
concentrations of the reactants and the diameter of the
rings. Ouchterlony presented two ﬁgures in his treatise of
1958 showing “halo reactions,” noting that time and the
concentration of antigen affected the size of the rings.
In a project to resolve some of the technical problems
with the Oudin single dimensional immunodiffusion
method (e.g., the long time to completion and the nonlin-
earity for large molecules), radial immunodiffusion (RID)
was conceived. In1963,2 Heremans, Mancini, and coworkers
(Ritchie and Graves, 1972; Mancini et al., 1964, 1965) pub-
lished work describing the single radial immunodiffusion
method (Fig. 7a,b). The method was immediately popular
and made possible quantifying protein concentrations inex-
pensively, reasonably quickly (36–48h) and even with anti-
serum that was of low potency.
No equipment was required other than illumination and
a simple yet accurate measuring instrument. The method
2When the question was asked of one of the original group that investigated ways
of quantifying serum proteins in serum, “How did it come about?,” he wrote
“Yes, I remember what led up to the Mancini method. At Joseph Heremans’ lab, and
with his advice, we were all trying to improve the ‘Oudin’ technique of speciﬁc
immunological titration of antigens, which was using small glass tubes containing agar
homogeneously mixed with speciﬁc antiserum against one protein. This was the ‘linear
immunodiffusion method’. If my memory is correct, it was initially the idea of Angelo
Carbonara (now deceased) who, together with Giuliana Mancini (they were not yet
married), were working with us at Heremans’ lab in Louvain, in 1962; but G.
Mancini devoted her Mémoire de Licence en Sciences Médicales to the subject, and
obtained her scientiﬁc degree in 1964.”
Jean-Pierre Vaerman, coworker, Brussels, Belgium, 2003
is still in use today in the developing world with commer-
cially viable products.
In 1965, a modiﬁcation of Mancini’s work was published
(Fahey and McKelvey, 1965) that became the standard in
the USA. By shortening the time to completion of RID to
less than 24h, the method was more attractive to laborato-
ries pressed for timely results, although the precision of
the shorter incubation time was less than the original
method and unacceptable by today’s standards. Commer-
cial ﬁrms also adopted this modiﬁcation. Fortunately, both
formats can be used, short vs long development time, to
suit individual preferences.
Laurell (1965) presented a new and more sophisticated
and complex method referred to as two-dimensional, or
crossed immunoelectrophoresis. A year later yet another
simpler format for individually quantifying many proteins
was introduced, based on electroimmunodiffusion (EID).
Eventually, the technique would be appropriately called
rocket electrophoresis (Laurell, 1966a,b) (Fig. 8). This
highly successful method had the advantage that antigen
migrated several centimeters rather than a few millimeters
as in other methods such as the recently introduced RID
technique of Mancini. Like RID, EID only required a
potent precipitating antiserum. This enabled any worker
with good quality antiserum the ability to quantify any
protein that was in reasonable concentration, about
10mg/L, and which had an isoelectric point committing
the protein to move in the electric ﬁeld. Most serum pro-
teins then identiﬁed, with the exception of the immuno-
globulins, were accessible to quantitative analysis by almost
any laboratory. The need for high-quality precipitating
antisera was a great stimulus to manufacturers of commer-
cial antiserum, some producing excellent material but oth-
ers failing to do so, as will be discussed below. A close
associate of C.-B. Laurell supplied this information for the
same question of how this method came about
FIGURE 7 (a) Relationship of antigen concentration (Y-axis) vs
diameter of precipitin ring (X-axis) (Mancini, 1964). (b) Image of actual
unstained gel in Mancini et al. (1965). Dilutions of human serum
albumin were added to each well as marked.
FIGURE 8 The gel ﬁlm contains a monospeciﬁc antiserum. Each well
was ﬁlled with samples containing the homologous antigen. After an
electric ﬁeld was applied (anode above, cathode below) for a speciﬁc
time, the height of the peaks is found to be proportional to the
concentration of the antigen (Laurell, 1966a).
347CHAPTER 4.1 The Foundations of Immunochemistry
“Joseph Heremans and Carl-Bertil Laurell agreed during
the autumn of 1965 that there was a need for
quantitative determinations of proteins as a complement
to agarose electrophoresis. In early 1966 Mancini with
Heremans reported about the success with the radial
immunodiffusion technique and CBL was stimulated and
stressed to modify the crossed immunoelectrophoresis ( for
use with a) monovalent antiserum. The quantitation of
alpha1-antitrypsin, which up to then had usually been
estimated from intensity of the electrophoretic band was
successfully demonstrated only a short time before (Störiko
and Schwick, 1963).
A series of diluted serum samples (3–4) was applied into
punched holes in an antibody containing agarose gel. The
same veronal buffer was used as before. The very ﬁrst
experiment showed precipitates like rockets and
proportional to the antigen concentration. The ﬁrst
publication was in Analytical Biochemistry (Laurell,
1966b) and the ﬁndings were also presented in Brugges,
May 1966 (Laurell, 1966a). A more detailed report of
electroimmunoassay used for different proteins was
published in a supplement in 1972 (Laurell, 1972).”
Jan-Olof Jeppsson, Malmö, Sweden, 2003
Various combinations of EID constituents were intro-
duced to better characterize the proteins being investi-
gated (Axelsen, 1973; Krøll, 1973; Weeke, 1973a,b).
Becker et al. (1968) used EID together with the photo-
metric method (Schultze and Schwick, 1959) and RID
(Mancini et al. 1965) for determination of serum proteins
and prepared a table showing normal mean values, stan-
dard deviations, and normal ranges for the concentration
of 25 proteins in human serum. Because EID required that
the antibody move cathodally, while the antigen moved
anodally presented problems in using EID for assay of any
protein in the gamma electrophoretic region which would
not be expected to migrate at pH 8.6. While EID had been
used by Laurell (1972) to quantify immunoglobulins by
measuring the physical dimension of the bidirectional pre-
cipitates, this was further resolved by modifying the anti-
body or the antigen, mostly IgG, by carbamylation before
mixing with the agarose gel (Weeke, 1968; Bjerrum et al.,
1973). However, with the ongoing development of instru-
ment-based immunoassays, this modiﬁcation did not
receive a great deal of use.
G Several scientiﬁc facts were combined to provide a
means of easily and inexpensively measuring the con-
centrations of proteins in liquid solution. No complex
instruments were required.
G The fact, that reactants diffusing toward one another in
transparent media produced visible halos, bands, and
rings, was known.
G It was appreciated that the position of these precipitates
could be related to concentration.
G By correlating immunoprecipitin ring diameter to con-
centration, a simple, widely applicable method of quan-
tifying proteins became available.
G The application of an electric ﬁeld accelerated the reac-
tion from days to hours meeting the practical needs of
Quantification of Antigens
Measuring amounts of a speciﬁc protein before the mid-
1930s was an arduous task and carried out by investigators
exclusively. Heidelberger and Kendall (1935a,b,c) pub-
lished careful analyses of the antigen–antibody reaction
using the amount of nitrogen as measured by the Kjeldahl
reaction in, for example, the precipitated complexes of egg
albumin and its homologous antibody. Few workers today
have an appreciation for the effort expended in measuring
dozens of such samples, but in 1935, there was no alterna-
tive. In 1938, Libby recognized the need for more direct
means of analyzing the immune reaction and published a
method for determining the amount of precipitate
between pneumococcal polysaccharides and the homolo-
gous antiserum using a new instrument, the photonreﬂec-
tometer. Measurement of the immunoprecipitate was
given in galvanometer readings, without the intervention
of secondary chemical analysis, for the ﬁrst time. This
instrument, basically a nephelometer, was an extension of
that used to measure sample turbidity as noted above
The introduction of numerical quantities progressed
slowly as listed above, but in 1921, a Duboscq colorimeter,
an early turbidimeter, was used for this purpose (Denis,
1921), to overcome the problems of non-proportional
results in biological systems derived from nephelometers.
The detailed work of Heidelberger and Kendall in 1935
(Fig. 2) used laborious Kjeldahl nitrogen analysis to give
numerical values to their work. However, in 1938, the
photonreﬂectometer was used to measure the turbidity of
BaSO4 suspensions (Libby) with answers given directly in
galvanometric readings. In 1943, Boyden and DeFalco
made the connection! They used the photonreﬂectometer
to quantitatively measure the turbidity resulting from the
reaction of antiserum to lobster hemocyanin and turkey
hemoglobin vs the homologous antibody. In their work,
they avoided an issue that compromised many of the
instruments measuring light scatter years later. They mixed
their reactant in a single tube and followed the change in
galvanometric reading, thus sidestepping the problems of
variability induced by differences among the reaction ves-
sels. The effect of the concentration of reactants, nature of
the antigen, pH, temperature, and reaction time was exam-
ined, and it was felt that the antiserum was the greatest and
most inconsistent variable. Different animals immunized
in identical fashion gave reagents that reacted differently
and unpredictably. Brief immunization produced more
speciﬁc antisera than prolonged programs. They also
noted that different antigens could show relative identity
when tested against a single antiserum, which expanded on
the extensive work of Landsteiner (1936). Using this prin-
ciple, they were able to show that species relationships
could be demonstrated, sometimes even distant phyla
could show a reaction of identity, albeit more weakly.
They appear to be the ﬁrst workers to observe the phe-
nomenon of antigen excess when they examined the effects
of varying concentration ratios of reactants.
348 The Immunoassay Handbook
About the same time, Pauling et al. (1944) applied
similar experimental methods to the inhibition of pre-
cipitating systems by haptens, and although instrument
measurements were obtained after traditional chemical
analysis, the concept of inhibition of immune precipita-
tion was to bear commercial fruit, only decades later.
Further, very detailed, instrument-based investigation
of the precipitin reaction by Bolton (1947) afﬁrmed the
theoretical value of quantitative methods when applied
to immune reactions. He studied the reaction of pure
bovine serum albumin and its homologous antibody and
measured the amount of precipitate in solution with the
photonreﬂectometer (Libby, 1938a,b) and demon-
strated that turbidity was directly proportional to the
amount of precipitate in suspension. He further found
that the proportions of each constituent affected the
amount of precipitate. Gitlin (1949) reviewed the obser-
vations that the measured mass of puriﬁed proteins
could vary as the result of the concentrations of three
amino acids: phenylalanine, tyrosine, and tryptophan.
Using ultraviolet spectroscopy, it was shown that the
dissolution of speciﬁc precipitates in sodium hydroxide
or acetic acid produced results that compared well
between species for puriﬁed proteins. He also applied
ultraviolet absorption spectroscopy to a detailed investi-
gation of the precipitin reaction presenting the now
time-tested concept of measuring protein concentration
by absorption at 280 nm (Fig. 9).
Until 1948, most quantitative protein investigation had
been at the academic level and published in the scientiﬁc
literature. But in that year, Kabat et al. (1948) published a
study of albumin and gamma-globulin in patient serum
and cerebrospinal ﬂuid, in a clinical journal. There had
been a previous paper approaching the topic (Goettsch
and Kendall, 1935), but Kabat’s paper was the ﬁrst to
approach a pathological state with immunochemical analysis
conﬁrming that antibody was in the gamma-globulin frac-
tion. Schultze and Schwick (1959) published a paper on
the quantitative analysis of nine human plasma proteins by
measuring immunoprecipitates in the Heidelberger mode.
Clinical interest in quantifying plasma proteins began as
early as 1948 (Kabat et al., 1948; Kabat and Murray, 1950).
The ability to quantitate antitoxins using early photomet-
ric devices was a forerunner of both quality assessment of
antisera and of the speciﬁc antigens. Not fully recognized
at that stage was the relative nonspeciﬁcity of immune
reagents. The nonspeciﬁcity of antiserum could not be
fully appreciated until the constituents of materials used to
immunize animals was understood. Standing in the way
was the method by which animals were immunized. See
box for more information on changes in immunization
practice, a crucial and often ignored issue.
The use of commercial instruments to measure the
immune reaction was extended to the immunoprecipitin
reaction directly by Boyden and Defalco (1943) using the
photonreﬂectometer. An extremely simple method using
this inexpensive and commonly available turbidimeter
was employed to quantify serum proteins in dilute solu-
tions, with reasonable accuracy. They avoided the difﬁ-
culty of sample concentration because of the increase in
sensitivity. Using more potent antisera allowed us to
measure protein concentrations as low as 0.5µg/L in
cerebrospinal ﬂuid. While this end point method
(Ritchie, 1967) did not come into wide use, it was the
FIGURE 9 The original Fig. 5.2 from Gitlin (1949) illustrating the
spectrophotometric spectra of crystalline human serum albumin
hydrolyzed with NaOH or Hac. The ordinate is wavelength (240nm at
the left) and optical density (OD) on the abscissa. The peak value at OD
280 is the compromise wavelength commonly used today.
Changes in immunization practice
Traditionally, immunization was effected by what we now know as truly
enormous amounts of immunogen. Tens and even hundreds of milligrams of
material were injected intramuscularly, intravenously, or into the footpads of
rabbits. Not surprisingly, antiserum produced by this protocol contained massive
quantities of irrelevant and unwanted antibodies, sometimes in concentrations
that bore little relationship to the immunizing, multispeciﬁc (contaminated),
antigens. In many ways, the complex patterns developed by diffusion-in-gel
methods (Fig. 4c) led to the efforts to purify proteins further thus producing
superior and less expensive antisera. The development that forced major
changes in antiserum production was the need for high-potency, precipitating
product in the mid-1960s.
At about the same time, protocols for purifying proteins with exchange
resins and fractional precipitation with various salts and alcohol greatly
improved the materials being used as immunogens. Hundred milligram amounts
of protein soon were replaced by single milligram amounts, and for certain
materials, even a few micrograms were sufﬁcient in large animals. We have
succeeded in making a usable rabbit antiserum with multiple intradermal
injection of less than 1µg of a rare antigen. Coupled to greater immunogen
purity was the awareness that lower levels of contaminants made immunoab-
sorption less rigorous. Eventually, solid-phase immunoadsorption became the
rule, making it possible to process antiserum in liter quantities with contami-
nant-bound columns. The subject of immunization schedules is a chapter in
itself and need not concern us further here.
349CHAPTER 4.1 The Foundations of Immunochemistry
forerunner of several commercial end point systems (e.g.,
Behringwerke’s BNA [Sieber and Gross, 1976], Hyland’s
laser nephelometer [Deaton et al., 1976]). These and all
end point systems experienced the same serious problem:
if the cuvette was rotated, clean or not, the results would
vary unacceptably, because of variations in the cuvette’s
In 1961, an immunochemical method to determine
β-lipoprotein using the immunocrit technique became
available (Heiskell et al., 1961). In this method, serum was
mixed with commercial anti-β-lipoprotein and aspirated
into a small glass capillary tube to a deﬁned height. The
precipitate that resulted was centrifuged, measured, and
compared against identically managed controls. Soon
after, a new approach came under development by two
groups, each without the knowledge of the other, and
amazingly, without the knowledge of the personnel within
the same company who supplied the equipment! Kahan
and Sundblad (1966) adapted the immunoprecipitin reac-
tion to the continuous ﬂow Autoanalyzer equipment man-
ufactured by Technicon Instruments Co. to quantify
β-lipoproteins. Our group developed an almost identical
system, for the analysis of several serum proteins in serum
and in cerebrospinal ﬂuid (Ritchie et al., 1969, 1973). The
latter development prompted the company to introduce
the automated immunoprecipitin (AIP) system to the mar-
ket. This approach used the bubble-segmented continuous
stream, introduced by Thiers and Oglesby (1967) and
Habig et al., (1969). In this mode, a single aspirated sample
is diluted to whatever ratio is desired, and air bubbles are
introduced, producing an evenly spaced series of sub-ali-
quots of the original. Since ﬂow dynamics and viscous drag
quickly reduce a single unsegmented aliquot to a concen-
tration distribution, introduction of bubbles halts further
diffusion by viscous drag. Each subsegment reacts inde-
pendently with identical doses of the provided antibody
until just before analysis, when the bubbles are removed
and immediately analyzed optically in a ﬁxed cuvette
(Fig. 10). Those on the leading and trailing limbs of the set
react appropriately (antibody excess) while those in the
center of the series may not react as expected if they are in
antigen excess (Fig. 11). The shape of the recorded curves
gives the operator a clue that the sample may have a high
concentration and must be further diluted and reanalyzed.
The AIP system was designed for rapid, high-volume test-
ing (40samples/h with total time per test of 20min vs
24–48h for RID); it was automated, reasonably precise,
and attracted worldwide attention. It did, however, require
considerable mechanical expertise, but produced results
that were signiﬁcantly more precise than previous meth-
ods. In 1973, Ebeling, and in 1974, Killingsworth et al.,
described optimization of buffers, ionic strength, enhanc-
ing additives, and other conditions for continuous ﬂow
immunoanalysis using the AIP system. In place of a con-
ventional light source, a laser was introduced in a kinetic
mode (Buffone and Savory, 1974). The kinetic approach
solved the problem of cuvette variability in previous static
systems by analyzing the signal being produced by a single
sample’s immunochemical reaction over time in a modi-
ﬁed centrifugal analyzer. It appeared that assay perfor-
mance in terms of precision was improved, as their data
The addition of nonionic polymers was a turning point
for automated instrument-based immunoanalysis. Early
attempts at light-scattering analyses were very successful
for most analytes; however, certain proteins presented
problems of an indolent reaction; most notably orosomu-
coid (α1-acid glycoprotein) and immunoglobulin M.
These were both crucial to introducing viable immuno-
chemistry to clinical laboratory programs. In 1964, Hells-
ing published work demonstrating that the addition of
dextran to the immune reaction increased the amount of
speciﬁc precipitate. In 1972, as AIP analysis was expanding
to include pathologic samples and additional serum pro-
teins, problems with the above-mentioned two analyses
FIGURE 10 AIP curve with unknowns to the left and a calibration
curve to the right (Ritchie and Graves, 1972).
FIGURE 11 The peak heights represent the antigen concentration of
each sample except when the peak conﬁguration becomes distorted.
With further increases in concentration, the peak height becomes
ﬂattened, then asymmetric, and ﬁnally, it becomes forked. The
explanation is that each peak represents many individual subsamples.
The early and late subsamples contain concentrations below antigen
excess while those that become distorted are beyond the point of
equivalence with the central samples in frank antigen excess, hence
diminished light-scattering ability (Ritchie and Graves, 1972).
350 The Immunoassay Handbook
was of great concern. At a European Technicon Sympo-
sium in Brussels in 1972, the late Dr Kristofer Hellsing
was introduced to the dynamics of the AIP system. With
his advice, polyethylene glycol (PEG) at various percent-
ages was added to the buffers (Hellsing, 1972). The results
were dramatic, and analysis of orosomucoid and IgM
became as vigorous as for the other protein species. PEG
Mm 6000 at 4–5% concentration was ideal (Hellsing, 1972,
1978). In theory, PEG’s effect could be explained by “ste-
ric exclusion,” i.e., each molecule of PEG is surrounded by
a hydration sphere which excludes other solute molecules
to various degrees. Small species diffuse more deeply while
large species, such as serum proteins, may be excluded
completely, thus, the total volume of solute exists as parti-
tions, one freely available to salts and the other only avail-
able to proteins. As a result, both antigen and antibody are
forced into more intimate contact than in a PEG-free
solution thus accelerating interaction.
The fact that the AIP was a large unit offered an oppor-
tunity for other manufacturers to design instruments
expressly for low-volume users. By chance, the senior sci-
entist of Beckman Instruments, Inc. and the author of the
AIP system met at a symposium in 1974. As a result, after
thorough research into the theory behind the method, a
device was designed, and within 18 months the ﬁrst oper-
ating rate nephelometric system was delivered to our labo-
ratory for a ﬁeld trial. The serum protein analyzer-1
(SPA-1) proved to be a remarkably precise and trouble-
free instrument and was ﬁrst stressed testing amniotic ﬂuid
alpha-fetoprotein. Precision was better than 5% coefﬁ-
cient of variation (CV), even in inexperienced hands
(Haddow et al., 1976). The architect of the instrument and
the rate analyzer was the late James Sternberg (1977). The
SPA-1 was followed by a family of ever more sophisticated
devices from Beckman Instruments, Inc. and their com-
petitors. Regrettably, the progress to more closed systems
corresponded with a gradual decrease in technical exper-
tise available in the majority of medical laboratories. At the
same time, requirements for greater precision and accu-
racy were coupled with the pressure to lower costs. For
those laboratories with access to highly qualiﬁed staff, the
development of cost-effective open systems has been
rewarding (Hudson et al., 1987; Ledue et al., 2002).
All serum protein analyzers face a common and worri-
some problem: antigen excess. While it was a familiar phe-
nomenon to the bench workers of the past, in the routine
delivery of serum protein values by automated instru-
ments, there was no obvious evidence that such a problem
existed. The AIP system presented a unique manner of
demonstrating the situation. As each sample was divided
into tiny aliquots by the bubble segmentation, with the
lowest concentration ﬁrst, progressing to the highest con-
centration, and continuing to the lowest again, the possi-
bility that the amount of serum protein antigen exceeded
the antibody content was announced by the change in the
shape of the recorded peak. In Fig. 11, peak conﬁguration
becomes blunted and then biﬁd when antigen excess super-
venes. Keep in mind that the stream being analyzed has
been held as multiple isolated segments until just before
measurement. Those aliquots that are in antigen excess
have diminished amount of immunoprecipitate, hence less
turbidity than expected. For systems that employ single
cuvette analysis, there is no detection device other than
preparation of more than one dilution of each sample, as
performed in an automated fashion by the AIP system. For
rate analysis by turbidimetry or nephelometry, a more
sophisticated approach is required (see Kusnetz and
Mansberg, 1978, for a complete description). It was recog-
nized very early in development of the Beckman analyzer
that samples with high levels of an analyte would develop
turbidity slowly, i.e., the rate was diminished yet usually
reached high levels. However, in samples with extremely
high Ag:Ab ratios, turbidity might not develop at all or be
very limited. The concern was most acute with the immu-
noglobulins, where the ﬁnal result could be contrary to the
actual fact, namely very high concentrations of protein
recovered no value at all. The clinical concern was well
placed, so an algorithm was devised to detect this. The
automated rate nephelometric system included an internal
dilution scheme triggered by the use of a “gate” that
instructed the instrument to insert a dilution different
from the usual: if the recovered value was very low a more
concentrated sample (less dilute) was submitted, for values
higher, but still below a low cutoff a more dilute sample
was submitted. Eventually, more sophisticated algorithms
were developed that included the addition of an aliquot of
antiserum and assessment of rate change.
Another interesting method is the kinetic turbidimetry
developed by Metzmann (1985) and Tuengler et al. (1988).
In this method, a determination of protein concentrations
is made through the simultaneous measurement on a
highly automated analyzer of two (interrelated) reaction
parameters: the maximum velocity (Vmax) of the immuno-
precipitation and the time required for the reaction to
Availability of such excellent devices characterized as
“Black Box,” “Walk away,” or similar marketing terms to
advertise their sophistication has allowed an increasing
number of laboratories to offer high-quality serum protein
analyses without the need for highly trained staff. As inti-
mated above, the downside is that bench workers have
been reduced to janitorial services as demanded by the
machines. The positive aspect of course is that results are
far superior than could be imagined 2 decades ago. A sin-
gle laboratory with one technologist can produce accurate
results on hundreds of tests a day, and in many depart-
ments, technical staff are never required to enter a result,
it all having been managed by electronic data transfer to
central computing facilities. Inevitably, good comes with
the bad, and progress toward more automation, higher
accuracy, lower per unit cost, and shorter turnaround time
is taking its toll. The loss of technical expertise in the clini-
cal laboratory is lamentable but irreversible. The refuge of
the expert technologist is now within industrial and
While accurate measurement of protein concentration
became available, results still required considerable time
to complete. Electronics moved from crude, often
homemade devices to commercially available instru-
ments with consequent improvement in precision and
351CHAPTER 4.1 The Foundations of Immunochemistry
G It was known that immunochemistry in liquid yielded
excellent meter-readable results, instruments needed to
G Serious problems in the methods of preparing antisera
became widely acknowledged, and major changes in
protocol were established largely by commercial
G Several publications appeared demonstrating that
instrument-based analyses of serum proteins were
G In clinical chemistry, early automation was extremely
successful and made clear that immunochemistry in
liquid could be designed for this application.
G The serious problem of erroneous results due to anti-
gen excess was appreciated and resolved operationally.
G End point analyses were supplemented by kinetic meth-
ods. Conversion of laboratories to automated devices
greatly improved performance.
Quantification of Antigens
by Particle Enhanced
Coincident with the development of automated immuno-
chemical analysis, the potential for adding a layer of tech-
nical sophistication came early. During a seminar on the
AIP system organized by Pierre Masson at the École de
Santé Publique in University Catholique de Louvain in
1972, attended by among others, the director, Professor
Joseph Heremans, I made a detailed description of how the
mechanics of the device were viewed.
I observed with concern that Prof. Heremans appeared
not to be listening, in fact I thought he was asleep. At the
close of the presentation, Prof. Heremans came to the
front of the room, fully alert, and in a rather disengaged
fashion laid out his vision of the workings of the AIP
system and then launched into a discussion of what
became inhibition immunoassay, the inhibition of
precipitation by the addition of non-precipitating antigen
such as hapten.
Robert Ritchie, Scarborough, Maine, USA. 2003
Soon after, workers at the Unit of Experimental Medi-
cine at Louvain, Belgium, were working with the theory
and practice of nephelometric immunoassay and inhibi-
tion immunoassays for haptens (Riccomi et al., 1972;
Cambiaso et al., 1974a,b). The practical application of
this was grasped by Gauldie et al. (Gauldie et al., 1975;
Gauldie and Bienenstock, 1978) of McMaster Univer-
sity, Hamilton, Ontario, who published an abstract
describing inhibition nephelometry of morphine and
digoxin by this method. These workers recognized the
difﬁculties of radioimmunoassay (RIA) at the time and
applied the knowledge that antisera could be produced
by immunization with antigen composed of an antigenic
macromolecule conjugated to a low molecular weight
non-precipitating compound. This haptenic antigen
generates antibodies to the hapten, the base protein, and
the complex. If the carrier protein is completely alien
(e.g., horseshoe crab [Limulus] hemocyanin) to the spe-
cies in which the hapten is to be measured (humans), it
can be assumed that any reaction is due to the presence
of the free hapten. By the addition of a concentration
series of free hapten, the light-scattering signal can be
attenuated. In a similar fashion, the addition of serum
samples from the test species containing the same hap-
ten attenuates the signal in proportion to the amount of
hapten present. In 1978, this group described the analy-
sis of dinitrophenol, progesterone, histamine, angioten-
sin, and other haptens.
The use of latex beads to enhance the sensitivity of
immunoassays dates at least to the landmark paper by
Singer and Plotz (1956) who observed that the serum of a
coworker had the ability to agglutinate latex particles. Fur-
ther investigation by Lospalluto and Ziff (1959) provided
the explanation for patient’s sera agglutinating particles
such as sensitized erythrocyte and latex particles. Further
work (Hall et al., 1958; Hall, 1961) set the stage for further
enhancements of assays based upon the inhibition of par-
ticle agglutination. Building on this well accepted diagnos-
tic method and the awareness that inhibition by hapten was
possible, the Louvain group (Cambiaso et al., 1977, 1978;
Masson and Holy, 1986) presented a unique approach of
immunoanalysis. Using small polystyrene beads coated
with antibody allowed measurement of the homologous
antigen in serum samples. Called PArticle Counting
ImmunoAssay (PACIA), they were able to use a particle-
counter instead of a nephelometer. By the addition of
diluted antibody to the surface of the particles, antigen in
solution causes the particles to agglutinate, i.e., this reduces
the absolute number of discrete particles counted. How-
ever, if the particles are coated with an antigen, i.e., non-
speciﬁc IgG, rather than speciﬁc antibody, then incubated
with a solution of the homologous antibody (rheumatoid
factor—anti-IgG), agglutination again diminishes the
absolute number of particles counted. In the former man-
ner IgE, human placental lactogen, alpha-fetoprotein, T3,
and T4 were effected. Later C-reactive protein (CRP) was
quantitated by the same technique in serum, cord serum,
and cerebrospinal ﬂuid (Collet-Cassart et al., 1983). Assays
for serum ferritin (Limet et al., 1982), α2-microglobulin in
urine (Bernard et al., 1981), theophylline (Liedtke, 1984),
and pregnancy speciﬁc α1-glycoprotein (Fagnart et al.,
1985) were developed. Sensitivity was in the order of tens
of micrograms per liter for serum proteins. A key issue
with PACIA was the ability to decrease nonspeciﬁc agglu-
tination as a result of unwanted serum factors. The use of
F(ab′)2 fragments for coating particles in the rheumatoid
factor assay reduced nonspeciﬁc agglutination as a result of
patients having anti-rabbit or anti-goat antibodies. Various
permutations of this system with haptens and inhibition of
agglutination were successfully devised for over 100 appli-
cations. The instrument was commercially introduced, but
because of nonscientiﬁc circumstances, this promising
technology received only limited use.
Kapmeyer et al. (1988) described a particle-enhanced
immunoassay that was further improved by developing
180nm particles of novel composition. A polystyrene core
surrounded a shell of chemically reactive material to which
antibody could be easily bound in a more stable fashion.
352 The Immunoassay Handbook
This method was used to develop an assay for CRP. Sensi-
tivity was comparable to existing RID plates with a range
from less than 1 to over 300mg/L. Assay time was about
6min. The assay for CRP with sufﬁcient sensitivity to give
precise answers in the low ranges (0.5–2.0mg/L) spurred
manufacturers to make available high-quality assays that
were particle-enhanced. Ledue et al. (1989) compared a
particle-enhanced assay to a ﬂuorescence immunoassay
and concluded that the automated particle-enhanced assay
was superior in efﬁciency and precision to previously avail-
able methods. A CRP particle-enhanced assay for three
serum proteins: CRP, serum amyloid A, and mannose-
binding protein was evaluated (Ledue et al., 1998), and
precision in a normal population was found to be equiva-
lent to traditional chemical and colorimetric assays.
The subject was reviewed thoroughly by Galvin in 1983.
Available from the plastics industry, “latex” beads of
extraordinary constant size and a bewildering variety pro-
vided an investigative tool for immunochemists. Modiﬁca-
tion of the constitution of the beads, which measured only
a few microns, also made the attachment of reactive ele-
ments routine. A large step forward was taken in the quest
for increased sensitivity and precision.
G Polystyrene particles coated with either antibody or
antigen provided a powerful tool for immunochemical
G Several instruments, including those used previously
for traditional light-scattering analyses, were successful
in lowering sensitivity by at least an order of magnitude
to micrograms of antigen (or antibody) per liter.
G A wide array of low-level analytes of interest became
easily and precisely analyzable.
Progress toward immunoassays employing antibodies con-
jugated with ligands of a wide variety of materials such as
enzymes, radioactive nuclides, and ﬂuorescent tags is cov-
ered elsewhere in this book and is summarized in extensa in
PRINCIPLES OF COMPETITIVE AND IMMUNOMETRIC ASSAYS by
Davies and will not be pursued to any degree here. As with
all advances, we stand on the shoulders of those before us.
For these labeled immunoassays, we have the pioneering
work of the following scientists to thank for what we now
take for granted:
G Avrameas and Uriel (1966): antibodies labeled with
enzymes, Avrameas and Guilbert (1972): peroxidase-
labeled antibodies, Avrameas (1976): method review.
G Blanchard and Gardener (1982): solid-phase ﬂuoroimmu-
noassay (FIA) of IgE.
G Coons and Kaplan (1950): ﬂuorescent-tagged antibody.
G Coons (1956): ﬂuorescent antibody and histochemistry.
G Dandliker and Saussure (1970): theory of polarization
ﬂuorescence assay, Dandliker et al. (1973): polarization
G Masseyeff et al., (1973): rapid enzyme labeling of anti-
body, Masseyeff et al. (1981): immunoenzymatic analy-
sis of immune complexes.
G Nakane and Pierce (1967): localizing tissue antigens
with enzyme-labeled antibody, Nakane (1971): peroxi-
G Voller et al. (1974): microplate enzyme-linked immu-
nosorbent assay [ELISA] method to detect malaria,
Voller and Bidwell (1976): ELISA detection of viral
infection, Voller et al. (1978): review.
G Wang et al. (1980): immunoglobulins.
G Yalow and Berson (1959): RIA assay of insulin.
In those days before grants-in-aid, sophisticated instru-
ments and reagents, and the Internet with its powerful
search engines, we can only be thankful that those gaps in
knowledge were ﬁlled with intellectual brilliance, persis-
tence, and vision.
The need for improved sensitivity to encompass many of
the clinically important analytes prompted many workers
to seek ways of modifying antibodies to provide higher
detectability. Powerful contributions from radiochemistry,
enzymology, and ﬂuorochemistry provided these tools.
Instrument engineering has kept pace with changing
needs, and the scope of methods and materials seems to be
Early work in immunochemistry, before the time when
accuracy and precision were issues of paramount concern,
proceeded in relative isolation. Each worker performed
assays as best they could with what equipment and
reagents were obtainable or could be made. The concept
of quality assurance was not to develop fully in the devel-
oped world until the 1980s. As a result comparative stud-
ies for reference intervals, clinical cutoff points and the
like were not comparable. With the development of
devices that could perform large numbers of assays under
nearly identical conditions, this matter became address-
able. Results from widely dispersed laboratories had to be
harmonized for clinical utility. This was a desirable goal
but expensive. It was not until programs were introduced
that required satisfactory performance in government
mandated exercises for continuation of licensure to oper-
ate, that the full value of automated testing was appreci-
ated. Immunochemistry testing was early into the
By the 1970s, the availability of several systems of pro-
tein immunoassay raised the concern for reagent qualiﬁca-
tion. Implicit in several of the older papers was the
comparison of one product with another and the semi-
quantiﬁcation of materials such as vaccines (McFarland,
1907; Libby, 1938; Boyden and Defalco, 1943; Becker,
1969). As interest in automated immunochemistry was
building, a group from the Centers for Disease Control
353CHAPTER 4.1 The Foundations of Immunochemistry
(CDC) and Prevention published a comparison of com-
mercial antisera (Phillips et al., 1971). Using a panel of 20
different preparations of human serum IgA, the 13 major
suppliers of anti-IgA were analyzed which showed at least
a sevenfold difference in titer. Findings such as lack of spe-
ciﬁc antibody, presence of interclass speciﬁcity, other con-
taminant antibodies, and low titer (one had none) by the
CDC group resulted in a fairly rapid improvement in the
quality of commercial antisera. A year later, our concern
for unrecognized contaminants in reagents used in the AIP
system prompted a method to demonstrate these impuri-
ties (Ritchie and Stevens, 1972). Perhaps the most effective
means of improving reagent antibody was the institution in
the late 1970s by the College of American Pathologists of
the Quality Assurance program for serum proteins. Manu-
facturers that supplied kits for these assays found them-
with embarrassing results. To assist with this upgrading of
products came a method of qualifying each batch of
reagents in a system that performed in close parallel with
others of the time (Hudson et al., 1981, 1983). Some man-
ufacturers still use this protocol, while others have devised
other but equally demanding protocols. Hand in hand with
improved antiserum requirements was the unavoidable
realization that there were no easily available and authori-
tative reference materials. This last link in the harmoniza-
tion of laboratories was to be the product of the European
Union in the form of the Bureau de Communitaire de
Référence, in Brussels. The European community, heavy
users of serum protein assays, instituted a program to pre-
pare and distribute a universally accepted and highly stud-
ied material, Certiﬁed Reference Preparation 470 (CRM
470), also known as Reference Preparation for Human
Serum Proteins in the USA, distributed by the College of
American Pathologists (Baudner et al., 1992; Whicher
et al., 1994; Blirup-Jensen et al., 1993, 2001 Bilrup-Jensen,
2001a,b,c; Dati et al., 1996). Crucial to the generation of
these types of reference materials are immunoassays that
can perform with a high degree of precision. Several highly
automated instrument packages were used in this value
transfer exercise (Johnson et al., 1996) and, to the surprise
of all involved, immunoassays preformed as well as, or bet-
ter than, traditional chemistries. Precision with CVs of less
than 5% was the norm.
From the very earliest studies, workers were inevitably
faced with differences in the quality and concentrations
of their reagents. When improved instruments became
available, the effort to compare products, especially
those on which assays depended, was a meaningful
G As a result of various reports demonstrating that prod-
uct labels could be very misleading, product quality
G Hand in hand with reagent veriﬁcation and quality
assurance came the need for a globally recognized ref-
erence material for human serum proteins. This was
I am indebted to our librarian, Linda Talamo, who has
been tremendously helpful in ﬁnding and obtaining the
necessary documents, many of which fall into the category
of ancient archives. I am also indebted to colleagues, most
of whom “were there,” for providing important insights
that have faded from most of our memories. Namely:
Soren Blirup-Jensen, Cesar Cambiaso, Francesco Dati,
Jan-Olof Jeppsson, A. Myron Johnson, Pierre Masson,
Thomas Ledue, Miroslav (David) Poulik, Jean-Pierre
Vaerman, John Whicher. Lastly, I am grateful for the
opportunity to assemble this narrative, which encompasses
the better part of many of our lives, a privilege I attribute
to David Wild.
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