SUMMARY

1
SPECIFICITY of EIA IMMUNOASSAY for COMPLEMENT
FACTOR Bb TESTING
Igor Pavlov,1 Nikol deForest2, Julio C.
Delgado1 1ARUP Institute for Clinical and
Experimental Pathology, Department of Pathology,
University of Utah School of Medicine, Salt Lake
City, Utah 2 QUIDEL Corporations, Santa Clara, CA
SUMMARY
Table 1. Results of cross-reactivity of the Bb
assay with purified factor B
RESULTS
Factor B, µg/mL Measured Bb concentrations, µg/mL Cross-reactivity
200 0.116 0.06
100 0.093 0.09
50 0.066 0.13
25 0.038 0.15
12.5 0.016 0.13
6.25 0.000 0.00
3.125 0.000 0.00
1.5625 0.000 0.00
During the alternative complement pathway
activation, factor B is cleaved in two fragments,
Ba and Bb. Concentration of those fragments is 2
logs lower than of factor B present in the blood,
which makes fragment detection vulnerable because
of potential crossreactivity. Immunoaffinity
purified factor B demonstrated 0.15
crossreactivity in Quidel Bb EIA assay. It was
shown that Bb concentration in serum is higher
than in plasma due to complement activation
during clot formation. To investigate the effect
of crossreactivity, we run 109 healthy donor
plasmas as reference population for Bb assay, and
80 sera samples (representing complement
activated state) with factor B immunodiffusion
and Bb EIA assays. Our study demonstrates that
despite the potential 0.15 crossreactivity
between factor B and cleaved Bb molecule,
measuring plasma concentrations of factor Bb
alone (without factor B assessment) is adequate
to evaluate the activation of the alternative
complement pathway.
The results of B and Bb measurements in EDTA
plasmas and sera in 40-paired samples
demonstrated that factor Bb concentrations in
sera were approximately 5 times higher than in
plasma, whereas factor B levels in paired sera
and plasma samples were similar. Based on these
results, we conclude that the differences in Bb
level between paired serum and plasma are not due
to the differences in factor B concentrations
between those two matrices. As it was shown in
number of publications, high levels of serum Bb
are most likely due to the activation of factor B
cleavage during the clot formation. This finding
let us use sera samples as representatives of
alternative complement pathway activated state.
EDTA plasma samples were used as reference
population. Maximum crossreactivity with
immunoaffinity purified factor B was measured to
be 0.15 in physiological range of factor B.
Reference intervals calculated with and without
accounting for the effect of 0.15
crossreactivity provided identical discrimination
between normal samples and specimens representing
alternative complement pathway activated state.
INTRODUCTION
Figure 1. Complement factors Bb and B in serum /
EDTA plasma paired samples
Biological functions of the complement system are
achieved through the chain of production of
activation fragments 1, 2, 3. During the
alternative complement pathway activation, factor
B bound to C3 is cleaved by factor D into two
fragments, Bb (60 kDa) and Ba (33 kDa) 4, 5.
The Bb fragment bound to C3b initiates formation
of the membrane attack complex. In the absence of
Bb generation, no alternative pathway activation
is possible. Thus, by quantifying the
concentration of Bb, the extent of alternative
pathway activation at the time of sample
collection can be measured. Objective To
investigate the effect of potential
crossreactivity of complement factor Bb EIA assay
with endogenous factor B.
CONCLUSIONS
Our study demonstrates that despite the potential
cross-reactivity between factor B and cleaved Bb
molecule, measuring plasma concentrations of
factor Bb alone is adequate to evaluate the
activation of the alternative complement pathway.
Quidel MicroVue Bb Plus EIA Kit used with human
EDTA plasma can be effectively implemented to
assess alternative complement pathway activation
at the time of sample collection.
Figure 2. Concentrations of factor Bb in sera and
EDTA plasmas
MATERIALS and METHODS
REFERENCES
109 de-identified EDTA plasma samples from ARUPs
healthy donor collection and 80 serum samples
were tested for factors B and Bb concentrations
to estimate the effect of crossreactivity with
endogenous factor B, and to investigate the
distribution of factor Bb in normal population.
Forty pairs of serum EDTA plasma samples were
run with the Bb Plus assay during matrix
evaluation study. Detection of complement factor
Bb was performed using the Quidel MicroVue Bb
Plus EIA assay. Factor B concentration was
measured by The Binding Site radial
immunodiffusion assay. Purified factor B sample
was developed at Quidel Corp. using
immunoaffinity purification
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Legend for Figure 2 open circles Bb
concentration in plasma open triangles Bb
concentration in serum grayed circles and
triangles represent adjusted Bb values in plasma
and serum, respectively dotted lines represent
correspondent upper levels of the reference
interval of Bb values in the normal population
with 90 confidence limits.