MPDTM Enhanced Immunoassays for Cytokines
ABSTRACT: BioTraces has developed a significant new assay protocol, IA/MPD, which provides quantitative measurement of biological substances at levels as low as a few femtogram/ml. Unprecedented sensitivity of SIRMAs for interleukins (IL-1 beta, IL-4, IL-6, IL-10, IL-11 and IL-12) has been documented.
Cytokines are glycoproteins which are regulators in cells of many lineages. They have a wide range of activities in physiological and pathophysiological processes. They play a very important role in maintaining proper balance in immune responses, especially in response to malignant growth, bacterial and viral infections and in autoimmune disorders. Our data obtained for a plurality of cytokines, IL-1 beta, IL-4, IL-6, IL-10, IL-11 and Il-12, demonstrates a capability to develop quantitatively accurate MPD immunoassays whose sensitivity is up to 500-fold better than currently used ELISA. Limits of detection (LOD) below an 0.5 attomole per ml has been achieved.
Significance of cytokines: Many proteins are available in physiologic fluids in extremely low concentrations. For example, cytokines are produced in very small quantities and act locally in paracrine, juxtacrine, and autocrine roles. Cytokines, especially the interleukins, serve as chemical messengers, i.e. they modulate and coordinate the activities of the immune system. The circulating blood levels of interleukins are not static, but constantly adjust to changes in a progressing immune response, disease, or physiological state of the individual. Quantitation of the interleukins depicts the state of the immune system, and may be useful in the diagnosis and prognosis of a number of diseases, particularly immune disorders.
The predictive power of measurement of serum levels of interleukins and other cytokines has been hindered by unreliable quantitative methods. Both polyclonal and monoclonal antibodies against interleukins are commercially available, and a number of ELISA, EIA, and RIA kits are available. Unfortunately, the standard immunoassays are not sensitive enough to determine the normal serum levels of most interleukins. Typically, the blood levels of many interleukins are much lower than 1 pg/ml except during conditions of exaggerated immune response and in certain diseases. The lower limit of detection (LOD) of enzymatic immunoassay kits for many cytokines is above the estimated mean value of serum concentrations of healthy individuals. Thus, even the best EIA systems are already at the detectability limits and often show false positives and false negatives.
MPD enhanced assays for Cytokines:
Coupling MPD technology to an immunoradiometric assay
(IRMA) has resulted in the development of a new class of ultra-sensitive immunoassays which
we call MPD enhanced immunoassay (IA/MPD) The IA/MPD is an antibody
sandwich capture assay and uses similar reagents. One antibody is immobilized and serves to
capture a ligand and a second antibody, which binds to a different epitope on the ligand, is
labeled and used to quantitate the ligand.
Specialized blocking and assay reagents have been developed to reduce
nonspecific biological background (NSBB). The set of techniques used to optimize the SIRMA
typically results in up to 500-times greater sensitivity than a classical IRMA or ELISA.
IA/MPD assays for several interleukins (IL-1 beta, IL-4, IL-6, IL-10,
IL-11 and IL-12), achieved limits of detection (LOD) as low as 0.005 pg/ml. IA/MPD for IL-1
beta, IL-6, and IL-10 have been used in the recent studies of AML patients, and are described
in detail in the following.
Interleukin-1:
Excellent results have been obtained for interleukin-1 beta.
Using a microtiter plate format, the standard curve of this IA/MPD ranged from 7 fg/ml to
10 pg/ml (Figure 1).
This is a factor of about 100 increase in sensitivity over a standard
ELISA. We studied the influence of different components of nonspecific background on the
performance of MPD enhanced immunoassays. The background is strongly
dependent on the assay conditions, especially the washing conditions. Also, optimization of
pH and use of appropriate blocking procedures is mandatory. Only about 30% of background is
true non-specific biological background (NSBB) which appears when serum is added.
Our experiments permitted further NSBB minimization by changing the temperature and the pH
at which the biotinylated antibodies are bound. Even though NSBB is important in the serum
samples, it only marginally influences the reproducibility of the assay.
Figure 1.Comparison of ELISA and IA/MPD
for IL-1 beta. Note, that BioTraces' ELISA have sensitivity of 120 fg/ml, i.e. about
10-fold better than best prior-art immunoassay. The limit of detection (LOD) of IA/MPD
is 0.007 fg/ml,i.e. 500-fold better than typical assays.
Interleukin-6:
Figure 2 shows that the standard curve of this IA/MPD ranged from 5
fg/ml to 1,000 pg/ml. We demonstrated that about 30% of healthy individuals have IL-6
levels which are not quantifiable using an prior-art ELISA but are reliably measured by a IA/MPD.
Even in the presence of serum, we achieved good
CV's down to a 5 fg/ml, i.e., down to about 0.2 attomole/ml. We used IA/MPD for IL-6 on about
50 samples from both AML positive and AML negative individuals. We demonstrated a clear
up-regulation of the IL-6 in AML patients.
Interleukin-10: For IL-10 achieved 6 fg/ml sensitivity. The CV's for this assay using six measurements in parallel are quite impressive, i.e.,neproducibility is better than 20%. An important aspect of the development of a IA/MPD for IL-10 has been a comparative study between different monoclonal capture antibodies obtained from a few commercial vendors. We observed that for a given amount of capture antibody, there is only a small difference between avidities, but a rather large difference in background. The less specific antibodies led to about three-fold higher background. Our results suggest that in the case of IL-10, the limit of quantitation is due to cross-reactivity between the capture and detector antibodies. Another important contributor to the background is 125I-streptavidin nonspecific capture on plastic. This factor is batch dependent and in some cases limits assay sensitivity.
Other cytokines: The MPD enhanced immunoassays have been developed for three other cytokines, IL-4, IL-11 and IL-12. The standard curves for all six cytokines mentinoned above are depicted in Figure 3. We consistently achieved the sub-attomole/ml sensitivity, i.e. for all these cytokines we achieved 100-500 better sensitivity than prior art immunoassays. Note that even at the 10 fg/ml level, the CV's of the assay are better than 20%. The comparison of MPD enhanced immunoassays and prior-art assays is presented in the Table 1.
Note, that the development of MPD enhanced immunoassays required a development of methods permitting reduction of non-specific biological background (NSBB). Thus, we have been able to formulate the proprietary ELISA procedures which reached 100 fg/ml for some cytokines. This is a factor 5 better sensitivity than prior-art ELISAs for these targets.
Applications of MPD enhanced immunoassay to AML patients: We used our MPD enhanced immunoassays for detection of IL-1 beta, IL-6 and IL-10 in serum of patients with AML. We analyzed about 100 clinical samples and documented:
Significantly, for the first time the level of IL-6 has been quantitated in all serum samples, both AML positive and AML negative. Previously only the up-regulation of IL-6 (about 30% of cases) could be reliably quantitated.
The data for IL-1 beta are compatible with the hypothesis that AML induces the large secretary modulation. This suggests that AML leads to important post-translational modulations in IL-1 beta. We hypothized that the processing of IL-1 beta by caspases (ICE) is impared.
Summary: The MPD enhanced immunoassay solves the problems of both signal to background ratio and radioactive waste disposal. The IA/MPD consists of the same components as a standard ELISA or IRMA: antibodies, ligand standards and antibody tracers. However, the format, volumes, and amount of tracer antibody are scaled down to reduce nonspecific sticking and the amount of radioisotope needed. The MPD enhanced immunoassays for cytokines achieved the sub-attomole/ml sensitivity. The further improvement by factor five seems possible when the SuperTracers are used to replace 125I- streptavidine.