Often biological processes are known to occur by protein interactions. Protein interaction in serum and plasma analysis carries a great wealth of information in detection, and prognostics of several diseases like cancer1. Identification of the serum and plasma proteomes in blood circulation has substantial relevance as biomarkers that predict the different stages of a disease2. Still, the abundance of serum and plasma proteins composes a dynamic range of at least 12 orders of magnitude, thus increasing the complexity of immunoassays to investigate these interactions1,2. Hence, new assay formats are needed to ameliorate sensitivity and specificity with good precision and prediction to measure the proteomes in the given samples.
Vascular endothelial growth factor (VEGF-A) a 165-amino acid residue form, is a regulator of angiogenesis. It also includes platelet-derived growth factors and stem cell factor3. It has been shown that enormous blood vessel growth occurs during cancer, rheumatoid diseases, and ocular diseases by the disruption of VEGF protein-signalling cascade. VEGF-A binds to receptor tyrosine kinases and induces proliferation, survival, and vascular permeability4. Interleukins (IL) are cytokines which regulates inflammation are expressed by white blood cells. Respiratory distress syndromes are results of inflammatory-cell segregation and neutrophils are mainly responsible; hence receive much attention5. IL-8 is the neutrophil attracting chemokine present in cystic fibrosis patients. These high levels are secreted airway epithelial of these patients6. The new analytical tool has to be developed in order to measure these biomarkers in the patients.
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Most conventional immunoassays have drawbacks in nonspecific binding of the secondary reporter, which will leads to false positive signal and also ineffective while handling multiplex reactions. To minimize this measurability and multiplexing the samples, a novel protein detection technology; the proximity ligation assay was performed7. The new immunoassay technique for in vitro analysis of proteins, termed proximity ligation was first described by Fredriksson et al.8 in 2002 The proximity ligation assay (PLA), is an affinity-based technology used to detect protein molecules through DNA ligation and amplification. This method enables sensitive and specific detection by help of antibodies coupled with specific DNA sequence and on proximal binding; the DNA strands are ligated with connecting oligonucleotide to form a template which is later amplified for quantification. In PLA, pairs of affinity probes directed against the same target molecule are modified by attaching short single-stranded oligonucleotide by making PLA probes. On identifying a common target molecule by a pair of modified antibodies, the coupled DNA strands are brought in proximity, allowing their free ends to be hybridized to a connector oligonucleotide by enzymatic ligation. The newly synthesized DNA strand that forms upon ligation can be amplified and quantified by methods such as quantitative real time PCR1,9.
A solid support-bound affinity reagent offers high possibility to search for target molecules in larger sampling and to remove excess probes and nonspecific binding before the ligation and amplification steps. In this approach a solid-phase PLA (SP-PLA) was modified by biotin-streptavidin interactions between antibody-oligonucleotide probes and solid support7. Here, the biotinylated antibodies were conjugated on the surface of streptavidin-coated magnetic beads and antigen was captured. Upon capturing biotinylated oligonucleotide PLA probes was added and ligated by connecting oligonucleotide. On proximal binding of PLA probes the signal was detected using RT-PCR (Fig 1). It has been showed by Darmanis S et al., (2010) SP-PLA is more advantage that of conventional sandwich ELISA in terms of sensitivity, specificity and dynamic range. They showed SP-PLA was able to detect antigens âˆ¼100 times lower concentrations compared with sandwich ELISA and dynamic range up to 2 orders of magnitude. Also, SP-PLA required very low volume of 5 Î¼l, whereas the comparable ELISA needed 100 Î¼l of sample1.
Figure 1. The pictorial representation of solid-phase PLA. In SP-PLA the analysing target protein molecule is first captured from a sample by immobilized antibodies on solid surface. After a washing, a pair of PLA probes with attached oligonucleotides binds to the antigen, if the probes bind in close proximity, then the ends of the oligonucleotides are ligated to each other by a connecting oligonucleotide. The amount of ligation product is quantified by RT-PCR, and the results measure the amount of the analyte. The image is modified from Tim Conze et al.10 (2009).
Materials and Methods
PLA probes preparation and beads capturing
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The biotinylated antibody (1 Âµl) was diluted in antibody dilution buffer in 1:10 ratio. Equal volume of diluted antibodies (100 nM) was mixed with streptavidin conjugated oligonucleotide (100 nM) containing free 3' end (SLC 1) and 5'end (SLC 2) was prepared separately in two different tubes. Mixture was incubated at room temperature (RT) for 1 hour. Simultaneously the washed magnetic beads were captured onto biotinylated antibody (50 nM) by incubating at RT for 1 hour under rotation. After incubation the beads were washed with washing buffer and stored it in 200 Âµl of assay buffer.
Antigen VEGF capturing and PLA probe binding
Serial dilutions of VEGF from 1 nM to 0.1 pM including blank of 0 pM was prepared using assay buffer are made in 180 Âµl assay buffer from 1 nM to 0.1 pM , including the assay buffer as 0 pM. To each tubes 5 Âµl assay buffer stored magnetic beads and 45 Âµl of diluted VEGF were added. The antigen capturing was done in triplicates in PCR tubes and was labeled appropriately. The wells firmly covered gently and incubated at RT for 1 hour under rotation. After incubation the tubes were briefly centrifuged and washed with washing buffer. Both SLC 1 and 2 was diluted in 1:100 in assay buffer and mixed together. From the mixture, 45 Âµl was added to each tube and incubated at RT for 1 hour under rotation.
Ligation & PCR
After incubation, antigen captured beads were washed twice and to each tube 50 Âµl PCR ligation mix was added. The tubes were firmly covered, following 5 min incubation at RT the plates was transferred to a real time PCR machine. The connector oligonucleotide was amplified by adjusting following parameters: The cycle was programmed for 45 cycles, with initial denaturation at 95â°C for 2 min, following by denaturation at 95â°C for 15 sec, annealing at 60â° C for 1 min.
Results and Discussion
The solid-phase PLA's sensitive and limit of detection (LOD) for VEGF was determined by using calibration curve. As mentioned in previous paper by Kamali-Moghaddam M et al.9, The SP-PLA has lower limit of detection compared with standard sandwich ELISA assay. By capturing VEGF in solution by primary antibodies on immobilized magnetic particles. Afterwards, the captured VEGF was detected using a pair of PLA probes thus, this assay ensures that only VEGF detection.
The concentrations of two unknown samples were also determined by using standard curve equation. The limit of detection is the minimum amount of analyte can be detected in the assay. Limit of detection is the two folds the standard deviation from the background signal. It was approximately found to be 0.3 pM/ml for VEGF detection (fig 2).
The Ct value of the two unknown samples was found to be 32.94 and 35 respectively. Substituting these values in the equation, the concentrations were calculated. It was found to be 4 pM/ml and 3 pM/ml (fig 2).
The Ct value of calibration curve is shown in the table 1. As the experiment was carried out in triplicates, the mean Ct value was plotted and the standard deviation was used as error bar.
Table 1. The Ct values from RT-PCR for various concentrations (pM/ml) is tabulated alomg with mean value and its standard deviation.
Figure 2. The calibration curve for VEGF detection by SP-PLA. X-axis shows the VEGF concentration in pM/ml and Y- axis shows the mean threshold frequencies (Ct values) of the RT-PCR. The vertical line shows the limit of detection of PLA and it was approximately found to be 0.3 pM. The error bars indicates the standard deviation from the mean triplicates values.
The advantage of using SP-PLA is the tight requirement of one captured probe and two PLA probes, thereby ensuring low nonspecific background. Another characteristic of SP-PLA is the use of DNA-based signal amplification by RT-PCR which increases the detection signals. Thus the combination of specific recognition and real time analysis of detected proteins enhances the sensitivity when compared to ELISA. Finally we can conclude the PLA technique increases the efficiency of protein detection by providing high specificity, sensitivity and a broad range of detection. This enables the SP-PLA as an important tool for diagnostics in various protein involving diseases like neurodegenerative9 and cardiac diseases.
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