Matrix Metalloproteinases household has grown to include 30 members since its first description in 1962.
MMPs are zinc and Ca dependant enzymes that are capable of modulating many physiological maps by hydrolysing the extracellular matrix under physiological pH ( Banerjee et al. 2010 ) . However, to forestall the inordinate dislocation of extracellular matrix, Tissue Inhibitor metalloproteinases ( TIMPs ) inhibit the action of MMPs to keep extracellular matrix homeostasis.
These enzymes are involved in procedures such as tissue fix, angiogenesis ; in add-on they take portion in a figure of pathological procedures such as metastatic malignant neoplastic diseases, shot, cardiovascular diseases, periodontic diseases ( Lombard et Al. 2005 ) . Many old ages of research has been dedicated to understand the functions of different MMPs so that they can be used as curative marks for drugs. However, in first clinical tests, MMP inhibitors in malignant neoplastic disease therapy were unsuccessful. To better understand the exact function of MMPs, as they are the marks for possible drugs, different techniques for MMP checks are used to test MMPs in tissue specimens and biological fluids. Some of the techniques discussed are immunohistochemistry ( IHC ) , Enzyme Linked ImmunoSorbant Assay ( ELISA ) , zymography, flow cytometry, Western Blotting ( WB ) , ( Di Girolamo, N 2010 ) .Immunohistochemistry ( IHC ) techniques are handily used to place MMP production in cells of local tissues under pathophysiological conditions ( Okada, Y 2001 ) . There are two types of labeling MMPs, direct and indirect labeling.
Indirect method is preferred over the direct method because it is more convenient and sensitive. In the direct method the primary antibody is applied straight to the tissue. The indirect method, the primary antibody in unlabeled and a secondary antibody will label the primary antibody. The secondary antibody is raised against the Ig of the species supplying the primary antibody ( Okada, Y 2001 ) . In IHC the specific ‘antigen ‘ should be known, for illustration in pterygium the antigen is MMP but the MMP figure should be known ( Di Girolamo, N 2010 ) . Specific acknowledgment of MMP by primary antibodies without antibody cross-reactivity with other molecules is indispensable for IHC, and monoclonal antibodies are suited for this procedure ( PHAR 3102, 2010 ) . In fact, monoclonal antibodies are recommended over polyclonal antibodies even though they have been used for the IHC of MMPs, nevertheless there are many different types of MMPs and therefore it is non an easy undertaking to raise polyclonal antibodies, which are monospecific to individual species of MMP ( Okada, Y 2001 ) . Before immunostaining the tissue, the arrested development with methanal and particularly glutaraldehyde sometimes cross-links proteins so strongly that antigenic site becomes vague and immunoreaction can non take topographic point.
So a via media has to be reached between the handiness of the antigen and the saving of the tissue. Monoclonal antibodies can be used for IHC in immunostaining tissues fixed with paraformaldehyde fixative. However, with formaldehyde fixed tissue, each antibody should be checked. Human tissues have to be preserved decently, so it should be handled carefully and processed at the latest within 1-3 H after surgery ( Okada, Y 2001 ) . To observe really little sums of MMP antigens the most sensitive manner to observe them in human pathological tissues is to immunostain by the Avidin-Biotin-Peroxidase Complex ( ABC ) and Immunogold-Silver Staining ( IGSS ) paraffin subdivisions of the tissue.
ELISA kits chiefly for human MMPs are widely available. The advantages of ELISA are that low sample sum is required and it can besides be used to mensurate the sum of MMP protein, so it is a good quantitative method. However, this method can non be used in mensurating the MMP activity. Furthermore, MMP complexes/dimers are believed to play a function in MMP-associated diseases and possibly can hold an deduction in the future rational drug design and the ELISA method is unable to observe the MMP dimers ( Kupai, K et Al. 2010 ) . Another job is that the antibodies, which coat the 96-microtitre home bases used in ELISA, can cross-react with the sample types from other carnal species.
Besides for ELISA soluble proteins are required, so the cells, which make different types of MMPs for illustration fibroblasts, macrophages, neutrophils, synovial cells, and some epithelial cells will non be identified ( Kumar, V 2007 ) .A widely used analytical technique is western blotting ( WB ) . WB is a semi-quantitative method used for the sensing of specific proteins such as MMPs in a sample of tissue homogenate or infusion ( Towbin, H 1979 ) .
The stairss of WB are shown in figure 1. One advantage of WB is that merely a little sum of other reagents are needed to add to the protein sample extracted when lading the sample into the gel, hence this is an efficient technique. Compared to zymography processs, the membranes used in WB are easy to manage, but similar to zymography is that both the active signifier and the zymogen of MMPs can be detected by separating on the footing of molecular weight ( Kupai, K 2010 ) . ‘WB is a good tool to observe and qualify a many of proteins, particularly those proteins that are of low copiousness. The proteins immobilized on the membrane are readily and every bit accessible to different ligands and multiple reproduction of a gel is possible. Last, drawn-out storage of transferred forms, prior to utilize, becomes possible and the same protein transportation can be used for multiple consecutive analyses ‘ ( Kurien, BT & A ; Scofield, RH 2006 ) .
The disadvantages of WB are that it is technically demanding and expensive process. The consequences are capable to reading, for illustration the presence or absence of sets and the strength of those sets ( Babu, VR 2010 ) . Besides another disadvantage is that the entire sum of the detected MMP of involvement in the membrane can be impeded as a consequence of the competition between the MMP of involvement with other MMPs of a similar size within the sample ( Mollica, JP 2009 ) .Substrate zymography is besides widely used to observe MMPs. Zymography involves the cataphoresis of enzymes through polyacramide gel. Electrophoresis is performed most normally utilizing gelatin as the substrate copolymerized with the polyacrylamide gel ( Hawkes, SP 2001 ) .
Gelatin detects MMP-2 and MMP-9 and the bound of sensing of MMP-2 in gelatin zymography is really little, 10I?g hence it is really sensitive ( Kupai, K 2010 ) . The protein samples are under denaturing status ( SDS ) without boiling or cut downing agent. After cataphoresis, the SDS is removed from the gel or ( zymogram ) and is replaced by a non-ionic detergent ; such as Triton X-100 this helps to renature ( unfold ) the proteins ( Hawkes, SP 2001 ) . The gel is incubated in a particular solution for the peculiar proteases under survey. The zymogram is later stained with Coomassie Blue and proteolytic activities are detected as clear sets against a blue background where the enzyme degraded the substrate.
A peculiar advantage of this system is that both the active signifier and the zymogen of MMPs can be detected by separating on the footing of molecular weight. Other substrate zymographies follow the same protocol ; nevertheless a different specific substrate is used for the MMP isoenzymes for illustration collagen zymography is used to observe MMP-1 and MMP-13 and casein zymography it is used to observe MMP-1, MMP-7, MMP-12 and MMP-13 ( Kupai, K 2010 ) . Recently Yu and Woessner ( 2001 ) developed a heparin-enhanced zymography technique for sensing of certain MMPs, which are hard to observe at low degrees by conventional zymographies such as gelatin zymography. They are able to observe matrilysin ( MMP-7 ) and collagenases ( MMP-1 and MMP-13 ) .
A disadvantage of zymographies is that the protocol takes 2 yearss to follow which is rather long ( Kupai, K 2010 ) . However, the protocol is easy to follow and inexpensive. Another disadvantage of this method is that the consequences should be confirmed by another protein method such as WB. Besides the consequences are semi-quantitative and non wholly quantitative and this method can non be used to place cells bring forthing the MMP unless utilizing cells in civilization media, or spinal fluid, serum, synovial fluid or piss or even aqueous temper, ( Di Girolamo, N 2010 ) . Zymography is used to observe MMPs in biological fluids, but similar to ELISA the cells that MMP activity is coming from are unknown.
Finally another used method for both clinical and research intents of testing MMPs, is flow cytometry. This process involves, the analysis of biological fluids incorporating cells such as synovial fluid. The primary system of the flow cytometry has two major constituents, the fluidic system which presents atoms or cells one at a clip to the question point and takes off the wastes ( Introduction to flux cytometry 2010 ) . The optical maser is the light beginning for spread and fluorescence, and the sum of forward and sideward spread straight indicates the size and internal complexness of the cells ( Pedreira, CE et Al. 2008 ) . Flourocrome-conjucated monoclonal antibodies recognize the MMPs expressed in specific populations of the cell. The look of several MMPs for each cell can be analyzed with the presently available clinical flow cytometers ( Pedreira, CE et Al. 2008 ) .
The primary advantage of flow cytometry is that multiple parametric quantities can be evaluated in 1000000s of cells in a few seconds and the information about the cell can be stored digitally. Besides, like ELISA and IHC little sum of the sample is required. The major disadvantage of this system is that it is really expensive to run and the readying of the samples requires reagents, which are expensive as good ( Curren, AD 1999 ) .There is no uncertainty that the above mentioned techniques have been, and go on to be valuable experimental tools for the sensing of MMPs, and later, the find of cognition in assorted Fieldss of research. There are advantages and, ofcourse, disadvantages in all of the techniques discussed above, and those that have non been discussed such as ELISA.
Until more betterments are made to these techniques, hence, research workers should take the method harmonizing to their involvements, every bit good as execute the techniques with preciseness and attention.There is no uncertainty that the above mentioned techniques have been, and go on to be valuable experimental tools for the sensing of MMPs, and later, the find of cognition in assorted Fieldss of research. There are advantages and, ofcourse, disadvantages in all of the techniques discussed above, and those that have non been discussed such as ELISA.
Until more betterments are made to these techniques, hence, research workers should take the method harmonizing to their involvements, every bit good as execute the techniques with preciseness and attention.
Western Blotting technique
Taken from: Thomas, S 2010