Biomarkers Are Often Molecular In Nature Biology Essay

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Today, biomarkers are often molecular in nature that can be detected through the use of advance techniques and methods such as PCR Polymerase chain reaction, Luminex and ELISA (Enzyme-linked immunosorbent assay) in HLA (human leukocyte antigen system) typing. This paper discusses the three HLA typing techniques, their attributes, characteristics, cost effectiveness and efficiency. Most studies deals with the development of these techniques for diagnosis as drug resistance detection which mainly involve the use of polymerase chain reaction method (Brancato, 2006). PCR is a technique or procedures which allows the in-vitro selective amplification of a certain HLA/DNA replication. The application of PCR in this study will help determining its potential development of diagnosis and drugs. This technique is often improved importance to the drug effect or disease that can be took through the multiple biomarkers' use. Moreover, the Luminex technology is one of the most widely utilized multiplexing technologies for the various biomarkers use of simultaneous measurement. Lastly, the retrospective analysis of HLA and serum samples, utilizing a new ELISA HLA screening technique, revealed that the rejection crisis and the subsequent graft loss were due to a specific pre-sensitization. This paper will examine which technique in HLA typing is more efficient and cost effective.

Introduction

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The advent of techniques in recombinant DNA identification and classification in the mid-70s and 80's facilitated the molecular characterization and cloning of the class I and class II genes, and the polymerase chain reaction (PCR) introduction in the mid-80s made the analyses of the extensive allelic sequence possible with the diversity of these loci. In addition, ELISA, following the determination of HLA sequence polymorphism in human populations, allowed the advent and development of a simple and rapid variety of DNA sequence-based typing methods such as Luminex chain reaction. Here, we review the deviation, application and importance of such methods to the clinical areas.

Recent studies shows that many laboratories and pharmaceuticals used for the diseases treatment have been based largely on the relative productivity of small organic molecules which can be synthesized by microbes, animals or by means of organic chemistry (Ziomek, 2008). These productions include most the antibiotics to prevent the growth of bacteria, analgesics, synthetic hormones, and other pharmaceuticals products. In these areas of microbiology, these methods are useful and facilitate advance techniques in developing innovative advantages (Bringloe, 2008).

Nowadays, the increase attention regarding this issue has been focused on larger and more complex HLA molecules identification as alternative for more advance therapeutic agents (Mitchell, 2009). These molecules are larger in nature and composed of long chains of subunits called amino acids. Proteins are composed of different combinations of the 20 or more amino acids; the length of proteins is often 100 to 1,000 amino acids long (Ikeno, 2008). The functionality and structure of specific proteins is determined and identified by its sequence of amino acids, which, in turn, determines its three-dimensional conformation, its composition and structure (Hewson, 2006). Internal bonds (sulfur and hydrogen bonds) among the amino acids are the determinants of the final shape and form of the given protein (Ziomek, 2009). Complex HLA forms undergo further chemical processing such as the addition of phosphate groups or the process of phosphorylation or the carbohydrate molecules which commonly known as the process of glycosylation, which identify and modify the proteins' functions. Information stored in DNA directs the proteins in synthesizing machinery of the specific cell to provide the given proteins required for its structure and metabolism (Bringloe, 2008).

Nowadays, the increase attention regarding this issue has been focused on larger and more complex techniques as alternative for therapeutic breakthrough. These techniques are more advanced in nature and composed of long chains of subunits called tyechnology (Ghosh, 2008). The functionality and structure of specific methods in HLA typing is determined and identified by its sequence of, which, in turn, determines its three-dimensional conformation, its composition and structure. Internal bonds (sulfur and hydrogen bonds) among the methods are the determinants of the final shape and form of the given primers (Howell & Carter, 2010). Complex systems undergo further chemical processing such as the addition of phosphate groups or the process of phosphorylation or the carbohydrate molecules which commonly known as the process of glycosylation, which identify and modify the proteins' functions (Held, 2007).

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There have been primary studies conducted on the capability of these three techniques to serve as a host for molecular farming of HLA typing. It has been introduced for the provision of biomarkers and production of enzymes, antibodies and other products, including lactoferrin, collagen, gelatin, and vaccines (Grummt, 2007). The said methods has been an ideal choice for the identification of therapeutic means because of its importance in genetic manipulation for its relative tractability and an impending demand to explore other possible uses for the said technique (Ziomek, 2009). It has noted that it has been widely used to determine the suitability of expression systems based from HLA for therapeutic typing and production and other transgenes. In these methods, the transgenes are maternally inherited (Trull & Phares, 2005). Some of the important factors that have been produced through growth hormones, the human serum albumin, erythropoietin, human-secreted alkaline phosphatase, collagen, recombinant antibodies such as IgGI, IgM, SIgA/G, scFv-bryodin 1, recombinant subunit vaccines such as hepatitis B virus envelope proteins, E. coli heat-labile enterotoxin, diabetes autoantigen, cholera toxin B ubunit and porcine transmissible (Bringloe, 2008).

This study aims to investigate and explore the efficiency of ELISA, Luminex and PCR in HLA typing. The use of these techniques for the expression of HLA is an important and new discipline in different fields such as pharmacology and the applied molecular biology and biotechnology (Davies, 2007) This research study was a response to investigate and compare the three techniques;

To be able to investigate the potential capacity and cost effectiveness of each methods

To clearly illustrate and describe the mechanisms involved in the process of the HLA and gene expression

To devise a simple, yet credible methodology that could be replicated using simple laboratory techniques and equipment;

To recommend ways for commercial production of therapeutic techniques using the sample species;

To produce a credible and reliable written output that could be used by companies as a basis and reference for future studies.

Practical recommendations will be developed to ascertain the practicability and applicability of the findings in the real world. Action research method may be employed to solicit feedback from a real life implementation of the recommendations. Likewise, this paper will provide an insightful learning about how the research process developed the skills for critical thinking, time management and careful attention to details of the researcher. It is opted to assert how problems incurred in the process have been resolved and what lessons were learned from them by the researcher.

Methodology

A research design can be stated as the major constituent which structures the entire research project. Also there are many research designs that one could follow such as action research, ethnography research and the case study approach. When focusing on the research design which will be discussed further in this chapter it could create a major impact on the desired sample size of the research as well. This study will utilize the non-experimental design. This method also utilized to explore interesting fields for further investigation and gather more data in order to achieve the objectives of the study (Davies, 2008)

In a non-experimental design, the relationship or association between variables are explored based on given parameters and data. The independent variables are not manipulated and are therefore, not randomized. Here, the research is much easier to conduct compared to an experimental one. However, it should be clear that the relationship being tested here do not imply causality. Another term to describe this kind of design is correlation design since, basically, the reason for the relationship is left unclear (Davies, 2008)

The evidences from a non-experimental design are not as powerful as that of experimental designs. If an experiment is designed and executed properly, not only can it determine relationships, it can also determine causality. However, a flawlessly executed experimentation is very difficult to achieve. It requires a lot of ingenuity, resources, experiences and time (McNee, 2008).

This process which has a wide range of techniques requires gathering primary as well as secondary data. When considering data collection the information which is been gathered will first be arranged according to a systematic form where it will then be analyzed.

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In addition to data gathering procedure the researcher will utilized the following technique to obtain the best possible data;

Archival Research

Books and journals from different libraries will be used to collect articles and information for the study. Other reference materials such as newspaper will also be utilized by the researcher for added information. The researcher will chose to review books and other written paraphernalia from the library sources as they hold information regarding the subject of the study (Davies, 2008)

Internet Research

The researcher opted to use computer-based research for the gathering of latest information and data, given that significant part of the research requires information from different part of the world (McNee, 2008).

The research method utilized to examine interesting areas for investigation and gather more data in order to achieve the objectives of the study. The researcher will also utilize derivatives to be computable with international law. In this study, the researcher will also employ case study to involve a comprehensive and extensive examination of a particular event and issue involving International trade theories in response to the development of biotechnology. This study also involves descriptive record from previous research.

In summary, the primary and secondary data will be gathered. Secondary data will be used to establish trends and to establish observations that lead the researcher to the research topic and how it will be conducted. Recommendations will also be given based on the results of the study.

The data gathered and the result will include based on materials related to the standard assessment policies including directives on standardized testing to check for validity of instrument. Also collected were samples of materials in assessment activities in for the purpose of reading such as worksheets, books, and paper and tests. The approach will be conducted in such a way that the answer found will be as meaningful and as accurate as possible. The researcher plans an approach that will best answer the research questions and to ensure the validity and reliability of the result.

Results/ Discussion

HLA or Human Leucocyte Antigen system

HLA forms part of the Major Histocompatibiblity Complex (MHC)

Found on the arm of chromosome six

Major Histocompatibiblity Complex antigens are integral to the functioning of normal immune response.

Important role of Major Histocompatibiblity Complex antigens depends in the control of self recognition and thus defence against surveillance and micro-organisms

HLA Alleles

TYPING METHODS

SEROLOGY used to be the 'gold' standard. Now being superseded by molecular techniques as they become more time efficient and robust (Hudson & Holdsworth, 2010).

CELLULAR rarely used now. Originally utilized for Class II typing

MOLECULAR is a fast becoming the method of choice. Choice of Many laboratories.

Description of Techniques

Polymerase Chain reaction Method

PCR Machine

Most studies deals with the development of the rapid techniques for diagnosis as drug resistance detection which mainly involve the use of polymerase chain reaction method. PCR is defined as the technique or procedures which permits the in-vitro selective amplification of a specific replication of HLA/DNA replication (Porth, 2009). The application of PCR in this study will help determining its potential development of diagnosis and drugs (Martin, et al, 2005).

PCR is a highly sensitive technique for the detection of bacteria and possesses a relatively useful potential for the development of a method of diagnosis. A novel approach is to establish a fast efficient, sensitive and specific means of diagnosis (Markham, 2009).

Typing Based on the Gel Mobility of the PCR Products

Other PCR-based methods incorporate in the utilization of multiple restriction endonucleases and gel electrophoresis (PCR-RFLP) to identify and characterize the PCR product polymorphisms; these approaches have been created and developed for class I and class II typing. The Conformation-based gel mobility analyses, such as PCR-SSCP (single strand conformation polymorphism) and PCR-DHA (directed heteroduplex analysis), have also been developed for class II typing, but these approaches, unlike SSO or SSP methods, are not widely used especially in clinical settings. A recent directed heteroduplex approach modification, termed double-stranded conformation analysis (DSCA), utilizes a fluorescent single-stranded probe specific for a particular HLA allele (i.e., HLA-A*0201) to hybridize to the PCR products (e.g., the two alleles of HLA-A) amplified from the sample, thereby generating heteroduplex labeled molecules. The mobility of these labeled molecules can be compared systematically to a standard set of markers for each allele (Arguello, 1998).

Typing by Hybridization

However, there have been also recorded limitations and shortcomings in some of the earlier devised and developed mechanisms of PCR methods. The common method of completing these methods with the use of microbial expression systems such as E.coli and the yeast system S. cerevisiae are very costly. Also, there is a high possibility of contamination of the fraction with human HLA and other samples found in human or animal sources (Ziomek, 2009). This leads to the limited availability of variety of therapeutically important DNA/ proteins. Also, production using plants and animal sources also has its limitations. Those who are using plants as the host organism which synthesize whole plants have several disadvantage in the intrinsic benefits of cultured cells, including the proper and precise control over production and growth conditions, group product consistency, a high level possibility of containment and the ability to provide the number of recombinant DNA analysis in accordance with good practice of manufacturing (Rosa & Socransky, 2010). In general, the advantages that can be gained from plants as the host for PCR production and analysis for commercial use is very promising and still needs to be explored using other species aside from those that have already been studied (Mullis, 2006).

Curently, new high-speed generation of thermal cyclers has been created on the basis of real-time PCR analyses. For example; Molecular Biochemicals developed by Roche which fluorometrically real-time monitor the formation of products utilizing PCR during thermal cycling. The sensitivity and specificity of the reaction can be enhanced greatly by amplification combination with identification of the melting behavior of the PCR double-stranded product.

Luminex

Luminex core

Luminex is the advance and effective use of biomarkers by medical practitioners to identify disease and improve therapeutic methods in the practice. In an old ways, blood pressure and pulse measurements are general techniques or methods for most physical examination - these general health biomarkers. Nowadayts, biomarkers are often on molecular nature that can be identified through the utilization of advance methods and techniques such as Luminex in HLA typing. This technique is improved be relevant to the diseases or drug effect that can be obtained through the utilization of multiple biomarker technology (Pozza & Ricci, 2010).

Enzyme-linked Immunosorbent assay (ELISA)

Microplate ELISA: coloured wells indicate reactivity

According to Coleman (2007) ELISAs are the most widely used method for antibodies screening in blood samples. The method's specificity and sensitivity provides current available commercial systems approaches with a result of up to 100% (Jensen, 2005). The occurrence of false negative and false positive reactions may also present. The sensitivity of this test systems are being improved presently by the utilization of recombinant antigens (Monien et al, 2009).

Comparative Analysis

PCR Results and Fact Sheet

According to Bringloe (2008), qualities of PCR such as its in-vitro sensitivity and specificity may increase the efficiency of local diagnosis and treatment. It may serve as a tool in the rapid screening for drug resistance along with the present drug susceptibility testing. By using PCR, one can simply amplify the desired portion of the gene under study and thus enhance the isolation of the important portion of the gene. Through this in-vitro amplification method it would b more efficient and easy to study the characteristics of gene mutations (Ziomek, 2008). Among all the condition needed for the success of the amplification procedure, the choice of the appropriate primers, the optimization of the temperature for annealing of primers as well as the molar concentrations and PCR reactions mixture are most essential. PCR including diagnosis can be performed in few hours and can be performed adjacent with the conventional diagnostic testing.

Based on the available literatures, the PCR database of class I and class II allelic sequence diversity have a variety of PCR-based typing methods which have been developed and applied to clinical HLA typing. It is more efficient yet costly (Salsberg, 2008).

Moreover, most typing methods involve the primer design pairs that are capable of amplifying every allele at the target locus with the sequence of polymorphic motifs localized between the sites of primers (Ziomek, 2008). The primer sequences between them subsequently characterized by analytic variety approaches, including oligonucleotide probes hybridization, restriction enzymes digestion, and termination of chain sequencing reactions, or are inferred from the mobility of conformation-based of the PCR products with gel electrophoresis. The other main approach to HLA typing uses the PCR itself as a distinguishing method of polymorphisms by specificity exploiting of oligonucleotide primer extension and places the primer's 3′ end of the at the polymorphic site (Bringloe, 2008).

Another PCR-based approach, based on the specificity of primer extension rather than that of probe hybridization, has also been applied to HLA typing. This method is known variously as allele-specific amplification (ASA) sequence-specific priming (SSP) and the amplification refractory mutation system (ARMS). Here, a specific primer pair is designed for each polymorphic sequence motif or pair of motifs and is based on the differential ability to extend a primer that is matched or mismatched with the template at the 3′ end. The presence of the two targeted polymorphic sequences in a sample is detected as a positive PCR product, typically identified as a band on a gel. In SSP/ARMS typing, if the PCR is negative and no product is detected, the sample is assumed to lack one or both of the specific motifs. Since inhibition of the PCR or absence of template also yields a negative result, each reaction should include a positive control, that is, PCR primers for an unrelated monomorphic target sequence that produces a fragment that can be distinguished (e.g., resolved by differential gel mobility) from the HLA PCR product (Mullis, 2006).

PCR is the original biomarker technology is based on the polystyrene microspheres. This technique utilized the vacuum filtration to release sample reagent excess and matrix in a way related to cell membrane-based experiments. The acceptable analytical performance in this method can be achieved through the process prone to individual clogged wells (Gibson, 2009) Magnetic microspheres have recently become available that have significantly developed and improved ease of use and make the biotechnology much more technology friendly. It demonstrate attributes of its performance through the a series of luminex automation of xMAP assays.

Luminex Result Table and Fact Sheet

Comparing to PCR AND ELISA, Luminex is continually creates and develops, manufactures and markets innovative biological testing technologies with applications throughout the biotechnology industry. The open-architecture technology of luminex enables huge numbers of biological tests (bioassays) to be analyzed and conducted quickly, cost-effectively and accurately (Washington & Hazbón, 2010). Systems using the luminex technology perform exclusive, effective and discrete bioassays on the color-coded surface beads known as microspheres, which are then analyze in a compact analyzer. Using high-speed digital-signal processors and lasers, the analyzer identifies the bioassay simultaneously and assesses the results, all in real time (Held, 2007).

Luminex have the multiplexing assays of this technology such as those provided by the biotechnology have proven to be highly effective and useful for identification and quantification of biomarker from tissues, cells and body fluids. The Applications of this technology include research on biological science, clinical diagnostics and drug discovery (El-Hamshary & Azazy, 2010). The classical technology based on the use of luminex tends to have complicated, manual work flows prone to errors and failure through cumulative system that can lead to problematic quantification precision. This technology demonstrates improvements and development in ease-of-use and precision for both polystyrene and HLA typing (Madrigal, 2009).

ELISA Experiment Result and Fact Sheet

ELISA, according to Madrigal (2009), the human leukocyte antigen (HLA) antibody accuracy typing and results on HLA screening is dependent and based on the technique involved. In the study conducted by Nanni-Costa, Scolari and Iannelli of, St. Orsola University Hospital, Institute of Nephrology in Italy, samples from patients awaiting transplantation of kidney were tested by two various enzyme-linked immunosorbent assay (ELISA) methods. The results of the study presented indicate that a single ELISA method does not invariably exclude or prove the HLA antibodies present in the samples, and that additional testing is required (Mullis, 2006).

According to Monien et al (2009), ELISA is effective in terms of retrospective analysis of HLA and serum samples, utilizing a new ELISA HLA screening technique, revealed that the rejection crisis and the subsequent graft loss were due to a pretransplant donor-specific pre-sensitization caused by a non-complement-fixing antibody of IgG2 class. The case illustrates the clinical significance of non-complement-fixing anti-HLA antibodies (Leah & Williams, 2007). In addition it is shown that ELISA methods are suitable for detecting potentially harmful donor not detectable by standard lymphocytotoxicity techniques. Hence ELISA could be an alternative to flow cytometry for this purpose rather that of PCR and Luminex. It is concluded that screening and cross-matching techniques which detect non-complement-fixing anti-HLA antibodies could improve graft outcome, and should form part of the immunological monitoring of kidney transplant waiting-list patients (Held, 2007).

Efficiency

HLA genotyping

PCR have one virtue of SSP/ARMS typing is that it can "set phase," in that the two polymorphic sequence motifs detected by the primers must be linked on the same allele. SSP/ARMS typing methods have been developed for both the class I and class II loci, and the introduction of robotics has, in some cases, simplified this procedure (Chung & Sidney, 2010). Detection methods that are not based on visualizing a band in a gel and can therefore eliminate the gel electrophoresis step have been developed recently (Bringloe, 2008). Although informative and relatively fast for small numbers of samples, the SSP approach requires many separate PCR's to achieve intermediate or high level typing and, in its current format, is not well suited to rapid throughput of a large sample of numbers (Inga, & Knutson, 2005). As noted above, allele-specific amplification can be used in conjunction with SSO probe typing for high-resolution typing by allowing the separate amplification of the two alleles in a heterozygote. PCR is more efficient that the other techniques when it comes to amplification and polymorphic sequences (Craig, 2008).

In contrast, the Luminex biomarker technology is one of the most recent and commonly used technologies in multiplexing for the simultaneous measurement of multiple biomarkers. There are recently a Luminex technique is efficient in producing reagent systems based on research biotechnology, drug discovery, clinical diagnostic applications and HLA typing methods (Markham, 2009).

ELISA technique involves in immune responses and some of which exhibit extensive genetic polymorphism (Nguyen, 2008). The genes encoding the HLA class I (A, B, and C) and the class II (DR, DQ, DP) molecules are the most polymorphic loci in the human genome, with some loci (e.g., HLA-B or DRB1) having more than 300. Initially, genetic variation at these loci was analyzed by HLA serologic typing using reagents derived from sera of individuals or multiparous women who had received multiple blood transfusions (Santi, 2008). In addition to such serological typing methods, class II polymorphism was analyzed, in some cases, by cellular typing methods, such as the mixed lymphocyte reaction (MLR). ELISA is efficient in determining antigenic differences were typed based on recognition by T cells rather than by the products (antibodies) of B cells (Monien et al, 2009).

Summary

Table Comparing the Three Methods

HLA Typing Methods

Efficiency

Cost effectiveness

Sensitivity and Accuracy

PCR

This method provided and permits users in an efficient and faster data collection and randomized results

More costly than other techniques but produces the same effectiveness when it comes to reult

The amount of variability that typically occurs in this method does not usually detract from the test's value and statistically is insignificant and have comprehensive quality control and assurance procedures

Luminex

Documents and results are analyzed and organized so they are readable requiring mijimal amount of interpretation and associated style sheet

Requires Higher cost than other techniques because of its avanced assay and tecniques

Contribute to small but measurable variations in results. Provide target values for test results and provide evidence for the expected ranges

ELISA

Utilize scripting languages and numerical values to display results, or to create interface laboratory elements, the information provided are functional and can be read by assistive technology.

Different types of this methods have various cost effectiveness, usually moderate cost in contrast to PCR and Luminex

Have an inherent ability to distinguish whether the specimens have the testing condition or do not have the condition and undergone multiple evaluations and comparisons

In summary, the allelic diversity study at the HLA loci as well as the simple development and rapid typing of DNA-based methods was facilitated dramatically by the amplification development. Generally, the sequence-based allows a more sophisticated and accurate result of PCR (fewer errors) and much more efficient and precise (more discriminating) method than cellular or serological methods (ELISA). For example, there are less than 300 alleles at the locus but only seventeen distinct specificities serology or serotypes. In addition, unlike reagents in serological typing, probes, the primers, and thermo stable polymerases DNA used in HLA typing via PCR-based that can be produced as standardized reagents is more efficient and cost effective (Grummt, 2007). PCR amplification also facilitates the HLA typing of very tiny or minute samples such buccal swabs, hair, dried blood spots, or even very tiny individual cells. It is better and less costly than Luminex (Chung & Sidney, 2010). Finally, the data of PCR-based typing from which the sequence of nucleotide for the amplified locus can be revealed or inferred how and where alleles differ, allowing the role of specific polymorphic amino acid residues analysis in peptide presentation and building as well as in clinical transplantation and disease association outcomes (Bringloe, 2008).

Conclusions

Recent studies shows that many pharmaceuticals used for the treatment of diseases have been based largely on the productivity of relatively small organic molecules which is synthesized by microbes, animals or by means of organic chemistry (LoBiondo-Wood, 2006).These productions include most the antibiotics to prevent the growth of bacteria, analgesics, synthetic hormones, and other pharmaceuticals products (Santi, 2008).

HLA typing methods and identification is really important. Firstly, PCR, its major advantages of applying its system include a very accurate and simple medium growth and the possibility for production of large-scale result. Having the target DNA in an appropriate vector in the expression system of choice is the first step in optimizing production of the heterologous proteins. Within a given system the transcription and translation processes leading to the heterologous protein production are a complex set of reactions. Each process is carried out and controlled by several enzymes/factors. In recent years we have learnt that the following few key steps or reactions are critical in determining the ultimate outcome (Ikeno, 2008). Secondly, the Luminex Chain reaction can produced a gene expression system is frequently used and regulated at the transcription level and it is generally assumed that the steady-state mRNA level is a primary determinant of the final yield of a foreign protein. The mRNA level is determined both by the rate of initiation and the rate of turnover (Sylvester & Kron, 2010). In most cases the yield of a foreign protein expressed using a host promoter has been much lower than the yield of the homologous protein, using the same promoter (Ghosh, 2008) The open-architecture of luminex technology facilitates a large number of biological testsings (bioassays) to be analyzed and conducted accurately, quickly and cost-effectively (Lipstein & Browning, 2010). On the other hand, ELISA is concluded that screening and cross-matching techniques which detect non-complement-fixing anti-HLA antibodies could improve graft outcome, and should form part of the immunological monitoring especially in transplant waiting-list patients (Nguyen, 2009).

PCR-based methods of HLA class I and class II typing have been considered as developed yet simple. It is also rapid, automatable and highly informative that can be carried out at either intermediate or high levels of allelic resolution in a clinical diagnostic setting as well as for research studies (Santi, 2008). For solid organ transplants, the data indicate that the degree of HLA matching, in terms of the number of mismatched A, B, and DR antigens, significantly affects the survival of kidney grafts, for both cadaveric and living unrelated donors, even with the most recent protocols of immunosuppresion (Nguyen, 2009).

The Luminex on the other hand have the degree of HLA matching which also have significantly affects the survival of heart transplants but not of liver transplants (Thomas et al, 2008). For bone marrow transplants, where the HLA typing was carried out at the allele level, the comparison of DNA-matched with DNA-mismatched among serologically matched donor and recipient pairs indicates that both graft rejection and graft versus host disease occur more frequently in allele-mismatched transplants (Rinshol, 2007). Acute graft versus host disease is increased with recipient disparity for one DR or DQ allele or for two or more class I alleles. Bone marrow graft rejection was associated with donor disparity for two or more class I alleles. It is likely that the effect of mismatching on these various clinical outcomes will depend on the specific HLA alleles that differ between the donor and recipient (Mullis, 2006).

According to Monien et al (2009), ELISA on the other hand have the capability of increasing the use of DNA HLA typing in transplantation that should help define the "rules" that govern the clinical outcomes of mismatched transplants and allow the identification of "permissible" (relatively well-tolerated) mismatches (Markham, 2009).

In this paper, the researcher have demonstrated the ability of these methods, their effectiveness, cost and sensitivity to automate the HLA typing and processing needed for any quantification assays of biomarker, whether they are expressions of gene or protein biomarkers and whether the assay used are based on polystyrene or magnetic microspheres. These methods specifically PCR and Luminex Automation serves to speed up and simplify the method of processing, but it also tends to develop and improve bead recovery to elevate the efficiency, cost effectiveness improvement to reduce background and improve precision for a better performance of assay. The various methods provide the end-user with three different automation levels: with for lower throughput; full plate determination with higher throughput; and has all the attributes for biotechnology applications, but also the dispense ability of reagents for more automotive processing of sample processing (McCance, 2008).