MOLECULAR LLUORESCENT MULTIPLEX ASSAY TO IDENTITY ALLO- ANTIBODIES AGAINST MHC ANTIGENS

FIELD OF THE INVENTION:

The present invention relates to a molecular fluorescent multiplex assay to identify allo-antibodies against MHC antigens. More specifically, the present invention relates to a cost-effective, specific and sensitive molecular fluorescent multiplex assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally. The present invention also relates to an economical kit for molecular fluorescent multiplex assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.

BACKGROUND OF THE INVENTION:

The world’s total population is estimated to be 7.7 billion, as of November 2018, with the largest contribution of 4.4 billion by the Asian population. At the start of the 21 st century, Asian population witnessed an increase in the life expectancy and decrease in the number of years expected to be lived in poor health conditions. Improving healthcare facilities, accessibility and technologies are the backbone of this achievement. Organ transplantation is one such development of healthcare, significantly contributing to the increase in life expectancy and improving the quality of life of patients suffering from organ failure. Organ transplantation therapy involves replacing damaged or diseased organ with a healthy organ donated by another person. In the cases of irreversible or terminal organ failure, it is the most promising therapy adopted worldwide. Data reported to the Global Observatory on Donation and Transplantation (GODT) (World Health Organization), analysis from 2015 transplant activity worldwide, revealed that 126,749 solid organ transplants were performed worldwide: 84,347 kidney transplants (41.8% from living donors), 27,759 liver transplants (21% from living donors), 7, 023 heart transplants, 5, 046 lung transplants, 2,299 pancreas transplants and 196 small bowel transplants. However, South East Asia with the highest growth rate of the number of people suffering from organ failure, contributed to only 6.73% of the total number of organ transplants performed worldwide, alarming the need to undertake accessible, accurate and affordable organ transplant measures for the world’s largest population.

Organ transplantation is one of the most complicated therapies. Many challenges are associated with each stage of organ transplant. Limited availability of organs, counseling the matched donor (in case of live donor transplant) or donor’s family (in cases of deceased donor transplant) challenges the organ donation process. These challenges are followed by the high possibility of organ rejection by the recipient’s body, post transplantation. This happens when the recipient’s immune system recognizes the transplanted organ as foreign and destroys it resulting in failure of the transplantation.

There are two ways to minimize the risk of organ rejection post transplantation. Recipients undergoing organ transplantation are administered with immunosuppressive drugs. These drugs suppress the immune response in the recipient’s body to safeguard the transplanted organ from rejection. In addition to immunosuppressants transplant rejection risk can be minimized by determining donor-recipient compatibility before transplantation. Cell-based and solid-phase antibody screening assays are employed for the detection of allo-antibodies in recipient’s serum against donor specific MHC antigens and hence they help in determining donor-recipient compatibility for transplant. If allo-antibodies against donor specific MHC antigens are detected in the recipient’s serum, it is recommended to not undergo transplant with the tested donor as organ rejection risk in such a case is very high. During pre-transplant risk assessment, several cell-based assays are routinely performed for testing histocompatibility between donor’s MHC antigens and recipients’s allo-antibodies including complement-dependent cytotoxicity cross match test (CDC-XM), and flow cytometry crossmatch (FCXM). These assays are used to determine if a recipient has antibodies in their serum that are against the native lymphocytes of a prospective donor. During the assay procedure mononuclear cells are collected from the donor’s blood and incubated with recipient sera to determine the presence of allo-antibodies against donor’s MHC antigens. However, there are many limitations associated with the above described tests. False negative reactions are reported in cases of low MHC antigen expression on lymphocytes. In many cases, false positive results are reported due to detection of non-MHC or auto-antibodies. These limitations have created a need for a more specific and sensitive assay for detection of allo-antibodies in recipients sera against MHC antigens of potential donors, further reducing the possibility of graft rejection.

Solid-phase antibody identification assays shows increased sensitivity as compared to cell-based assays. There are two kinds of solid phase assays - ELISA (enzyme linked immunosorbent assay) and assay employing fluorescent beads coupled with MHC antigens. These assays involve adding recipient's serum on a fluorescently labelled surface coupled with MHC antigens. The antigen-antibody reaction between the allo-antibodies present in the recipient’s serum and MHC antigens coupled on the fluorescently labeled surface is analyzed, enabling the identification of allo-antibodies present in the recipient’s serum. Solid phase assay is reported to have high sensitivity, however limitation on the number of MHC antigens that can be coupled with the fluorescently labelled surface and high genetic diversity of MHC antigens poses the risk of missing out allo-antibodies present in recipient’s serum. This might further result in misinterpretation of potential donor- recipient incompatibility, increasing the chance of transplant failure by several folds. MHC antigens are genetically very diverse. Extensive genetic diversity of MHC antigens is witnessed in Indian population. The Indian population, comprises of 4693 communities with several thousands of endogamous groups, 325 functioning languages and 25 scripts. To understand the inherent genetic variability of Indian population, The Indian Genome Variation database (IGVdb) Consortium was established with the objective of building an SNP and repeat polymorphism database of the Indian population to identify susceptible biomarkers for any disease or understanding drug response in different subpopulations. Genetic diversity at similar scale exists in MHC antigens expressed in Indian population. MHC antigen based studies carried out in the Indian population by various groups have highlighted unique and extensive MHC diversity of the Indian population. For example, the HLA-A2 and A33 allele families in the Indian population show greater diversity with the existence of several‘unique’ alleles. The best example is the presence of A*02l l, an allele that occurs only in the Indian populations with almost complete absence in the Caucasoid groups.

Amongst the haplotypes, the three most common HLA haplotypes that occur in North India are HLA-A33-B44-DR7, HLA-A33-B58-DR3, and HLA-A2-B50- DR3. Of these, HLA-A33-B44-DR7 is also observed in most other Asian countries like Malaysia, Thailand, South Korea, Bangladesh, Pakistan, China, but is not reported from‘rest of the world’. Similarly, HLA-A33-B58-DR3 is one of the commonest haplotypes in the Chinese population, which occurs with significantly high frequency in the Indian subcontinent suggesting racial admixture from the East. On the other hand, haplotype HLA-A2-B50-DR3 is observed exclusively in India particularly in patients with type 1 diabetes and is therefore‘unique’ to this population. The most commonly encountered haplotype in western population showing a strong association with autoimmune diseases is HLA-A1-B8-DR3, often referred to as the ancestral haplotype 8.1 (AH 8.1). The population sharing Indian gene pool, on the other hand, does not follow the western trend, and AH 8.1 occurs either with a very low frequency or is absent in the Indian subcontinent. Instead, the haplotype HLA-A26-B8-DR3 designated as AH8.2 is the most frequently occurring haplotype in the population sharing Indian gene pool, and this shows strong association with several autoimmune diseases just as AH 8.1 does in the western population.

The limitation of the assays described above and genetic diversity of MHC antigens demands identification of commonly expressed MHC antigens to be coupled on microsphere beads to increase the accuracy, specificity and sensitivity of allo-antibodies detection and minimize the possibility of graft rejection. The present invention identifies common MHC antigens to be coupled with fluorescently labelled microsphere beads with the aim to increase the specificity of allo-antibodies detection in the population sharing Indian gene pool globally. The most common MHCclass I alleles in the Indian population were found to be HLA-A'02 (34.9%), A'24 (24.33%), A'l l (34.5%), A'33 (18.1%) and A'320l (10.2%) in the A locus and B*07 (20.12%), B'35 (23.78%), B*40 (17.68%), B'57 (9.74%) and B'58 (4.26%) in the B locus. There was complete absence of BΊ4 in this population. The alleles were identified with the aim to present a specific and sensitive multiplex assay to identify allo-antibodies against conserved MHC antigens in population sharing Indian gene pool globally.

Since Asian population is reported to have the highest growth rate of the number of people suffering from irreversible and terminal organ failure with a very small number of transplants performed in South East Asia as compared to the total number of transplants performed worldwide, as described above, there is an alarming need to adopt accurate and affordable organ transplantation measures. The present invention provides a cost-effective, specific and sensitive molecular fluorescence based multiplex assay to identify acquired allo-antibodies in patients’ serum against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.

OBJECT OF THE INVENTION: The main object of the present invention is to provide a molecular fluorescent multiplex assay to identify allo- antibodies against MHC antigens.

Yet another object of the present invention is to provide a cost-effective, specific and sensitive molecular fluorescent multiplex assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.

Yet another object of the present invention is to provide a kit for molecular fluorescent multiplex assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.

Yet another object of the present invention is to provide an economical kit for molecular fluorescent multiplex diagnostic assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides molecular fluorescent multiplex diagnostic assay to identify allo-antibodies against MHC antigens. More specifically, the present invention provides a specific and sensitive molecular fluorescent multiplex assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally. The present invention also provides an economical kit for molecular fluorescent multiplex diagnostic assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.

A design panel of MHC antigens, according to their frequency in population sharing Indian gene pool globally, has been created. The MHC antigens in the design panel have been coupled with fluorescently labeled microsphere beads and then these beads have been tested with patients’ sera for detection of allo- antibodies against identified MHC antigens using a standard flow cytometer.

The present invention comprises the steps of identification of antigens in the design panel specifically conserved in population sharing Indian gene pool, coupling of the antigens with a bead under predetermined conditions with predetermined reagents, determination of coupling efficiency in singleplex assay and multiplex assay employing Fluorescent Microsphere -based Immunoassay (FMIA) and testing cross -reactivity of allo-antibodies with the coupled MHC antigens. After determining the efficiency, diagnostic kit prototypes have been developed and its specificity and sensitivity has been validated with stored patient’s sera. The prototype was then scaled up as a molecular fluorescent multiplex diagnostic assay to identify allo- antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.

The present invention provides a molecular fluorescent multiplex assay for identifying specific allo-antibody which binds to a purified MHC antigen coated over microsphere bead. The process to obtain said purified MHC antigen comprises identification of MHC alleles conserved in population sharing Indian gene pool globally, manufacturing of MHC antigens specific to said MHC alleles and synthesis of monoclonal anti-MHC antibodies specific to said manufactured MHC antigens.

The invention utilizes fluorescently labeled microsphere beads coated with a coupling agent such as avidin, coupled with manufactured MHC antigen for detection of allo-antibodies against MHC antigens in human serum by a standard flow cytometer. According to the methods of the invention, mixture of different microsphere beads, each coated with manufactured MHC antigens is used to detect specific allo-antibody. The invention also provides an array of microsphere beads coated with different purified MHC antigens which can be distinguished on the basis of intensity of fluorescent labels. Such use of microsphere beads labeled with fluorophores allows the identification and/or separation of different beads by a standard flow cytometer.

In a preferred non-limiting embodiment, microsphere beads (also referred to as microbeads) coated with MHC antigen have been incubated with serum to be tested for 30 minutes at 20°-25° C on a rotating platform at a predetermined dilution, preferably in the range of 1:8. The microbeads were then washed with a buffer, preferably PBS and incubated with anti-Human IgG antibodies conjugated with phycoerythrin (PE) for 30 minutes at 20°-25° C on a rotating platform. The microbeads are then re-suspended with wash buffer and analyzed on a standard flow cytometer. Sera which contain allo-antibodies against the tested microsphere (MHC antigens) will show a high median fluorescent intensity in comparison to the negative controls.

BRIEF DESCRIPTION OF DRAWINGS:

Fig. 1 represents mean MFI values of the monoclonal antibodies detected against the identified MHC antigens

Fig. 2 represents mean MFI values of the pooled monoclonal antibodies, diluted in the ratios of 1:16, 1:32 and 1:64, detected against identified MHC antigens using the developed prototype.

Fig. 3 represents mean MFI values of allo-antibodies detected against identified MHC antigens in pooled sera, prepared from the serum collected from ten MHC sensitized individuals.

Fig. 4 depicts a heatmap representing the mean MFI values of allo-antibodies identified in ten MHC sensitized individuals against common MHC antigens, represented in the developed kit.

DETAILED DESCRIPTION OF INVENTION WITH NON-LIMITING EMBODIMENTS AND EXAMPLES: It should be noted that the particular description and embodiments set forth in the specification below are merely exemplary of the wide variety and arrangement of instructions which can be employed with the present invention. The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. All the features disclosed in this specification may be replaced by similar other or alternative features performing similar or same or equivalent purposes. Thus, unless expressly stated otherwise, they all are within the scope of present invention. Various modifications or substitutions are also possible without departing from the scope or spirit of the present invention. Therefore, it is to be understood that this specification has been described by way of the most preferred embodiments and for the purposes of illustration and not limitation.

Although the following description is provided in the context of an example, it should be understood that the disclosure is not limited by the examples or claims. None of the structures, steps, or other features disclosed herein is essential or indispensable; any can be omitted or substituted by an equivalent.

It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. The words "comprising", "having", "containing", and "including", and of other forms thereof, are intended to be equivalent in meaning and be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items or meant to be limited to only the listed item or items. The disclosed embodiments are merely exemplary methods of the invention, which may be embodied in various forms.

The assay of the present invention is capable of detecting allo-antibody against single MHC antigen, thus identifying donor specific antibodies in patient’s serum. This assay has promising potential to determine allo-antibodies against donor specific MHC antigens in recipient’s serum with high specificity and sensitivity. The assay involves incubation of recipient’s serum with fluorescently labelled microsphere beads coupled with MHC antigen, allowing allo-antibodies present in the added serum to react with the MHC antigen coupled to the fluorescently labelled microsphere beads. The antigen-antibody reaction can be determined via fluorescence (MFI) generated with the help of labelled secondary antibodies added post the antigen-antibody reaction.

The present invention also allows detection of multiple allo-antibodies in a unified multiplexed alloantibody detection assay where each microsphere bead is coupled to a unique MHC antigen and has a unique fluorescence, this helps in identification of all the different MHC antigens coupled on the beads in a single assay. Addition of fluorescently labeled secondary antibodies against the anti human IgG chain generates a fluorescence spectrum which helps in analyzing the antigen-antibody reactions.

The experiments described herein suggests that the identification of MHC antigens conserved in population sharing Indian gene pool globally is clinically important for optimizing the method of allo-antibodies detection in said population, and hence improving the transplantation outcome. The present invention provides molecular fluorescent multiplex assay to identify allo- antibodies against MHC antigens. More specifically, the present invention provides a specific and sensitive molecular fluorescent multiplex assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally. The present invention also provides an economical kit for molecular fluorescent multiplex assay to identify said allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally. The present invention provides multiplex bead based assay to detect acquired allo- antibodies in individuals who have been sensitized due to exposure to corresponding antigens. The assay is carried out with MHC antigens conserved in population sharing Indian gene pool globally. When sera of a particular individual, previously reported with the history of organ transplant, pregnancy or blood transfusions, is allowed to react with multiplex bead based assay of the present invention, the corresponding beads would hybridize with the allo- antibodies. On using fluorescently labeled secondary antibodies, fluorescence is generated which is detected by using a standard flow cytometer

The present invention involves the identification of conserved MHC alleles specific to the population sharing Indian gene pool globally. Genetic database of Genebandhu (Adult stem cell registry) is used through a formal Memorandum of Understanding (MOU). MHC alleles conserved in the population sharing Indian gene pool globally has been identified. The nucleotide sequences of said MHC alleles have been searched/identified using a freely available online tool (IMGT database). Said nucleotide sequences have been converted to amino acid sequences. Proteins have been manufactured from said amino acid sequences. Table 1 represents the list of conserved MHC alleles specific to the population sharing Indian gene pool globally.

Table 1

Said manufactured protein has been coupled with avidin or an alternate coated fluorescently labeled bead to form activated protein coupled beads. The manufactured protein coupled beads are incubated under predetermined conditions to obtain pellets. Said pellets have been re-suspended in a buffer and stored at 2-8 °C in the dark.

Sera of highly MHC sensitized subjects are taken for analysis. Samples of highly MHC sensitized subjects are collected from bio-bank of Chimera Transplant Research Foundation (CTRF) and obtained under a formal Memorandum of Understanding.

Sera taken from highly MHC sensitized subjects are pooled and mixed in a tube. The pooled sera is diluted in the ratio of 1:8 in buffer and then incubated with manufactured protein coupled with fluorescent beads under predetermined reaction conditions followed by washing with PBS buffer. Then goat anti-Human immunoglobulin conjugated with fluorescent dye labeled secondary antibodies are added to the tube, mixed well and allowed to react for a specific time. Then buffer is added to the tube and analyzed using a standard flow cytometer.

The coupling method is selected from biotinylation, carbodiimide coupling. The present invention is carried out using biotinylation to yield experimental data.

In a preferred embodiment, said MHC antigens are coupled with fluorescently labelled microsphere beads. The specificity of the coupled beads is validated by adding monoclonal antibodies specific to the coupled MHC antigen. Antigen - antibody reaction between the coupled MHC antigen and monoclonal antibodies nonspecific to the said MHC antigen is also carried out to rule out the possibility of nonspecific binding. The process of the present invention comprises of (a) coupling of said identified MHC antigen with the avidin or an alternate coupled- fluorescent labeled beads by incubating 1.0x105 avidin or an alternate labelled beads with 250 pi of 4000 nM diluted biotinylated protein (example B *40:06) and incubated at predetermined conditions on a rotating platform, (b) followed by washing with phosphate buffer saline (PBS) three times at >8000 g for 1-2 minutes to obtain the formed pellet, (c) resuspending the separated pellet in 500 pl PBS-Tris-NaCl Blocking buffer and storing it at 2-8 °C in the dark, (d) procuring monoclonal antibodies against said conserved MHC antigens labeled with fluorescent dye preferably, phycoerythrin (PE), (e) diluting the procured monoclonal antibodies in the ratios of 1:8 using PBS buffer (f) Incubating 50m1 biotinylated coupled proteins to react with 20 mΐ of monoclonal antibodies in a single plexing assay for 30 minutes at 20-24 0C temperature, (h) followed by, washing with PBS buffer, (k) diluting the pellet with 500 mΐ of PBS buffer, (1) loading the mixture on the slide for detection of antigen-antibody reaction using fluorescent microscopy analysis, followed by detection of fluorescent labels using flow based technique ( standard flow cytometer). The said procedure is then repeated with monoclonal antibodies nonspecific to the coupled antigen to rule out the possibility of nonspecific binding. The said procedure is carried out for all the identified MHC antigens represented in the prototype. The sensitivity of the developed prototype has been tested by following the above said procedure with pooled monoclonal antibodies (pooling the procured monoclonal antibodies specific to each identified, conserved MHC antigens together) diluted in the ratios of 1:16, 1:32 and 1:64.

Fig. 1 represents mean MFI values of the monoclonal antibodies detected against the said MHC antigens. X-axis represents conserved MHC antigens and Y-axis represents mean MFI values of the detected monoclonal antibodies. The figure represents MFI values of specific and nonspecific monoclonal antibodies added against the coupled MHC antigens. The MFI values are detected using flow based technique - standard flow cytometer. High mean MFI values corresponding to the detection of monoclonal antibodies, specific to the coupled MHC antigen and low mean MFI values corresponding to the detection of monoclonal antibodies nonspecific to the coupled MHC antigen suggests reliable specificity of the developed prototype. Fig. 2 represents mean MFI values of the pooled monoclonal antibodies, diluted in the ratios of 1:16, 1:32 and 1:64, detected against said MHC antigens using the developed prototype. X-axis represents conserved MHC antigens and Y-axis represents mean MFI values of the detected monoclonal antibodies. Mean MFI values of the detected monoclonal antibodies is significantly higher than the negative control even at 1:64 dilution suggesting high sensitivity of the developed prototype.

For the analysis, serum of 10 highly MHC sensitized subjects is used. 300pl of the sera is taken from all of the samples, pooled and mixed in a tube to obtain pooled sera. Said pooled sera is diluted up to 1:8 dilution using PBS buffer. Then 10 mΐ of the diluted pooled sera is incubated with 50 mΐ of the stored pellets for 30 minutes on a rotating platform at room temperature, followed by washing with PBS three times. Now 50 mΐ of Goat anti-Human IgG conjugated to phycoerythrin (PE) secondary antibodies diluted 1:10 in PBS are added and mixed. The solution is allowed to react for 30 minutes, followed by the addition of 100 mΐ PBS. The analysis is carried out by using a standard flow cytometer 200 analyzer.

Fig. 3 represents mean MFI values of allo-antibodies detected against said MHC antigens in pooled sera, prepared from the serum collected from ten MHC sensitized individuals. X-axis represents said MHC antigens and Y-axis represents mean MFI values of the detected allo-antibodies in individual’s serum. High MFI values of the allo-antibodies detected against identified, conserved MHC antigens - B*35:02 and B*40:06, suggests that the frequency of occurrence of the identified MHC antigens in the population sharing Indian gene pool globally is significant and hence it is essential to detect allo-antibodies against the said MHC antigens to reduce the chances of fallacious results.

In another embodiment, all the common biotinylated proteins are coupled with avidin coated fluorescently labeled beads. All the common biotinylated proteins coupled with avidin coated fluorescent-labeled beads are then pooled into one single tube to obtain multiplexed beads. Then the differentiation between the substrates is done according to the fluorescent coating on beads. The level of fluorescence is analyzed by solid state laser of 488 nm in a standard flow cytometer.

When said multiplexed beads are allowed to react with serum containing allo- antibodies against the coupled proteins, an antigen- antibody reaction takes place. The unbound antibodies are removed by washing with buffer. Then fluorescently labeled Goat anti-Human IgG is added which binds on the antigen-antibody complex and upon analysis through two lasers at the same time, (one for differentiation of substrate and other for detection of analytes) using a standard flow cytometer, a unique fluorescence is generated which is detected by using a specific filter and detectors.

According to a general method of the invention, a mixture of fluorescently labeled microsphere beads coated with MHC antigens are incubated with a serum of ten highly MHC sensitized individuals, for 30 minutes at 20°-25° C on a rotary shaker. The microbeads are then washed with a wash buffer (PBS) three times and incubated with anti-Human IgG antibodies conjugated with phycoerythrin, for 30 minutes at 20°-25° C on a rotary shaker. The microsphere beads are then re suspended with wash buffer and analyzed on a standard flow cytometer. The power of multiplexing allow us to differentiate between the microbeads according to their fluorescence and further the fluorescence of secondary antibodies helps in determining the presence of allo-antibodies against MHC antigens in the tested serum. Serum which contains allo-antibodies against the tested microsphere beads (MHC antigens) show a high median fluorescent intensity in comparison to the negative controls.

The present invention also provides an economical kit for molecular fluorescent multiplex diagnostic assay to identify allo-antibodies against common MHC antigens.. The kit comprises of fluorescently labelled beads coupled with said MHC antigens and containers for the following reagents - wash buffer (Phosphate Buffer Saline), secondary antibodies (anti-human IgG), negative control (sera with known absence of allo-antibodies, representing negligible MFI values for secondary antibodies) and positive control (sera with known presence of allo- antibodies against coupled MHC antigens, representing higher MFI values for secondary antibodies.

Fig. 4 depicts a heatmap representing mean MFI values of allo-antibodies identified in ten MHC sensitized individuals against common MHC antigens, represented in the developed kit. X-axis represents the common MHC antigens coupled with fluorescently labelled microsphere beads, Y-axis represents the anonymous id of MHC sensitized individuals tested for the presence of allo- antibodies and Z-axis represents mean MFI values of the detected allo-antibodies. Red colour represents the presence of allo-antibodies and blue colour represents the absence of allo-antibodies. The first column of the heat map is positive control and the second column of the heat map represents the negative control.

The solid phase multiplex assay of the present invention is capable of being conducted on multiplex solution comprising of different fluorescently labeled microsphere beads coated with different corresponding MHC antigens.