What is A-Cube®?
A-Cube® is a research test that can detect many types of autoantibodies at once from a small amount of serum.
A-Cube® is a research test using the indirect immunofluorescence that uses wet antigen proteins and can detect multiple types of autoantibodies in a single test using a small amount of serum (300μL).
This test can detect 67 types of autoantibodies associated with systemic scleroderma and dermatomyositis/polymyositis, which are collagen diseases.
The test results will be reported as a quantitative value (index value) of the detected antibody for each antigen and a positive/negative result.
＊This is a research test and cannot be used for diagnostic purposes.
What is collagen disease?
Collagen disease is not the name of a single disease, but a generic term for a multitude of diseases.
The cause of collagen disease is believed to be an abnormality of the “immune system”.Our body has an “immune system” that protects us by eliminating foreign substances such as bacteria and viruses.Conversely, an immune system that becomes hyperactive and erroneously identifies self-antigens, leading to an attempt to eliminate them, is known as “autoimmune disease.
Patients with autoimmune diseases are believed to have cells (autoreactive lymphocytes) and proteins (autoantibodies) in their bodies that attack their own body and cause inflammation of the skin, muscles, joints, internal organs, and blood vessels.
A list of typical collagen diseases include rheumatoid arthritis, systemic lupus erythematosus (SLE), Sjögren’s syndrome, polymyositis/dermatomyositis, and systemic scleroderma.
The majority of collagen diseases are rare, and their pathogenesis is not well understood. The Japanese government has classified these diseases as intractable.
There are different types of autoantibodies in collagen diseases. How bad the disease is and what complications depend on the type of autoantibody that is positive in a test.
For patients with scleroderma and myositis, knowing which autoantibodies they have can help predict which complications are likely to occur, how the disease will progress, and what treatments will be effective.
The presence or absence of autoantibodies is one of the diagnostic criteria, and measurement of autoantibodies is considered important in the diagnosis of collagen diseases.
Autoantibody test for collagen disease (blood test)
Measuring autoantibodies is considered important for the diagnosis of collagen diseases.Autoantibodies associated with the collagen disease have also been discovered one after another.
Some of these autoantibody tests are covered by insurance and can be performed at hospitals.However, in some cases (e.g., about 30% of systemic scleroderma cases), all insurance-covered tests are negative, and in these cases, medical research institutions use specialized tests (e.g., immunoprecipitation) that are not covered by insurance to measure autoantibodies.In recent years, the number of institutions performing immunoprecipitation has been decreasing, and the problem is that it takes more than three months (and in some cases more than a year) to obtain results for all autoantibody tests.
Efficacy of A-Cube®
A-Cube® can simultaneously detect 67 types of autoantibodies, including 20 types that can be measured by insurance-covered tests and 47 types that cannot be measured by insurance-covered tests.The problem with the 47 typesmeasured by immunoprecipitation and other research tests was that it took more than three months to measure all of them, but A-Cube® responds to all test results within 10 business days.
The accuracy of the test is also high, with a 95% correlation with insurance tests and a 98% correlation with immunoprecipitation methods.In addition, by simultaneously measuring 67 different types of antibody that were not previously measured at the same time, new data necessary to elucidate pathological conditions is also generated.
3 years since service started.Currently, 85% of hospitals with specific functions and more than 350 medical institutions are using the A-Cube. (As of January 2025)
＊This is a research test and cannot be used for diagnostic purposes.
Relationship between autoantibodies and symptoms
Scleroderma and myositis, both autoimmune diseases, have different symptoms, treatment, and prognosis depending on the autoantibodies the patient carries, even if the disease has the same name.Therefore, it is important to test for autoantibodies at an early stage of disease onset and formulate a treatment plan for each patient.
Relationship between autoantibodies and symptoms related to scleroderma
The birth of A-Cube®
―Innovative testing for the future of medicine―
Development of A-Cube® began with a desire to save patients with collagen disease.
In 2017, Goshima (currently our Executive Vice President and CSO) of the National Institute of Advanced Industrial Science and Technology (AIST) constructed the world’s largest protein expression clone library, the Human Proteome Expression Resource (HuPEX), and was exploring various ways to use the results to give back to society.One of these is the Human Protein Array.
The Human Protein Array is a tool for measuring “autoantibodies” (i.e., antibodies that attack one’s own tissues and cells) that exist in the human body from a small amount of blood.The human protein array was utilized in collaborative research with a number of medical institutions because of its coverage by HuPEX and high detection sensitivity.As part of these efforts, we received feedback from clinical sitefor collagen diseases, saying, “In the clinical siteof collagen diseases, we need a kit that can test for multiple types of autoantibodies quickly and simultaneously. Could this be achieved using human protein array technology?”
This encounter marked the beginning of A-Cube® development.
Issues facing collagen disease clinical site: diagnosis refugees.
Systemic scleroderma and dermatomyositis/polymyositis, a type of collagen disease, are nationally designated intractable diseases.
In the patient’s body, the immune system becomes abnormal for some reason, causing “autoimmune abnormalities” in which antibodies attack the patient’s own body.
Diagnostic guidelines for scleroderma and myositis include “the presence of disease-specific autoantibodies.
Sixty-seven autoantibodies associated with scleroderma and myositis have been reported in papers, but only 20 of these autoantibodies associated with collagen diseases have been tested for by insurance (according to our survey).
In addition, it is said that the percentage of patients for whom all insurance-covered autoantibody tests are negative is 30-40%, and the lack of a positive autoantibody test delays a definitive diagnosis, creating a problem of “diagnostic refugees” who visit multiple hospitals for a second opinion.
Development of “Human Protein Array” for business use, which was originally intended for academia.
At that time, the “Human Protein Array” had acquired technical POC, including biomarker discoveries in various collaborative studies.
Therefore, with the aim of commercializing human protein arrays, we selected 67 types of autoantibodies to be measured and produced a prototype (protein array) in cooperation With the cooperation of experts in the field of collagen diseases.
For the validation of the prototype, we used several hundred specimens fromPatient samples provided by cooperating medical institutions.In addition, to confirm the accuracy of the test, we conducted a consistency test with an autoantibody test and immunoprecipitation method covered by insurance, and obtained a high correlation as a result.
After two and a half years of development and testing,the “Research Test A-Cube®” was born, which can test for multiple types of autoantibodies simultaneously.
Rapid Autoantibody Testing Opens the Door to Early Diagnosis
3 years since service started.Currently, we have received more than 3,500 requests for testing from 350 medical institutions nationwide, including 85% of hospitals with specific functions (as of January 2025).Users have commented that the system “eliminates the problem of diagnosis refugees who were sent from one hospital to another because a diagnosis could not be made,” and that “case studies using combinations of autoantibodies have progressed, which is helping us understand the pathology of diseases.”
We are extremely pleased that our research test, the A-Cube®, is being so highly regarded in clinical settings.
Leading the world in autoimmune disease research. The passion behind MVV.
Proteobridge’s mission is to “elucidate the causes and pathology of autoimmune diseases and provide a hopeful future for people suffering from autoimmune diseases.”
To that end, we aim to collaborate with companies, physicians, and researchers around the world to become a leading team in the development of new drugs and diagnostic agents for autoimmune diseases.With this in mind, we will continue to work together as a team to provide high-quality testing services.
◎Origin of the service name
The name “A-Cube®” comes from the analytical method that combines the three elements of “Autoantibody,” “Array,” and “Assay.” It was named as an advanced autoantibody analysis technology that combines the three “A’s.”
Examples of use of A-Cube® antigen binding plates for research testing
User Utilization Results
TAFRO syndrome can be distinguished from idiopathic Castleman disease by anti-SSA/Ro60 antibodies.
Research Methodology
Selection of antigenic proteins
It has been reported that TAFRO patients may be positive for anti-SSA antibodies.
To measure SS-A/Ro60 and SS-A/Ro52 autoantibodies, these authors selected the research test A-Cube® (Systemic Sclerosis (SSc) Detection Kit).
The Systemic Sclerosis (SSc) Detection Kit contains 33 antigen proteins capable of detecting 28 autoantibodies associated with SSc.
The Systemic Sclerosis (SSc) Detection Kit contains 33 antigen proteins capable of detecting 28 autoantibodies associated with SSc.
Autoantibodies were measured using a systemic sclerosis (SSc) detection kit.The sera used were from seven TAFRO patients and ten idiopathic Castleman disease (iMCD) patients.
Detection of anti-SS-A/Ro60 and anti-SS-A/Ro52 antibodies.
In TAFRO patients, 5 samples (71.4%) were positive for anti-SS-A/Ro60 antibodies, and 2 samples (28.6%) were positive for anti-SS-A/Ro52 antibodies. No iMCD was detected.
It has been suggested that anti-SS-A antibodies are a disease marker for TAFRO syndrome.
Supplementary evaluation
Anti-SSA and anti-SSB antibodies were detected by immunoprecipitation. Pathological classification of lymph nodes or bone marrow was performed.
Results
◎ TAFRO syndrome has been considered a subgroup of iMCD, but it has recently been argued that the two are separate entities.For this reason, diagnosing TAFRO is complex, and pathological testing can be difficult, especially when the patient’s condition is unstable, so the ability to diagnose the disease with autoantibody testing is of great significance.
Paper information
Mirei Shirakashi et al. TAFRO syndrome is associated with anti-SSA/Ro60 antibodies, in contrast to idiopathic castleman disease
Sci Rep. 2024 Feb 5;14:2889. doi: 10.1038/s41598-024-53413-5
Correlation between epitope suppression of anti-RNA polymerase III antibodies and clinical symptoms of systemic sclerosis.
Research Methodology
Protein expression
To analyze autoantibodies in the sera of systemic sclerosis (SSc) patients, we synthesized partial proteins of subunits of the RNA polymerase III (RNAP III) complex and the RNAPIII major antigen RPC1.
Seventeen full-length proteins of RNAP III complex subunits and five partial proteins of RPC1 were synthesized as FLAG-GST fusion proteins using a wheat germ cell-free protein synthesis system.
Preparation of antigen-binding plates
The synthesized proteins were used to create antigen-binding plates using our proprietary technology.Each well of the antigen-binding plate (96-well plate) is coated with glutathione (GSH) to allow binding of FLAG-GST fusion proteins.
Detection and quantification of autoantibodies
Serum samples from 75 SSc patients, including 34 anti-RNA polymerase III antibody (ARA) positive SSc patients, were used to measure autoantibodies against 17 subunits of the RNAP III complex and five partial proteins of the major antigen RPC1.As a result, autoantibodies against various subunits of the RNAP III complex were detected in ARA-positive SSc patients, and showed different patterns in each patient.Therefore, we quantified and compared the intermolecular epitope spreading for subunits of the RNAP III complex and the intramolecular epitope spreading for the major epitope RPC1.
Statistical analysis and correlation with clinical features
We also analyzed the epitope distribution of the quantified RNAP III complex subunits and the major epitope RPC1 and its association with clinical findings.Interestingly, the greater the spread of the epitope across the subunits, the more significant the correlation with skin sclerosis (mRSS) and SP-D (a marker for interstitial pneumonia) in the Spearman correlation coefficient.
In addition, we found that the greater the epitope spread to the major epitope RPC1, the higher the risk of developing renal crisis.In addition, we found that the greater the epitope spread to the major epitope RPC1, the higher the risk of developing renal crisis.
Results
◎ We showed that antibodies targeting subunits of the RNAP III complex correlated with disease severity and organ damage (skin and lung) in SSc patients, suggesting that these antibodies may be useful biomarkers for early diagnosis and prognostic evaluation.
*Epitope spreading (ES) refers to the phenomenon in which, in autoimmune diseases, autoantibodies against a specific antigen are initially formed, and then autoantibodies against different antigens are gradually formed.
Paper information
Hirohito Kotani et al. Diversity and Epitope Spreading of Anti-RNA Polymerase III Antibodies in Systemic Sclerosis: A Potential Biomarker for Skin and Lung Involvement
Arthritis Rheumatol. 2024 Sep 1. doi: 10.1002/art.42975.
A-Cube® Related Papers
List of papers
Idiopathic Interstitial Lung Disease with Positive Anti-Zoand Anti-Ro52/SSA1 Antibodies: A Case Report
Intern Med Advance Publication. April 5, 2025 5062-24
ELISA・immunoblot 法で陰性もRNA免疫沈降法で複数抗体陽性となった抗合成酵素症候群の1例
臨床神経学 . 2025 年 3 月 65 巻 3 号 p. 211-217
Concurrence of bird-related hypersensitivity pneumonitis and systemic sclerosis-associated interstitial lung disease
Journal: Rheumatology Advances in Practice (IF 2.1) Type: Letter to the Editor :26 March 2025
Autoantibodies to nuclear valosin-containing protein-like protein: systemic sclerosis-specific antibodies revealed by in vitro human proteome
Rheumatology, Volume 63, Issue 10, October 2024, Pages 2865–2873,
Artificial intelligence and omics-based autoantibody profiling highlights autoimmunity targeting ligand-receptor interaction in dementia
medRxiv 2024.09.20.24313547