Chlamydia abortus serological diagnostic kit containing MOMP and CPAF recombinant proteins.

Abstract

The invention relates to a diagnostic kit developed for the accurate and specific serological detection of Chlamydia abortus infections, based on the antigenic epitope regions of the MOMP and CPAF genes. The invention presents an indirect ELISA-based system using antigens derived from highly virulent bacteria obtained from field isolates as coating antigens. This design allows for targeting only the specific epitope regions that cause disease, reducing false positive rates and providing high species-specific sensitivity and accuracy.

Description

Chlamydia containing MOMP and CPAF recombinant proteins. ABORTION SEROLOGICAL DIAGNOSTIC KIT TECHNICAL FIELD The invention provides a serologically accurate and specific method for diagnosing Chlamydia abortus infections. Developed for the detection of MOMP and CPAF genes in this manner. The invention relates to a diagnostic kit based on antigenic epitope regions from field isolates. Antigens derived from highly virulent bacteria are used as coating antigens. It offers an indirect ELISA-based system that is used. Thanks to this structure, only Specific epitope regions that cause disease are targeted, with a false positive rate. By reducing sensitivity, high sensitivity and specificity are achieved on a species basis. PREVIOUS TECHNIQUE Chlamydia is one of the most important factors causing abortion in farm animals. abortus (C. abortus), in small ruminants “Enzootic Abortion of Sheep (EAE)”, “Ovine The infection is called "Enzootic Abortion (OEA)" or "Ovine Chlamydiosis". This causes and leads to serious economic losses. The factor affects not only sheep and... Not just in goats, but in many animals including birds, reptiles, mammals and humans. It can cause this type of infection. Its zoonotic nature and resistance to antibiotics... Because it is difficult to treat, it also poses a significant threat to public health. It forms. Since C. abortus is an obligate intracellular bacterium, it can be cultured. Its isolation is extremely difficult, and therefore diagnosis under routine laboratory conditions is not recommended. It is generally based on molecular or serological methods. However, the culture method, This requires the use of embryonated chicken eggs or cell cultures, which is high. The level of biosafety necessitates specialized equipment and trained personnel. Therefore, culture-based diagnosis is not routinely applicable in laboratories. Current serological methods, while practical, have significant limitations. It includes, in particular, cross-contamination with other Chlamydia species or gram-negative bacteria. Reactions are observed; species-based differentiation is not possible, and the false positive rate is high. This situation affects the specificity and sensitivity of the tests. This limits their levels and reduces diagnostic accuracy. Molecular methods are more... Although it provides high accuracy, it is not widely used due to cost, expertise and equipment requirements. It cannot find an application area. 1 At the genetic level, the Major Outer Membrane plays a significant role in the pathogenesis of the pathogen. Protein (MOMP) and Chlamydial Protease-Like Activity Factor (CPAF) genes, species structures that differ based on their base and play a fundamental role in the development of infection. It has been defined that MOMP plays a role in the bacteria's evasion of the host immune system, The CPAF protein suppresses MHC production in host cells, thereby influencing the immune response. This reduces its effectiveness. Therefore, serological methods based on classical whole-cell antigens are not recommended. Tests are unable to adequately reflect these antigenic variations, leading to inaccurate or incomplete diagnoses. This can lead to consequences. In recent years, recombinant protein-based approaches have been used in serological diagnostic systems. These methods have come to the forefront. In these methods, controlled expression of specific epitope regions is used. This allows for higher specificity and sensitivity compared to classical whole-cell antigens. This reduces false positives caused by cross-reactions. The repeatability of the tests is being improved. However, the major drawbacks of existing commercial kits are... Most are single antigen-based, and several play a direct role in the development of the disease. It does not contain many epitopes; this is a limiting factor in terms of diagnostic accuracy. Therefore, multiple tests are required to reliably detect C. abortus infection. Next generation technology that targets multiple epitope regions, providing high specificity and accuracy. There is a need to develop diagnostic kits. THE PURPOSE OF THE INVENTION The invention provides for the accurate, specific, and reliable diagnosis of Chlamydia abortus infections. developed for diagnosis, containing multi-epitope antigens The invention relates to an indirect ELISA test kit. The aim of the invention is to improve the results seen in existing serological tests. eliminating key limitations such as low specificity, cross-reactivity, and false positives. by removing it, high levels of pathogenicity based solely on disease-causing epitope regions specific to C. abortus. The aim is to provide a sensitive diagnostic system. The developed kit is obtained from different field isolates. thanks to its inclusion of multiple epitopes that play a role in the pathogenesis of infection It provides a high level of specificity and sensitivity. Unlike existing kits, the system developed in this invention can eliminate all bacteria or just a single one. It targets not the antigen itself, but the antigenic regions that directly cause infection. This approach increases the accuracy rate of the test, minimizing false positives, and It also enables reliable diagnosis under field conditions. In addition, high accurate results, early identification of infection entry into the herd 2 By doing this, both animal health is protected and economic losses are minimized. It contributes to its reduction. In conclusion, the invention is based on a multi-antigen design with specific epitopes. It differs from diagnostic kits in that it offers high sensitivity, species-specificity, and clinical characteristics. by providing reliability, scientific and It presents an innovative solution from a practical point of view. DETAILED DESCRIPTION OF THE INVENTION The invention provides for the accurate, reliable and specific diagnosis of Chlamydia abortus infections. developed for diagnosis, with a multi-epitope structure. This relates to an indirect ELISA-based serological diagnostic kit containing recombinant antigens. The invention eliminates the limitations of existing tests by detecting only the infection. High-performance drugs targeting specific antigenic regions that play a direct role in its formation. It offers a system with specificity and sensitivity. Chlamydia abortus affects many farm animals, particularly small ruminants. It is an obligate intracellular, gram-negative bacterium that causes abortion in animals. Diagnosing the infection is difficult because the causative agent can be cultured in a laboratory setting. This is due to the high biosafety requirements and the need for specific equipment. It is difficult. Therefore, serological methods are generally used in diagnosis. However, Most current serological tests are based on a single antigen or test the entire bacterium for that antigen. This approach uses cross-reactions and species-level discrimination. This leads to the inability to perform the procedure and to false positives. This diagnostic kit, developed in this invention, contains two key features specific to C. abortus infection. Major Outer Membrane Protein (MOMP) and Chlamydial Protease- which are virulence genes Like Activity Factor (CPAF) is targeted. MOMP, the bacterium's immune response to the host immune system, is targeted. an outer membrane protein that allows escape from the system and exhibits antigenic variability It contains epitope regions. CPAF, on the other hand, is part of the Major Histocompatibility Complex in host cells. It weakens the immune response by suppressing (MHC) production and reduces the risk of infection. This causes the infection to progress. The epitope regions of these two genes cause the infection to progress. They are antigenic structures that play a direct role in pathogenesis and therefore have a high potential. It offers a high level of diagnostic specificity. The system developed within the scope of the invention is obtained from field isolates of C. abortus. Identification of the antigenic regions of these two genes from the strains and these regions by combining multiple epitope structures using recombinant technology 3 This has been done so that the targeted epitope regions are unique to C. abortus. It is a recombinant protein and does not cross-react with other Chlamydia species. Its production is carried out in a suitable expression system, and the structural characteristics of the antigen are determined. It is purified while maintaining its stability. The resulting multi-epitope recombinant antigen is coated onto the surface of the ELISA plate. It is used as a coating antigen. During the testing phase, the suspected animal or Serum samples taken from herds react with this antigen, producing specific antibodies. Measurement is performed using the indirect ELISA principle based on antigen binding. The method has been improved to show both high specificity and high sensitivity against C. abortus. It detects the presence of antibodies. The technical superiority of the invention lies in the fact that, unlike classic ELISA kits, it does not target all bacteria. the bacteria contains only epitopes directly related to the disease and is multiple antigenic It uses a combination of regions. This structure leads to inaccuracies seen in serological tests. Minimizing positivity while also providing highly accurate results against different field isolates. It enables its production. In addition, thanks to the recombinant protein-based production process. This facilitates standardization of the kit, increasing its repeatability and reliability. The current literature contains information on C. abortus infection involving multiple epitopes. There are no studies on the development of a recombinant protein-based diagnostic kit. This is not the case. This situation undermines the scientific originality and global reach of the invention in question. This clearly demonstrates its innovative value on a large scale. The invention is not only for domestic production. not only does it meet the need, but it also reflects the existing scientific advancements in the field of serological diagnosis. It fills the gap. In conclusion, the invention has high specificity and sensitivity, and is species-specific. Minimizing false positives, suitable for field conditions, reliable and domestically produced. It presents a Chlamydia abortus diagnostic kit with potential. The invention is currently available. to overcome the shortcomings of techniques, high accuracy, originality and reliability by providing significant contributions to veterinary health and herd management by working together to provide early and accurate infection detection in veterinary diagnostic laboratories This contributes to both the protection of herd health and the prevention of economic losses. It provides. 4

Claims

1. Accurate, reliable, and specific diagnosis of Chlamydia abortus infections. It is a serological diagnostic kit designed to enable this, and its feature is; Based on the indirect ELISA principle, on the surface of an ELISA plate coated, Major Outer Membrane, which plays a role in the pathogenesis of C. abortus. Chlamydial Protease-Like Activity Factor (CPAF) with protein (MOMP) gene created from antigenic epitope regions of the gene using recombinant technology It is characterized by containing a recombinant antigen with a multiple epitope structure.