C1 Inhibitor Test

C1 Inhibitor C1-INH Function Test Methods Applications Optimization

C1 inhibitor testing is a critical tool for understanding the complex roles of this multifunctional protein in health and disease. At Creative Biolabs, we are committed to providing cutting-edge C1 inhibitor testing services to support researchers and pharmaceutical companies in their quest for new insights and innovative therapies.


Understanding C1 Inhibitor

C1 inhibitor Structure (Zubareva, E. V., et al., 2023)

C1 inhibitor, also known as C1 esterase inhibitor or SERPING1, is an important complement inhibitor that regulates the complement system, a key component of innate immunity. This multifunctional serine protease inhibitor is encoded by the C1-INH gene and plays a crucial role in the control of various physiological processes.

C1-INH is a protein that is widely present in human serum, and its main function is to inhibit the activity of C1 esterase. C1 esterase is the first component of the complement system and plays an important role in the immune response and inflammatory response. C1-INH regulates the complement cascade by controlling the activity of C1 and thus protects the organism from excessive immune response and tissue damage.

Mutations or deletions in the C1-INH gene result in C1-INH deficiency, which can lead to a range of clinical problems such as hereditary angioedema (HAE). Therefore, testing the function and activity of C1-INH is essential for the study and treatment of such diseases.


C1 Inhibitor in Immunological Function

C1-INH is a core regulator of the classical pathway of complement, preventing the overconsumption of C4 and C2 by inhibiting C1 esterase and avoiding abnormal activation of the complement system. In addition, it regulates contact-activated systems (e.g., kinin release) and the intrinsic coagulation pathway to maintain the balance between the immune and coagulation systems.

The concentration, functional activity and mutation analysis of C1-INH in experimental tests provide key data for recombinant protein quality control, disease model construction and complement inhibitor drug development.

Functional C1-INH. (Staller, Kyle, et al., 2022)Fig. 2 The lack of functional C1-INH affects the complement, contact, coagulation, and fibrinolytic pathways.2,3


C1 Inhibitor Test Methods

Detection of C1 inhibitor activity is essential. Several methods are available to quantify and assess C1-INH function. These methods can be broadly categorized into quantitative and functional assays.

Table 1 Methods are available to quantify and assess C1-INH function.

Categories Methods Principle Advantages Limitations
Concentration Detection ELISA Concentration was determined by antibody specific binding to C1-INH, combined with chromatography.
  • Simple and well-established assay
  • Provides reliable estimates of C1-INH levels
  • Non-invasive and widely available
  • C1-INH functions are not evaluated, only their number
Dried blood spot (DBS) sample testing C1-INH was extracted from DBS and the functional activity was quantified by LC-MS/MS, suitable for high-throughput laboratories.
Function Detection C1q binding assay This assay measures the ability of C1-INH to block the interaction of C1q with C1r and C1s in the classical complement pathway.
  • Evaluate the activity of C1-INH in inhibiting its target enzymes in vivo
CH50 (hemolytic complement test) The CH50 assay assesses the overall functional activity of the classical complement pathway and indirectly measures C1-INH function by assessing the ability to lyse antibody-coated sheep erythrocytes.
Mutation Analysis Sequencing of the SERPING1 gene Analyze the presence of specific mutations or deletions in the C1INH gene that impair C1-INH synthesis or function using high-throughput sequencing or PCR-based methods.
  • May provide a definitive genetic diagnosis
  • Test is relatively expensive
  • Require specialized laboratory equipment

Experimental Procedures

Serum samples are incubated with anti-C1-INH antibodies. Binding produces a detectable signal whose intensity correlates with the concentration of C1-INH in the sample.

Samples are incubated with a C1q-coated solid phase carrier. After washing, the remaining C1q-C1r complex is quantified with a secondary antibody to measure the ability of C1-INH to inhibit this binding.

The CH50 assay uses diluted serum samples to assess complement-mediated lysis. The degree of lysis is inversely related to C1-INH activity and thus to classical pathway inhibition.

Serum C4 levels are measured before and after activation of immune complexes. A significant decrease in C4 concentration indicates that C1-INH is not properly controlling complement activation.

High-throughput sequencing or PCR-based methods are used to analyze the C1INH gene for specific mutations or deletions that impair C1-INH synthesis or function.

At Creative Biolabs, we offer advanced services and cutting-edge research tools for C1 inhibitor testing.


Experimental Applications of C1 Inhibitor Functional Assessment

C1 inhibitor assays play a critical role in the study of diseases associated with disorders of the complement system. Accurate and comprehensive assays (from antigen level measurements to functional activity assays) are essential in a variety of research applications.

Functional Validation of Complement Inhibitors
  • Complement activation inhibition assay: The inhibition efficiency of C1-INH is indirectly assessed by detecting C3/C4 catabolic products (e.g., C3a, C4b) after activation of the traditional pathway of complement.
  • Bradykinin level assay: C1-INH reduces bradykinin production by inhibiting C1 esterase, indirectly reflecting its role in the regulation of complement-mediated inflammation.
Protein Purity and Stability Studies
  • Polymer assay: SEC-HPLC method is used to detect the polymer content of recombinant C1-INH (e.g., 0.11%-0.60%) and verify protein purity.
  • C1 esterase inhibitor serum assay: Assesses drug stability by measuring the concentration and functional activity of C1-INH in serum.
Gene Mutation Modeling
  • SERPING1 gene editing: By introducing SERPING1 gene mutations (e.g., deletions or base substitutions), HAE type I (low concentration) or type II (functional defects) are modeled for functional defect research.
Complement Inhibitor Drug Development
  • Functional assay screening: Screening of novel C1-INH analogs with complement inhibitory activity by enzyme activity assays (e.g., LC-MS/MS quantification of C1s substrate conversion products).

Table 2 Methodological comparisons and challenges.

Application Scenarios Assays Advantages Limitations
Complement inhibition validation Complement activation inhibition assay Directly reflects the regulatory ability of the complement system Requires the combination of C3/C4 assay, complicated operation
High-throughput screening DBS assay Immediate, portable, suitable for large-scale samples Extraction conditions need to be optimized to improve the accuracy of activity detection
Recombinant protein purity verification SEC-HPLC Accurate separation of monomers and polymers Instrument dependent


Optimizing Directions for C1 Inhibitor Testing

Assay sensitivity and standardization

  • Problem: The accuracy of the C1-INH activity assay needs to be improved.
  • Optimization: Improve the sensitivity of activity detection by improving the extraction solution or incubation conditions.

Standardization of functional assays

  • Problem: Differences in C1-INH activity units and quality control standards among different laboratories affect the comparability of results.
  • Optimization: Develop uniform activity unit standards and reference intervals.

Sample handling and complexity

  • Problem: Complement assays require strict avoidance of sample activation in vitro, which can lead to spurious results.
  • Optimization: Reduce sample processing time and risk of isolated activation by using instant detection technology.

Recombinant protein purity verification

  • Problem: The SEC-HPLC method relies on high-precision instruments, and the polymer content detection requires strict control of column parameters.
  • Optimization: Develop portable molecular exclusion chromatography equipment to reduce laboratory equipment dependence.

High-throughput screening needs

  • Problem: Although there are available high-throughput methods, manual steps are still required.
  • Optimization: Integrate ELISA and HPLC technologies and develop a fully automated platform to achieve large-scale sample screening.

Stability assessment in drug development

  • Problem: C1-INH functional activity in serum of C1 esterase inhibitors needs to be monitored for a long period of time, and the existing methods are not sufficiently automated.
  • Optimization: Development of a real-time monitoring system.

Table 3 Challenges and optimizations for C1 inhibitor testing

Type of Challenge Specific Problem Optimization Strategy
Insufficient sensitivity Sample extraction conditions affect accuracy of activity assay Optimization of extraction solution and incubation conditions
Lack of standardization Large variations in units and standards between laboratories Development of QC standards
High throughput requirements Manual steps limit screening efficiency Development of fully automated platforms

The C1 inhibitor test is an important tool for evaluating the complement system, especially in the absence of C1 esterase inhibitors. As complement inhibitor research progresses, the C1 esterase inhibitor test will continue to play an integral role in the diagnosis and treatment of complement-associated diseases, offering new hope for the development of therapies.

Creative Biolabs offers a full range of complement-related services and products, including:

If you want more information, please feel free to contact us.

References

  1. Zubareva, E. V., et al. "Development and validation of a peptide-mapping procedure for a novel C1 esterase inhibitor." Biological Products. Prevention, Diagnosis, Treatment 23.2 (2023): 203-218. https://doi.org/10.30895/2221-996X-2023-23-2-203-218
  2. Staller, Kyle, et al. "Consider hereditary angioedema in the differential diagnosis for unexplained recurring abdominal pain." Journal of Clinical Gastroenterology 56.9 (2022): 740-747. https://doi.org/10.1097/mcg.0000000000001744
  3. under Open Access license CC BY 4.0, without modification.
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