Hemolytic Inhibition Assay

Background Services Overview Protocols Related Products Published Data Q&A Resources

To support the complement system clinical and pre-clinical research, Creative Biolabs provides rapid hemolytic assay and hemolytic inhibition assays for complement function or activity assessment.

Background

Mechanism of Hemolytic Assay

Generally, the complement system mainly functions by cracking the target cells through the formation of the cytolytic membrane attack complex (MAC). Based on this property, the hemolysis assay has been developed as the major approach for total complement system activity assessment.

Usually, serum isolated from immunized rabbits and SRBC is used in vitro for hemolytic assay. Specific antibodies, namely hemolysin, will be produced after the sheep red blood cells (SRBC) are injected into rabbits as antigens. When SRBC is added to the tube with isolated serum, the hemolysin immediately reacts with the antigen on the surface of SRBC forming antigen-antibody complex. If a complement sample with normal activity is added to the tube, the complement will attack the antigen-antibody complex on the surface of SRBC, leading to the SRBC lysis and hemolysis. Common hemolysis assays are CH50 and AH50 (i.e., diluted serum complement concentration at 50% lysis of SRBC), which are respectively used for assessment activities of complement classic pathway and complement alternative pathway.

Fig. 1 Hemolysis assay process. (Fornaguera et al., 2017)

Fig. 1 Hemolysis assay process.1, 3

Hemolytic Inhibition Assay for Complement Activity Assessment

With the deepening of research and the development of complement therapeutics, a variety of complement inhibitors, such as CD55, CD59, anti-C5 antibody, C1 inhibitor, have been approved and developed for human disease treatment. This requires a method to evaluate the inhibitory activity of these inhibitors, namely hemolysis inhibition assay. The inhibition activities can be determined by comparison of the hemolysis inhibition between normal complement samples with that of complement samples with inhibitors. Two references can be used as a measure of the activity of complement inhibition, one is the value of CH50 or AH50, the other is the hemolysis inhibition curve of complement samples with gradient dilution.

Besides, hemolytic assay or hemolytic inhibition also can be applied for the detection and identification of complement system deficiency as well as deficiency or function verification of a single complement component. These kinds of assay usually are based on the depleted serum of individual components. By adding the missing individual component, the complement activity can recover.

Services Overview

Hemolytic Inhibition Assay in Creative Biolabs

As a leading diagnostic service biotech company, Creative Biolabs has armed with a powerful and standardized complement assay platform, aiming to offer the best and convenient complement related services. At present, we provide custom hemolytic inhibition assays for the rapid complement activity assessment. For the samples that are not suitable for mailing, we also provide ready-to-use hemolytic assay products and guidance. So what you need to do is just feel free to contact us or send us your specific demands. Our professional scientist will reply to you as soon as possible.

Our hemolytic inhibition assay service includes an array of advanced features designed to meet the unique needs of our clients.

Our team consists of highly qualified scientists and professionals with specialized training and experience in conducting such tests. The laboratory is equipped with state-of-the-art equipment and software to ensure reliable and detailed results.

We end up providing detailed and easy-to-understand reports of laboratory results, highlighting key findings and explaining their potential impact. A dedicated team is available to answer any questions you may have about the testing procedure, results or next steps.

Protocols

Related Products

Cat Product Name Form Application
CTP-456 Native Cynomolgus Monkey Complement C3b Protein Cynomolgus Monkey Functional Assays
CTP-457 Native Human Complement Factor I Protein Human Functional Assays
CTP-458 Native Human Complement Factor H Protein Human Functional Assays
CTP-459 Native Human Complement C4b Binding Protein Human Functional Assays
CTP-460 Native Human Complement C1 Complex Protein Human Functional Assays
CTP-461 Native Human Complement C1q Protein Human Functional Assays

Published Data

Binding of complement inhibitor FH on platelets and leukocytes, hemolytic assay and complement activation.Fig.2 Complement inhibitor FH binding on platelets and leukocytes, with hemolytic assay and complement activation analysis.2,3

Hereditary thrombocytopenias are a genetically diverse cause of heightened bleeding risk. Sialic acid serves as a crucial ligand for complement factor H (FH), a key regulator that shields host cells from unintended complement attack. FH binding to erythrocytes was indirectly assessed by measuring complement-mediated hemolysis rates. The proband showed significantly reduced sialic acid expression on platelets and leukocytes, resulting in markedly diminished FH binding and moderate activation of both alternative and classical/lectin complement pathways, along with increased erythrocyte lysis. Reduced sialylation on platelets, leukocytes, and erythrocytes diminishes FH binding, prompting moderate complement activation and higher hemolysis rates.

Resources

References

  1. Fornaguera, Cristina, and Conxita Solans. "Methods for the in vitro characterization of nanomedicines—biological component interaction." Journal of personalized medicine 7.1 (2017): 2.
  2. Smolag, Karolina I., et al. "Severe congenital thrombocytopenia characterized by decreased platelet sialylation and moderate complement activation caused by novel compound heterozygous variants in GNE." Frontiers in Immunology 12 (2021): 777402.
  3. Distributed under Open Access license CC BY 4.0, without modification.

Questions & Answer

A: In hemolytic inhibition assay, the test sample, typically serum or plasma, is mixed with red blood cells and complement proteins. If the complement system is functional, it will cause lysis of the red blood cells. The addition of an inhibitory substance, such as an antibody or drug, can prevent or inhibit this lysis, which can be quantified to determine the level of inhibition.

A: Hemolytic inhibition assay has various applications, including assessing complement deficiencies, diagnosing autoimmune disorders such as systemic lupus erythematosus (SLE), evaluating the effectiveness of complement-targeting drugs, and researching the role of complement in various diseases.

A: Some limitations of the hemolytic inhibition assay include the need for specialized laboratory equipment and technical expertise. It requires careful handling of complement proteins, which are heat-labile and can be affected by storage conditions. Interfering substances present in the test sample, such as hemolysis inhibitors or complement inhibitors, can impact the assay results and should be considered. Standardization and quality control measures are important to ensure reproducibility and accuracy of the assay results. Our services can meet these requirements.

A: Proper storage and transport of samples are crucial to maintain their integrity. Generally, you should freeze your samples at a recommended temperature (usually -80°C) if they're not being sent immediately. For transportation, samples should be well-packed in a leak-proof, insulated container with ice packs. However, specific storage and shipping instructions may vary depending on the nature of your samples.

A: The turnaround time depends on the nature of the study and the total volume of samples. Depending on these factors, it could vary from a few days to a couple of weeks. Rest assured, we prioritize providing high-quality, accurate results in a timely manner.

A: The raw data from the assay is typically analyzed using statistical techniques to distinguish significant results. Parameters such as percent hemolysis and concentration of inhibiting antibodies are used for quantification. Our team of experts will analyze the data and provide you with a detailed report.

A: We follow strict quality control guidelines for our hemolytic inhibition assays. Protocols are meticulously followed and each step gets validated and recorded. Known positive and negative controls are run alongside unknown samples to validate the accuracy of results. We also regularly participate in proficiency testing to ensure our assay performance remains at its peak.

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