AlloDx CompliNeF™ Assay: Reading C3NeF Function Through Bb Retention

A reliable C3NeF assay should not only reflect the functional changes of C3 convertase, but also minimize the interference of anti-IgG signals. If you test C3NeF as an ordinary autoantibody, you are likely to encounter two types of problems: one is that it can only prove that the antibody binds the invertase, but cannot prove that it stabilizes the invertase [4]. Anti-human IgG secondary antibodies are often used in binding experiments to read patient IgG, and anti-complement factor B (CFB) antibodies and other IgG that can bind to C3bBb will also be read, easily forming false signals [2]. The other type can only read downstream effects and cannot distinguish the source of amplification. The hemolysis reading of functional experiments is affected by multiple complement components and is an indirect inference [2].

The ideal platform's readout should directly reflect the formation and stabilization of C3 convertase by C3NeF and minimize reliance on the patient's IgG signal. What AlloDx does is detect the retention of Bb, not IgG.

CompliNeF™ Assay: reads Bb retained on magnetic beads after washing

The design core of CompliNeF™ Assay is to construct a mini-invertase reaction on magnetic beads. First, based on C3b-coated magnetic beads, complement factor B (CFB) and complement factor D (CFD) are added to form C3bBb. The presence of C3NeF in the plasma to be tested will stabilize C3bBb across the interface between C3b and Bb[1,3]. Then after extensive washing, the Bb remaining on the beads is read.

The reason for choosing to read Bb rather than reading IgG is that Bb is the component that is actually left after the invertase is stabilized. The amount of retention corresponds to the comprehensive effect of C3NeF on the formation and stability of invertase. This step bypasses the anti-human IgG secondary antibody and in principle reduces the interference caused by anti-CFB antibodies and other IgG that can bind to C3bBb [5].

Verification process for selecting magnetic beads as reaction carrier

The choice of magnetic beads as the reaction carrier is supported by previous experiments. During the exploration of raw materials and reaction conditions, we observed two points:

First, the purity of raw materials is verified by SDS-PAGE. The α' chain of C3b is about 105 kDa, the β chain is about 75 kDa, complement factor B is about 93 kDa, and complement factor D is about 24 kDa. Each raw material has a main band near the corresponding molecular weight, which can be used for subsequent exploration of C3bBb formation and cleavage conditions.

Second, there are system differences in CFD cutting. In the enzyme plate solid-phase system, CFB is difficult to be effectively cut by CFD; in the liquid-phase and magnetic bead solid-phase systems, the cutting can be effectively performed [5]. CFB needs to bind C3b before it can become a substrate for CFD. This feature also shows that the magnetic bead solid phase is closer to the natural assembly conditions in the liquid phase. Plate immobilization may affect C3b conformation, CFB binding, or CFD access to the substrate. The determination of the platform route is based on the facts about these reaction conditions.

Preliminary comparison of CompliNeF™ method and indirect ELISA

Preliminary observations based on methodological direction showed that the Bb-sparing method based on CompliNeF™ compared with indirect ELISA showed higher signal values for positive patient samples [5]. Indirect ELISA reads C3NeF with anti-human IgG, while this platform reads Bb retention; the signal in positive samples is higher, indicating more adequate capture of functional C3NeF.

Another design goal of CompliNeF™ Assay is to convert the Bb retention amount into a comparable and quality-controllable signal to form a formation index (FI) to express the relative role of C3NeF to facilitate intra-batch comparison and quality control [5].

Back to the original criteria: the readout points to functionality (formation and stability), is independent of the anti-IgG channel, and can be quantified and therefore can be incorporated into quality control. All three point to the same goal, that is, each value corresponds to a clear and interpretable convertase event.

References

1. Corvillo F, Okrój M, Nozal P, et al. Nephritic Factors: An Overview of Classification, Diagnostic Tools and Clinical Associations. Front Immunol. 2019;10:886. PMID: 31068950

2. Michels MAHM, Volokhina EB, van de Kar NCAJ, van den Heuvel LPWJ. Challenges in diagnostic testing of nephritic factors. Front Immunol. 2022;13:1036136. PMID: 36451820

3. Welsh SJ, et al. Monoclonal nephritic factors reveal insights into C3 convertase dynamics and dysregulation. bioRxiv [Preprint]. 2026 Jun 12:2026.06.11.731599.

4. Zhao F, Afonso S, Lindner S, et al. C3-Glomerulopathy Autoantibodies Mediate Distinct Effects on Complement C3- and C5-Convertases. Front Immunol. 2019;10:1030. PMID: 31214159

5. AlloDx (AlloDx). CompliNeF™ Assay methodology validation and performance exploration data.

The methodology and internal research data involved in this article are in the research and development/verification stage and do not constitute a performance commitment for marketed products; relevant statements must be based on the final registration certificate, instructions and the latest guidelines. The internal exploratory data such as "higher signal value" in the article are not conclusions on clinical efficacy or diagnostic performance.