Isotype-Specific Agglutination-PCR (ISAP): a sensitive and multiplex method for measuring allergen-specific IgE

To the Editor

Component-resolved diagnostics (CRD) are a rapidly-growing class of in-vitro tests for evaluating individuals thought to have allergic diseases. In contrast to using whole-allergen extracts, CRD employs individual, molecularly pure allergen components to measure allergen-specific IgE (sIgE), thereby helping to assess the risk of allergy to such allergens with improved accuracy¹. For example, the presence of sIgE against certain peanut components (e.g., Ara h 1, Ara h 2 and Ara h 3) indicates a higher risk for anaphylaxis than does anti-Ara h 8 sIgE (which can reflect cross-reactivity with birch pollen)¹. While ELISA-based CRD tests may lack analytical sensitivity, the alternative more sensitive assays currently employed clinically (e.g., ImmunoCAP) are not multiplexible and may omit certain critical allergens². These problems can be mitigated by collecting more blood for additional tests. However, this approach can be problematic when studying small animals or testing young children.

Here we report Isotype-Specific Agglutination-PCR (ISAP), a highly sensitive and multiplexible approach for measuring allergen-specific immunoglobulins (Igs) in 1 μL of sample (Fig 1, A). ISAP employs chemically-synthesized allergen-DNA and secondary antibody-DNA conjugates (see Fig E1 in this article’s Online Repository at www.jacionline.org). Each DNA conjugate bears either the 5’ or 3’ portion of a split DNA barcode (Fig 1, A). Upon binding to the target Ig in the sample, the allergen-DNA and secondary antibody-DNA conjugates are agglutinated into close proximity. The addition of a short complementary bridge oligo and DNA ligase reunites the two halves of the barcode to create a full-length DNA amplicon, which can then be quantified by qPCR. The amount of the amplicon directly reflects the quantity of analyte within the sample. Importantly, in the absence of the specified allergen-specific Ig, the two DNA conjugates will neither ligate nor amplify by PCR. By requiring the presence of the analyte to generate signal, this “turn-on” mechanism circumvents the washing or DNA purification steps needed to remove unbound secondary reporters in other formats such as immuno-PCR^3,4. In principle, these features allow ISAP to represent a CRD assay with enhanced sensitivity, multiplex capability, and an operationally simple workflow.

Figure 1. Principle scheme of PCR-based antibody quantification methods.

(A) ISAP for quantification of sIgE. Samples containing sIgE are first incubated with allergen-DNA and antibody-DNA (in this case, anti-IgE antibody-DNA) conjugates. Upon formation of immune complexes consisting of sIgE, allergen-DNA and anti-IgE-DNA, the DNA components of the conjugates are brought into close proximity, and the addition of a short bridge oligo and DNA ligase joins the two halves of DNA into a full length amplicon. Real-time qPCR is then used to quantify the abundance of the ligated DNA amplicons, which reflects the level of sIgE in the sample. (B) PLA for quantification of total IgE (tIgE). Samples are incubated with two anti-IgE-DNA conjugates that each bear half of a full-length DNA amplicon. (C) ADAP for quantification of all anti-allergen immunoglobulins (IgE, IgG, IgM, etc.). This procedure is performed as before, but with two allergen-DNA conjugates. (D) Integration of ISAP, PLA and ADAP into a single assay, termed CLIQ. The sample is incubated with two allergen-DNA and two anti-IgE-DNA conjugates followed by ligation. The reconstituted DNA amplicons can be interrogated independently by dedicated primer pairs in the different wells of a 96- or 384-well plate.

To establish proof-of-concept for the detection of allergen-specific IgE (sIgE), we prepared a dilution series of: 1) anti-OVA sIgE, 2) anti-OVA sIgG and 3) total IgE (control). The three samples were then analyzed with an ISAP assay designed to detect anti-OVA sIgE. As expected, concentration-dependent signal arose only for the sample containing anti-OVA sIgE (Fig 2, A). Next, we performed a head-to-head comparison of ISAP with a standard ELISA, which showed a markedly (~800-fold) increased analytical sensitivity of ISAP compared to ELISA (Fig 2, B).

Figure 2. Multiplex and sensitive detection of allergy-related immunoglobulins by PCR.

(A) Serially-diluted anti-OVA sIgE, anti-OVA sIgG and total IgE (control) analyzed with ISAP ( Ct [Delta Cycle threshold], a standard way to report qPCR signal=Ct value of buffer-only control minus that of sample). (B) Aliquots of the same anti-OVA sIgE dilution series assayed by ISAP ( Ct) and ELISA (AU=arbitrary units). (C) CLIQ profiles of immunoglobulin responses to peanut sensitization in BALB/c wild type mice, BALB/cRag^−/− (Rag KO) mice and BALB/c-Jh^−/− (Jh KO) mice (n=5/group) sensitized to peanut epicutaneously. CLIQ results are expressed as the relative fold change (value after sensitization divided by value before sensitization) (* P < .05 for values before vs. after sensitization). The h1, h2 and h3 labels in the x-axis refer to Ara h1, Ara h2 and Ara h3, respectively. (D) ELISA analysis of sera from the same 5 BALB/c mice analyzed by ISAP in C. No statistically significant differences in levels of anti-peanut sIgE were observed by ELISA before vs. after sensitization. (E) Correlation of ISAP and ImmunoCAP results (R=0.83–0.96, P < .05). 1 μL each of 20 baseline (pre oral immunotherapy) plasma specimens from participants in the POISED trial analyzed by ISAP (x-axis) vs. ImmunoCAP (y-axis).

In addition to sIgE, total IgE and total anti-allergen sIg levels (of all Ig classes) are also of interest in characterizing allergic responses. To create an assay capable of measuring these parameters, we integrated ISAP with two other PCR-based methods: the Proximity Ligation Assay (PLA)⁵ and Antibody Detection by Agglutination-PCR (ADAP)⁶ (Fig 1, B and C). PLA employs two secondary antibody-DNA conjugates to detect total IgE (tIgE) whereas ADAP uses two allergen-DNA conjugates to detect all allergen-specific Igs (e.g., IgE, IgG, IgM). The integrated assay, termed CLIQ (Comprehensive Ligation-based Immunoglobulin Quantification), converts sIgE, tIgE and all sIgs of any isotypes into distinct DNA amplicons that can be independently interrogated with unique primer pairs (Fig 1, D).

To validate this concept, we re-analyzed the anti-OVA sIgE, anti-OVA sIgG and total IgE (control) dilution series with CLIQ (see Fig E2 in this article’s Online Repository at www.jacionline.org). For instance, Fig E2, A shows that both the PLA (which measures total IgE, solid squares) and the ISAP (which measures sIgE, open triangles) portions of CLIQ lacked signals when used to assess anti-OVA sIgG; only the ADAP portion of CLIQ (which measures total anti-OVA sIgs, solid circles) shows concentration-dependent signals. The data in Fig E2 indicate that each of the three PCR-based methods selectively detected its specified target in an integrated assay without cross-reactivity.

Next, we sensitized mice with OVA⁷ and analyzed their antibody responses using CLIQ. CLIQ demonstrated elevations in serum and blood levels of tIgE and total anti-OVA sIg at d7 and sIgE at d14 in OVA-sensitized mice, but not in PBS-treated control mice (see Fig E3, A and C, and Fig E4 in this article’s Online Repository at www.jacionline.org). ELISA also detected an elevation in serum sIgE at d14 in the same OVA-sensitized mice (see Fig E3, B in this article’s Online Repository at www.jacionline.org). These results demonstrate substantial equivalence between the CLIQ and ELISA methods in this setting, in which strong sIgE responses are induced.

To probe the sensitivity of CLIQ in settings with low levels of serum sIgE, we sensitized mice epicutaneously with peanut oil. The sensitized mice were later challenged with peanut extract to attempt to induce anaphylaxis. Despite the strong systemic reaction to peanut challenge (see Fig E5 in this article’s Online Repository at www.jacionline.org), peanut sIgE was undetectable by ELISA (Fig 2, D and Fig E6). However, when we assayed serum from these mice with CLIQ (using multiple peanut components as antigens: Ara h 1, Ara h 2 and Ara h 3), CLIQ, but not ELISA, detected increases in tIgE and Ara h 1 sIgE after peanut oil sensitization in BALB/c or C57BL/6 mice (Fig 2, C and see Figs E7 and E8 in this article’s Online Repository at www.jacionline.org). By contrast, CLIQ did not detect such changes in antibody-deficient Jh^−/− and Rag^−/− mice after we attempted to induce peanut sensitization⁸ (Fig 2, C). These results show that the enhanced analytical sensitivity of CLIQ can detect potentially disease-relevant levels of sIgE which may be undetectable by traditional means like ELISA.

Finally, we employed CLIQ to analyze plasma obtained from 20 peanut-allergic subjects upon their enrollment into the IRB-approved POISED trial (ClinicalTrials.gov Identifier: NCT02103270)⁹. CLIQ results displayed substantial positive correlations with ImmunoCAP data from the same specimens (Fig 2, E and see Figs E9, E10 and E11 in this article’s Online Repository at www.jacionline.org).

It is important to emphasize that no assay for sIgE can be used, in isolation, to establish the diagnosis of a clinically relevant allergy. However, such testing, if used appropriately, can identify those who have been sensitized to specific allergens or their components and therefore can confirm that these individuals are potentially at risk for exhibiting sIgE-associated clinical allergies. Although it is not possible to fully prevent the misuse of any diagnostic assay, and this is a particular concern with highly sensitive assays, our data suggest that, as with the ImmunoCAP assay, appropriate selection of the assay cutoff value will permit the CLIQ assay to be used to identify potentially clinically relevant levels of sIgE against allergen components.

In summary, ISAP is a sensitive and specific method for multiplex detection of isotype-specific sIgE against specific allergen components in very small (1 μL) sample volumes. Integration of ISAP with PLA and ADAP to create the CLIQ assay greatly expands the information which can be obtained in a single assay. We envision that the much lower sample consumption and improved sensitivity of this assay will prove useful for allergy research and diagnostics, and for the management of allergy patients.

MATERIALS AND METHODS

Materials and Methods are described in the “Supplemental Material” section.

ABBREVIATIONS USED

CRD

Component-resolved diagnostics

sIgE

Specific IgE

tIgE

Total IgE

Ig

Immunoglobulin

ISAP

Isotype-specific agglutination-PCR

PLA

Proximity ligation assay

ADAP

Antibody detection by agglutination-PCR

CLIQ

Comprehensive ligation-based immunoglobulin quantification

POISED

Peanut oral immunotherapy study:safety efficacy and discovery

ELISA

Enzyme-linked immunosorbent assay

qPCR

Quantitative PCR