Use of a Novel Technology for the Detection of Human IgG in Non-Human Primate SerumRenée Riffon, Karine Dumaresq-Doiron, Valérie Lavastre and Iohann Boulay

ABSTRACTPurposeDeveloping a rapid TK assay suitable for the detection and quantitation of an analyte in non-human primate serum, specifically human IgG monoclonal antibody (mAb) Test Items, would be of great interest in preclinical research. Historically quantified by traditional ELISA, here we propose an alternative assay with increased efficiencies. MethodA SPARCL® (Spatial Proximity Analyte Reagent Capture Luminescence) assay was used herein to qualify a human IgG quantitation method in non-human primate serum. SPARCL is a novel technology that allows rapid and cost effective immunoassay development, qualification and sample analysis. The principle of the assay lies on reagent proximity in the homogenous phase. Two affinity purified IgG-specific antibodies are used, one conjugated to HRP and the other to Acridan, a chemiluminescent substrate. When these two antibodies are brought into close proximity due to the specific binding and when a solution containing hydrogen peroxide is added, HRP catalyzes oxidation of the proximal Acridan molecules causing a chemiluminescent reaction, which is captured by a plate-based luminometer.

ResultsFollowing adaptation of a commercially available human IgG SPARCL assay, method qualification was performed. In order to better characterize the performance of the assay, a Test Item-specific standard curve was prepared, ranging from 0.4 to 100 ng/mL. Alternatively, a standard curve can be prepared using the human IgG provided with the kit. A series of Positive Control (PC) samples (High-Medium-Low) were used to define accuracy and precision, dilution linearity and hook effect (important for homogenous assay formats) and minimum required dilution.

ConclusionA novel method for the quantitation of human IgG in non-human primate serum samples was successfully qualified. The SPARCL assay is found to present short assay run time since no wash steps are required. It allowed a high throughput for method qualification and sample analysis, with an analytical range that covered relevant concentrations suitable for a monoclonal antibody TK assay.

METHODOLOGYSPARCLTM (Spatial Proximity Analyte Reagent Capture Luminescence) technology is a proximity dependent, non-separation, chemiluminescent detection method, in which a chemiluminescent substrate (Acridan) is brought into the proximity of an oxidative enzyme (horseradish peroxidase, HRP) through the specific antigen/antibody interaction. A flash of light proportional to the quantity of analyte present in the sample is generated on the addition of a trigger solution containing H2O2 and para-hydroxycinnamic acid (pHCA). There is no need to remove excess reactants, as Acridan conjugated antibodies distant from HRP produce no signal. Furthermore, to enhance signal to noise ratio, a background reducing agent can be added to minimize the background signal from unbound reactants (1).

Dilution LinearityDilution linearity and hook effect samples were prepared with TI-1 and TI-2 in Rhesus and Cynomolgus monkey serum, respectively. Two hook effect samples were tested for TI-1 to better characterize this critical parameter for this homogenous assay. A hook effect started to be observed for TI-1 at 1,000 ng/mL, corresponding to 10X the ULOQ concentration. To overcome the hook effect observed, one should carefully determine the dilution fold used during sample analysis based on the Test Item administered concentration. TI-2 is an example where dilution linearity would be used to establish the MRD of the assay. In this case, an MRD of 1/500 should be used to overcome the dilution linearity recovery issue at dilutions between 1/625 and 1/2,500.

INTRODUCTIONTest Items Test Item 1 (TI-1) is a monoclonal antibody (IgG1) targeting a viral protein that was used in a Rhesus monkey preclinical toxicity study. TI-1 was previously measured by ELISA with a quantitation range from 0.4 to 100 ng/mL and an assay time of 4 hours (preceded by overnight coating incubation). Test Item 2 (TI-2) is a monoclonal antibody (IgG2) for the treatment of a kidney disease that was used in a Cynomolgus monkey preclinical PD study. TI-2 was previously measured by ELISA with a quantitation range from 1.1 to 70 ng/mL and an assay time of 3.5 hours (preceded by overnight coating incubation).

For both Test Items, the minimum required dilution (MRD) was 1/100 for the SPARCL and ELISA methods. In the SPARCL assay evaluated herein, the standard curve range tested for both Test Items was 0.4 to 100 ng/mL and the assay time was 1 hour. Assay WorkflowThe human IgG SPARCL kit from Life Diagnostics, Inc. was selected and slightly adapted for use.

- human IgG specific antibodies, one conjugated to HRP and the other to Acridan are mixed with standards, positive controls or samples in a 96-well white plate

- the whole mixture is incubated at room temperature for 60 minutes

- a background reducing agent is added

- once placed in the luminometer, the trigger solution is injected into each well and luminescence is immediately measured.

RESULTSPrecision and Accuracy For this short qualification, precision samples at high, medium and low concentrations were prepared by spiking TI-1 and TI-2 in Rhesus and Cynomolgus monkey serum, respectively and then diluting to the MRD using kit diluent.TI-1 and TI-2 were tested at final concentrations of 75, 8.0 and 2.0 ng/mL.

Figure 1. SPARCL key components. A. HRP labeled Antibody. B. Immunoassay target (analyte). C. 96-well low binding white plate. D. Acridan labeled Antibody. E. SPARCL Kit. F. Luminometer.

Figure 2. A representative SPARCL assay. Specific antibody and antigen interaction brings Acridan and HRP into close proximity, and the addition of a trigger solution then causes a flash of light.

Table 3. Dilution Linearity of Test Item 1 in Rhesus Monkey Serum Table 4. Dilution Linearity of Test Item 2 in Cynomolgus Monkey Serum

REFERENCES 1. Text and Figures 1 and 2 courtesy of Beckman Coulter Inc. 2. Guidance for Industry: Bioanalytical Method Validation (2001), CDER and CVM.

3. Organisation for Economic Co-operation and Development OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring No 1. Paris, France: ENV/MC/CHEM (98)17.4. deSilva et al., Pharmaceutical Research, 2003, 20:1885. 5. Findlay et al., J. Pharmaceutical and Biomedical Analysis, 2000, 21:1249.

6. Viswanathan et al., Workshop/Conference Report, AAPS Journal, 2007, 9:E30. 7. Kelley M, DeSilva B., AAPS Journal, 2007; 9 (2) Article 17.8. Smolec J. et al., Pharmaceutical Research, 2005, 22:1425

SPARCL is a trademark of Lumigen, Inc. and Beckman Coulter, Inc.

Standard Nominal Concentration (ng/mL)

0.1 1 10 1000

200000

400000

600000

800000

1e6

1.2e6

1.4e6Graph

4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2Std Test Item 1 (Std Test Item 1: Concentration vs ... -9.38e+03 1.17 16.9 1.44e+06 0.999Std Test Item 2 (Std Test Item 2: Concentration vs ... 1.32e+04 1.47 38.3 9.72e+05 0.999

__________Weighting: Fixed

Time (secs)

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 10

200000

400000

600000

800000

1e6

1.2e6

1.4e6

1.6e6

1.8e6

2e6

2.2e6

2.4e6

2.6e6

2.8e6

3e6

3.2e6

3.4e6

Well A1Area Under Curve 1.28e6

Figure 4. Example of a SPARCLTM flash luminescence signal for the highest Test Item 1 standard.Luminescence was measured every 0.02 seconds for 1 second.

Figure 3. Example of Test Item 1 and 2 standard curves using a 4-PL curve fit.

Sample ID

Dilution fold

Nominalconcentration

(ng/mL)

Mean RLUvalue

Mean Result(ng/mL)

RE (%)

Comment

Hook 1 100 500.0 1,069,679.0 1,765.8 > ULOQ A No hook effect

Lin 1 625 80.0 877,700.0 104.8 31.0 -

Lin 2 1,250 40.0 667,486.0 50.6 26.5 -

Lin 3 2,500 20.0 411,653.0 24.9 24.4 -

Lin 4 5,000 10.0 180,108.0 10.7 7.3 -

Lin 5 10,000 5.0 75,763.0 5.0 -0.6 -

Lin 6 20,000 2.5 33,558.0 2.3 -8.8 -

Table 4. Dilution Linearity of Test Item 2 in Cynomolgus Monkey Serum

Note A: ULOQ is 100 ng/mL

Sample ID

Dilution fold

Nominalconcentration

(ng/mL)

Mean RLUvalue

Mean Result(ng/mL)

RE (%)

Comment

Hook 1 100 10,000.0 630,170 13.8 < ULOQ A Hook effect

Hook 2 1,000 1,000.0 1,075,142 42.8 < ULOQ A Hook effect

Lin 1 13,333 75.0 1,265,321 91.9 22.6 -

Lin 2 40,000 25.0 936,230 28.9 15.7 -

Lin 3 120,000 8.3 479,687 9.5 14.1 -

Lin 4 360,000 2.8 138,123 2.6 -6.8 -

Table 3. Dilution Linearity of Test Item 1 in Rhesus Monkey Serum

Note A: ULOQ is 100 ng/mL

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CONCLUSIONA novel method for the quantitation of mAb Test Items in Rhesus and Cynomolgus monkey serum was qualified. The SPARCL assay gave short assay run time since no wash steps are required. It allows for rapid TK method development using the specific mAb and high throughput for method development and qualification with an analytical range comparable to the ELISA method.

Table 1. Test Item 1 Intra-Assay Precision and Accuracy

Table 2. Test Item 2 Intra-Assay Precision and Accuracy