DCN Diagnostics

Summary 

A new method of conjugating antibodies to particles was compared to covalent conjugation in a lateral flow assay for human Chorionic Gonadotropin (hCG). Anteo Technologies will be launching a new Lateral Flow Coupling Kit at AACC 2016 that contains Anteo’s patented Activation Reagent with all necessary buffers for conjugating antibodies to a range of particles used for lateral flow applications. Here we demonstrate the performance advantages of using activated magnetic particles, only requiring the addition of antibody solution and blocking. Keeping all other variables constant, this kit was directly compared to covalently (EDAC chemistry) conjugated magnetic particles of the same type using visual detection as the readout. In this hCG study, this coupling kit achieves five times more sensitivity than the covalently conjugated magnetic particle based assay using half the amount of antibody.

 

Comparison of Anteo’s Activated Magnetic Particles and Covalently Conjugated Magnetic Particles in a Lateral Flow Assay for Human Chorionic Gonadotropin (hCG)

Method                      

The hCG assay has previously been developed using colloidal gold at DCN Diagnostics, Carlsbad, CA for internal use and demonstration purposes. The same assay materials (membrane, pads, and antibodies) were used with Anteo’s technology and covalently conjugated magnetic particles.

                            

All materials and tests were performed in DCN’s laboratories using magnetic particles conjugated with anti-hCG antibody using Anteo’s protocol and DCN’s standard covalent conjugation procedure. hCG concentrations from 0 to 250 mIU/ml were prepared in 1%BSA, 0.1%Tw- 20 1xPBS and in negative pooled human urine. Tests were performed using 100ul of sample and test strips with the covalently conjugated and Anteo’s conjugated particles dried onto a conjugate pad. Five replicates for each concentration were tested and results were recorded 15 minutes after the addition of the sample using DCN’s visual grading scale and an ESE Quant (Qiagen Lake Constance, Germany) reflectance reader.

                            

The amount of antibody conjugated to the different particles and the amount of particles used per test were optimised to produce the highest sensitivity. Anteo’s conjugate was tested with an antibody loading of 25ug of anti-hCG antibody per mg of particles at 0.05% solids. The covalently conjugated particle was tested with an antibody loading of 50ug of anti-hCG per mg particles at 0.05% solids.

Results                            

The results for the visual grades and the values recorded by the reflectance reader, including standard deviation and %CV are shown in Table 1 for each of the two tests. The limit of detection for the Anteo assay was ~10 mIU/ ml hCG in buffer and ~25 mIU/ml in urine for the visual and reader based results. The limit of detection for the covalently conjugated hCG assay was ~50 mIU/mL in buffer and ~100 mIU/mL in urine. As a reference the limit of the detection for the optimised colloidal gold assay is at ~2.5 mIU/mL in buffer and ~25 mIU/mL in urine.

                            

NOTE: A visual grade of 2 or higher at the test line is considered positive, while the cut-off for the reader is at 30mV.

 

 

 

 
 
 
 
 
 

Conclusion

The direct comparison of the two tests show:                                                    

  • The Anteo technology based hCG assay is 5 times more sensitive than the covalently conjugated magnetic particle based test using the same critical reagents.

  • The estimated limit of detection for the Anteo assay is ~10 mIU/ mL of hCG in buffer and ~25 mIU/mL of hCG in urine while the covalently conjugated hCG assay has limits of detection of ~50 and 100 mIU/ml of hCG in buffer and urine respectively.

  • The hCG assay using the Anteo particles used half the amount of antibody to achieve better sensitivity than the covalently conjugated assay.

  • The variability of the Anteo technology based hCG assay is lower than the covalently conjugated hCG assay. There is also less non-specific binding with the Anteo particles.

"The Anteo technology based magnetic particle hCG lateral flow assay produced a comparable limit of detection to the optimised colloidal gold reference assay of ~25 mIU/mL in urine. This confirms that particles conjugated with the Anteo Activation Reagent can rival traditionally used gold colloids in lateral flow applications and bring with it benefits associated with cost, handling and manufacturability improvements."