NONISOTOPIC IMMUNOASSAY

New Approaches to Synthetic Calibrators and Controls

for NONISOTOPIC IMMUNOASSAY
by: Dr. Anand Akerkar

INTRODUCTION:
The use of synthetic calibrator matrices in Immunoassays have been previously reported.1

Manufacturers of Immunodiagnostic kits instruments reagents, etc. have regulatory requirements that involves providing controls material to the end user that will assure consistently precise and accurate potential results. Calibrators also sometimes refer as standards and controls with long term, preferably stable at ambient temperature and appropriate but accurate concentration of analyte and accurate assignment and target values are absolutely critical for this purpose. Unfortunately, even though calibrators prepared from human sources, lot to lot variation in their makeup make it very difficult if not impossible to make them behave as patient samples. These variations or properties are sometimes referred to as matrix effects.

Since non-isotopic immunoassay introduced in the early 70s, a number of manufacturers have made several attempts to manufacture or synthesize various matrices, that includes human blood, animal serum and other endogenous or non-endogenous proteins, or protein-like substances in various configurations to mimic human serum. Although some manufacturers were able to synthesize formulation that works well for one or two analytes, lthey were unable to make universal “cocktail” which could be substituted as “matrix solution.” In addition, they encountered another problem associated with protein based calibrators as shown in Table #1′. The presenter and author of this article has developed synthetic matrices under SeraSub~ and UriSub~ which are substitute matrices for serum and urine respectively.’

MATERIALS AND METHODS:
The test kits used were obtained directly from the manufacturers, that included Abbott Laboratories, Chicago, IL, Technicon Corp., Tarrytown, NY, Becton Dickinson, Orangeburg, NY.
The salts and other buffer material was obtained from Sigma Chemicals, St. Louis, MO.

DISCUSSION:
SeraSub(r) is nonbiological organic polymer material which can be used as a base for either standards (calibrators) or controls. Both SeraSub(r) and UriSub(r) are stabile at ambient temperature (20-330C) for at least five years if not more. More interestingly, when analyte such as hapten or antigen is added to the SeraSub(r) or UriSub(r), the stability at room temperature increases tremendously.

We undertook extensive study to determine the usefulness of SerSub(r) and UriSub(r) and their stability after the particular analytes were spiked in. The Study Protocol was as follows: The standard calibrates were prepared using SerSub(r) or UriSub(r) as required. Each container was exposed to 450C, 370C ambient temperature (20-300C) +40C and -200C for one week (to simulate shipping condition) and then stored at ambient temperature for the entire study period which lasted from 11 months to 36 months. The analysis was performed periodically. In all cases it showed no degradation of the product during the study period. The data is shown in Tables 2 to 5.
The data presented in Table No. 2 and Table No. 3 include comparison of patient sample results calculated based on standards generated using SerSub(r) and standards supplied by the manufacturers. The study was conducted after at least four weeks of ambient storage of the calibrators made using SeraSub(r). All the other components were stored according to the manufacturer’s guidelines.

SUMMARY AND CONCLUSION:
Synthetic matrices reported in this paper not only offer stability, compatibility, safety but several other features which are listed below:
1. Safety: Because the material is nonbiological, it does not have the HIV, hepatitis, and other infective possibilities of HSA and other materials. Benefits for the manufacturer are safety, reduced testing and handling cost, and reduced liability. Benefits for the laboratorian are safety and reduced handling cost.
2. Multianalyte calibrators and controls: Because the material has no endogenous substances, it is possible to produce multianalyte calibrators and controls and simple “labeled subsets” as needed without concern about interferences. Therefore, one multianalyte lot will suffice for a complete range of “subset” standards (i.e., B12/folate, thyroid panel, or complete panel as required), and the same matrix can be used for either standard or control without stripping.

Benefits for the manufacturer are:
Single stock, various labels
Interchangeable material for standards and controls
Ease of manufacture of full line
Lower cost of manufacture and quality control

Benefits for the laboratorian are:
Both subset and complete standards or controls can be the same
Reduced cost
Reduced variability

3. No endogenous anal ytes: Because the material is nonbiological, it has no endogenous interfering substances. Hence, the matrix has the following attributes:
No “stripping of unwanted substances” required Easy to make “0” standard
Very low (NSB) nonspecific binding
Benefits for the manufacturer are reduced quality control efforts, improved yield, and reduced cost. The benefit to the laboratorian is simplified user calculations or adjustments.

4. Stability without lyophilization: Because it is nonbiological, the material is not susceptible to degradation due to elevated temperature exposure during typical handling.
This produces the following benefits.

For the manufacturer:
No lyophilization process required
No special packaging
Stable in liquid form
Storable at room temperature
Shippable at ambient temperature
Large single lots possible
Reduced cost of manufacture storage,
packaging, quality control, and shipping

For the laboratorian:
Ready to use without reconstitution, reducing errors and improving ease of use
Easy to store
Reduces lot change problems

5. Consistent properties: Because the material is not derived from a living host, it can be produced in large quantities with consistent properties, therefore eliminating lot-to-lot variations commonly associated with biological matrices. Benefits to the manufacturer include higher yield, lower cost to produce, less inspection required, and the fact that it takes only 1/2 hour to produce a homogeneous solution. The principal benefit to the laboratorian is continuity of quality control due to consistency between lots.

TABLE 1

Disadvantacres of Protein-Based Calibrators

1. Potential shortages in the supply of human blood products due to fewer donors, high donor rejection rates, and improved whole blood utilization resulting in fewer outdated blood units becoming available for in vitro use.
2. Large and expensive inventories must be maintained to minimize screening and validation expenses.
3. The relatively high cost of human and animal products, particularly of purified blood fractions.
4. The variable and frequently diminishing endogenous enzyme levels and their unpredictable effect on calibrator performance.
5. Protein matrices are excellent growth media requiring aseptic manipulation and/or use of effective antimicrobial agents.
6. Presence of viruses and infectious agents in some approved lots due to the limitations of current testing procedures 2.
7. Potential contamination with, and the need for testing for, pathogenic or nonpathogenic microorganisms and viruses, including hepatitis virus and HIV.
8. Potential contamination of protein matrices with unacceptably high levels of endogenous analytes, necessitating in-house processing or purchase of more costly “stripped” protein matrices.
9. Inherent instability of proteins or protein solutions requiring storage at freezer temperatures.
10. Lot-to-lot variability.

 

TABLE 2

ACCURACY STUDY – THERAPEUTIC DRUGS

 

 

TABLE 3

Accuracy Study — Drugs of Abuse

Accuracy Study – Antigen

TABLE 4

Level of Theophylline Recovered

TABLE NO. 5
Stability Recovered
Level of DiGOxin Recovered

REFERENCES:

1. 1.A. Akerkar, Synthetic Calibrator Matrices in Iminunoassays. Recent Developments in Therapeutic Drug Monitoring and Clinical Toxicology, edited by Irving Sunshine,677-682(1992).
2. S.J. Strainer, J.S. Heller, R.W. Cooxnbs, J.V. Parry, D.D.Ho, and J.P. Allain, Markers of HIV infection prior to IgG antibody seropositivity. J.Am.Med.Assoc,262:64-69(1989).