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Critical quality attributes of rapid test kits – A practical overview

Article  in  Journal of Critical Reviews · August 2020

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 JOURNAL OF CRITICAL REVIEWS
ISSN- 2394-5125 VOL 7, ISSUE 19, 2020

“CRITICAL QUALITY ATTRIBUTES OF RAPID TEST

KITS – A PRACTICAL OVERVIEW”
Prince Manta1*, Deepak N Kapoor2, Gurmeet Kour 3, Manpreet Kour 4, Abhishek Kumar Sharma5
1,2,5
School of Pharmaceutical Sciences, Shoolini University, Bajhol, Solan, Himachal Pradesh 173229, India.
3
Department of Zoology, Savitribai Phule Pune University, Ganeshkhind Pune, Maharashtra 411007, India.
4
Division of Veterinary Medicine, Faculty of Veterinary Sciences & Animal Husbandry, SKUAST-Jammu, Jammu
& Kashmir 181102, India.
Email:- princemanta@gmail.com1

ABSTRACT: Over the last two decades, various Immune assays utilizing gold nanoparticles have been developed
for diagnosis of infectious diseases such as SARS-CoV-2. These tests are very sensitive but sometimes deliver
nonspecific results that lead to diagnostic errors. Thus, reliability of these test kits needs to be examined. Such test
kits are composed of protein conjugated uncut sheets labelled with markers (mostly AuNPs) and nitrocellulose
membrane (NCM) coating. In most countries, the un-cut sheets are purchased from vendors and are assembled to
compose final test kits. Due to specific antigen-antibody-protein design and stability, these sheets often have
specificity issues. For the development of an accurate rapid test kit various factors to be addressed are evaluation of
un-cut sheets, quality attributes during sheet cutting and scale up studies to ensure the effectiveness of kits. Present
review presents a discussion on various critical quality attributes essential for kit assembly in order to enhance the
reliability of the kits and fulfil the regulatory scrutiny.

KEYWORDS: Rapid test kits, immunochromatographic diagnostic kits, lateral flow, gold nanoparticles, COVID-19
Kit.

1.0 INTRODUCTION

In recent years, various methods have been employed for the clinical diagnosis of diseases, including high-
performance liquid chromatography (HPLC), gas chromatographic-mass spectrometry (GC-MS), liquid
chromatographic-mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA) [1]. Although,
these techniques offer several advantages concerning selectivity and sensitivity, most of these techniques are very
time-consuming and require expensive equipment and skilled workers, which limits their practical application. The
gold nanoparticle-based immunochromatographic test kits are an attractive means for the development of stable, easy
to utilize and inexpensive biosensors. These features make the immunochromatographic test kits an ideal candidate
for performing primary screening of samples on-field [2]. The immunochromatographic sheets components are
assembled over the polyvinyl chloride backing [3]. These sheets have various components viz. sample pad, conjugate
pad, nitrocellulose membrane (NCM) and absorbent pad [4]. Figure 1 represents the various components of strips.

Figure 1: A representation of strips and its component

Antigen-antibody specific proteins are conjugated with signal markers such as gold nanoparticles (NPs) on the
conjugated pad [5,6]. The chromatographic test assay is of two types, i.e. Sandwich model and competent model [7].
Conjugated antigen-antibody proteins react with their counter-antigen-antibody proteins on a nitrocellulose
membrane (NCM) at the test and control line [6, 7]. The nature and stability of antigen-antibody proteins and its

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conjugation with gold NPs are critical factors to determine the performance of the kit. A typical sheet is shown in
figure 2.

Figure 2: A typical sheet of size 300 mm X 60 mm and its strips.

In most countries, immunochromatographic un-cut sheets are purchased from another vendor. The process flow chart
for the manufacturing of rapid test kits from sheets assembly to the final release in the market is shown in Figure 3.

Figure 3: Process flow chart for manufacturing of rapid kits from sheets assembly.

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The time frame for interpreting the results vary from 4 minutes to 20 minutes [8]. This itself is a source of variation
in the results optained from the kits.

These strips sometimes give false-positive results for nonspecific binding and sometimes give false-negative results
because of the non-binding of the antigen-antibody complex [9]. It is also seen that on the initial run, a negative
result was interpreted and whereas after 30 minutes a positive colour band was developed on the test line and vice
versa. This leads to a plethora of conflicts about the correct interpretation of the results [9,10]. However, these strips
are recommended for screening then to conformity. The kit must be designed and manufactured in such a way that
they are opportune for the intended purposes. In particular, the kits must achieve performance with sufficient
analytical sensitivity/specificity, diagnostic sensitivity/specificity, precision, repeatability, reproducibility including
regulation of known germane interference and detection limits.The traceability of values assigned to calibrators or
control materials must be assured through reference quantification procedures and reference materials of a higher-
order [10,11]. Two key performance attributes which affect the output of the strips are manufacturing and quality
testing.Thease attributes are discussed below.

2.0 MANUFACTURING ATTRIBUTES: Specific critical parameters to be used in the validation protocol ahead
of sheet cutting , strip assembly into a device and final packing are discussed below.

2.1 Sampling: The sheets are usually wrapped in bags of aluminium. The initial surface screening can be achieved
by 100% aluminium sampling [12]. Each packet should have one sheet sampled. Sample sheets should be assessed
on each of the quality standards as outlined in the attributes of quality testing. The packets should be opened under
controlled environmental conditions and should be re-sealed after sampling the packets. During processing (in-
process) and on finished products the consistency checking should be carried out. Monitoring critical parameters
during manufacturing is very necessary for improved kit efficiency.

2.2 Sheet assembly and its properties analysis: The sheets are mostly 300 mm long and 60 mm wide. Size mostly
depends on the type of device. The sheet consists of a sample pad, red-coloured conjugate pad, nitrocellulose
membrane (NCM) coated with proteins on the test and a control line [13]. These tests are Bi-line (e.g., pregnancy kit)
or Tri-line (e.g., HIV 1/2 kit). These all components are assembled such that specimen sample is dropped in sample
pad and should move by capillary action to conjugate pad [13]. The conjugate pad is composed of protein-gold
colloids, uniformly bonded to glass fibres to compose the most paramount part of the kit [6,13]. The conjugate pad is
red to pink in color. The uniformity of binding can be visually perceived from the intensity of colors. Non-uniformity
of the conjugate pad causes invalid device results. In un-cut sheets, binding of gold colloids to glass fibres is done by
a manual process (dipping and spreading) which sometimes causes faulty conjugation. Ergo, the un-cut sheet with a
non-uniform gold conjugate pad should be rejected.

The second important parameter to be identified in the un-cut sheet is the performance of the nitrocellulose
membrane. The biological marker (antigen and antibody) present in the sample reacts with its counterparts at the test
and control line [1,5]. The nitrocellulose membrane (NCM) should be such that it can have an appropriate pore size
that can facilitate specimen reaction on the test line and control line [4,13]. It is important to locate the spot in a
Nitrocellulose membrane (NCM) where protein coating is absent at the test and control line (mostly located at start
& endpoint). During sheet cutting these portions should be abstracted. The nitrocellulose coating on the membrane
should be smooth without a scratch which could otherwise impact the sample flow through the membrane.

All the above-mentioned parts are mounted on the backrest of a PVC pad [1,4,13]. A cutter should punctiliously cut
the sheet backing into narrow iotas; otherwise, it will cause all sheet components to dislocate.The absorbent pad or
wick is the last parts of the un-cut sheet should be capable of abstracting the extra specimen for obviated the
backflow to stop unspecific binding [4,13].

2.3 Sheet Cutting and assembly: The sheet cutting to individual strips is done by utilizing cutter. The strips are
placed in plastic moulded cassettes [4,13]. The strips should be placed in cassettes with its corresponding indication
(figure 4). The strips are placed in cassettes facing sample pad towards the sample window. Placing the strip in
inversion order causes the malfunctioning of the kits, as the specimens lack capillary flow. Figure 4 represents Bi-
line & Tri-line test strips placed in cassettes showing various indications

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.
Figure 4: A Bi-line & Tri-line cassettes assembled test kit showing its various indications.

2.4 Kit assembly room environment condition: The device should be assembled in a room having low relative
humidity (% RH) [14,15]. Mostly, the percentage of relative humidity below 20% is optimal [14,15]. The
temperature of the room should be maintained below 25 0C [14,15]. Most of the antigen-antibody proteins are stable
in these conditions.

2.5 Strips packing: Kit must be designed, manufactured and packaged in aluminium pouches to minimize the risk of
environmental exposure, and the devices should be stable during conveyance and storage. The thickness and sealing
of the aluminium pouches are very consequential to keep the strips stable for a long period. The aluminium pouch
should be a minimum of 4 millimetres (mm) thick and should be sealed without any leakage. A good quality silica
sachet with optimum weight should be placed along with the cassettes to forfend the kit from humidity exposure
[16].

3.0 QUALITY TESTING ATTRIBUTES: During the in-process and on finally assembled kits the product
evaluation for its consistency should be carried out. Below are the different criteria the kits should be tested on.

3.1 The Reference Specimen Standard: The reference specimen standard should be appropriately characterized
and documented for its purity, identity, sample population, and stability. Therefore the reference standard should be
evaluated for its stability, cross-reactivity, binding and performance with a standard curve. The reference should be
yare in the same matrix in which study samples are to be assayed [10,17,18]. The sample stability should be checked
in the matrix for all the concentration range under stress by a validated method [10]. During method development,
the reference standard should be selected from the quantitation range of the assay, and the concentrations of the
calibration standards are selected on the substratum of the concentration range expected to be identified in particular
kits [19,20]. The simplest model should be chosen that adequately describes the concentration-response relationship..

3.2 The strips specificity and selectivity analysis: Specificity is the ability to quantify the analyte unequivocally in
the presence of other components. In order to avoid any interference, it should be verified that the substance being
quantified is the intended analyte. Generally, the selectivity of the method is routinely demonstrated by analyzing

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blank samples of the congruous biological matrix (e.g., plasma) from multiple sources [10]. Depending on the
intended utilization of the assay, the impact of hemolyzed samples, lipemic specimens or specimens from special
populations can be included in the selectivity assessment [19,20]. Internal standards should be assessed to avoid
interference with the analyte sample. Potential interfering moiety in a biological matrix includes endogenous matrix
components such as metabolites, decomposition products and from the authentic study concomitant medications and
other xenobiotics. If a stabilizer or enzyme inhibitor is utilized during sample amassment, the evaluation of the
potential for interference on the quantitation of the analyte should be done [21]. The percentage specificity of kits
can be calculated by the formula:

(True Negative)
Specificity = X 10𝟎
(True Negative + False negative)

3.3 The strips Sensitivity analysis and dilution linearity: The kits sensitivity is the ability of strips to detect the
biological markers in the specimen [19,20,21]. The percentage sensitivity of strips can be calculated by the formula:

(True Positive)
Sensitivity = X 100
(True Positive + False positive)

The sensitivity analysis can sometimes be utilized as a quantitative implement by calculating the dilution linearity. A
sample with a higher concentration causes profound test bands, whereas a sample with a lower concentration of
biological markers causes a lower band intensity, e.g. in pregnancy test kits, the test line is less profound when hCG
concentration in urine is 25 mIU/ml, and band intensity increment is noticed as the concentration of hCG increases
from 20 mIU/ml to 500 mIU /ml [8]. The band intensity of the test and control line should correspond with a
concentration of marker in specimen and accordingly rating is defined to test device. The calibration curve between
band intensity versus marker concentration in specimen should be plotted. The device should comport accordingly
for its entire shelf life.

3.4 The cut off value: The lower working range (LR) and upper working range (UR) of the kit is the inhibition
within which kit can detect the biological marker in specimens [10,19,20,21]. The kit insert should contain cut off
value information. The kit should perform within the ranges corresponding to the calibre of biological markers
present in infected humans. Many biological markers in the body are sometimes present in very low concentrations
in the absence of disease infection, and any other physiological condition, e.g. β-hCG at 5 mIU/ml in female urine is
considered negative for pregnancy [19,21]. If pregnancy strips are oversensitive to detect 5 mIU/ml β-hCG, then the
results indicated by the pregnancy test strip is considered nonspecific.

3.5 The strip uniformity analysis: Uniformity is the character of strips that produces same results for same
specimens samples when the test is reapeted [19,21]. The kits sensitivity and specificity result should be uniform for
a kit manufactured from particular un-cut sheets. There are quality variability factors associated with un-cut sheets
that affect the uniformity of results given by kits, e.g. protein conjugation process, nature of proteins used, variability
due to gold nanoparticles (NPs), the stability of strips over shelf life. The manufacturer should perform the un-cut
sheet quality assessment before cutting the un-cut sheet into strips. The probability of uniformity of un-cut sheets can
be predicted by calculating the percentage of positive predictive value and percentage negative predictive value [10].
The percentage of positive predictive value is calculated as

(True Positive)
Positive Predictive Value = X 100
(True Positive + False positive)

Similarly, percentage of negative predictive value is calculated as

(True Negative)
Negative Predictive Value = X 100
(True Negative + False negative)

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The kits results are considered to be uniform if the percentage of positive predictive and negative predictive value is
not less than 99.9 %.

3.6 The strips Capillary flow analysis: The specimen sample runs by capillary action after integrating a specimen
to the sample window (fig.4) [1,22]. The different immune-chromatographic test kits operate using various
specimens, e.g. Blood, Serum, Saliva, urine specimens. The specimen's density and its flow through nitrocellulose
membrane (NCM) are critical factors to determine the kit's performance. Sometimes, the specimens do not flow
across the nitrocellulose membrane (NCM) and cause in-valid or false-negative results. Sometimes, blood specimens
illuminate colour on test band resulting in erroneous interpretations (positive) by the operator [10,22]. Hence, the
strip background should become white after the specimen passes through a nitrocellulose membrane (NCM).

3.7 Time limit of results analysis: The immune-chromatographic tests are known for their rapid results, but the time
frame for results interpretation should be well defined in kit inserts to avoid any misinterpretation of results by the
operator. In many cases, it has been perceived that operators interpret results too early or too late, causing
misinterpretation of results. However, the antibody and antigen reaction process in an immunoassay is dynamic, and
the binding of antibody–gold conjugate and the immobilized antigen is perpetual on and off the process in a
particular time window. Perpetual exposure of specimen samples (during the test) may sometimes destabilize the
antigen's hydrophobic binding composing a more tenebrous band or may become lighter depending on the test
causing erroneous results [10]. Consequently, the manufacturer should be particular about the optimal time window
to read the test result. This information should be documented and should be included in the product insert for the
operators to follow while testing.

3.8 The specimen’s quantity required: The manufacturer must validate the kits for the optimal volume of specimen
samples required for antigen-antibody reaction. The specimen volume details should be mentioned in the product
insert. The low sample volume cannot facilitate capillary flow and lacks in forming an antigen-antibody complex.
Similarly, the excessive volume inside the nitrocellulose membrane can bypass the active components [10]. This can
cause flooding of the test kits.

3.9 The buffer study: The lateral flow immunoassay uses the buffer as a specimen diluent and pH stabilizer, e.g.
Covid-19 and HIV test kit. The buffer concentration and pH is a very important factor for kits antigen-antibody
reaction [10,17]. The effect of buffer pH, buffer concentration and buffer stability on device performance should be
well-known to operators.

3.10 Robustness or Ruggedness analysis: Robustness or ruggedness is the ability of kit performance to remains
unaffected by small changes in test parameters. The results of lateral flow immunoassay are interpreted visually. The
laboratory's environmental conditions like lab lux condition and relative humidity ( % RH) affects the kit
performance. The kits qualitative result interpretation entirely depends on the ability of the operator training [10,17].
The kits result from interpretation should remain unaffected for different operators, laboratories condition and inter-
day analysis.

4.0 THE KITS STABILITY STUDY: The shelf life of kits is related to its stability study. Mostly, commercially
available kits have 24 months of expiry. The kit should be validated for its stability at real-time temperature (300C)
and accelerated stability conditions (40 0C and 50 0C). Stress stability and transportation stability should be
performed at -200C and -20C. The stability conditions vary in accordance with different stability zones. The kits
stability studies should have to be performed before market release.

5.0 THE KITS EFFICACY: The kit's efficacy is the competency of the kit to determine the presence and absence
of biological markers in the specimen. Based on validation results, efficiency is calculated by utilizing the given
equation given below.

(True Negative + True Positive)

Efficiency = X 100
(True Negative + True Positive + False negative + Flase positive)

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The false results in the kit do occur from the purchased un-cut sheet. However, the false results can be minimized by
adequately regulating all the manufacturing, quality and stability parameters.

6.0 CONCLUSION: The quality of immuno-chromatographic test kit is defined by merit of its specificity,
sensitivity, speed of analysis and uniformity/precision of results. The immuno-chromatographic test kits
manufactured from the un-cut sheet requires analysis for its accuracy/precision and proper functioning during
production. Manufacturers and operators should have training for all critical quality attributes. The decision of kit
commercialization should be predicated on statistically validated data and kit efficiency. Un-cut sheet, not meetings
the criteria should be rejected.

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